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1.  Development of Tectal Connectivity across Metamorphosis in the Bullfrog (Rana catesbeiana) 
Brain, Behavior and Evolution  2011;76(3-4):226-247.
In the bullfrog (Rana catesbeiana), the process of metamorphosis culminates in the appearance of new visual and visuomotor behaviors reflective of the emergence of binocular vision and visually-guided prey capture behaviors as the animal transitions to life on land. Using several different neuroanatomical tracers, we examined the substrates that may underlie these behavioral changes by tracing the afferent and efferent connectivity of the midbrain optic tectum across metamorphic development. Intratectal, tectotoral, tectotegmental, tectobulbar, and tecto-thalamic tracts exhibit similar trajectories of neurobiotin fiber label across the developmental span from early larval tadpoles to adults. Developmental variability was apparent primarily in intensity and distribution of cell and puncta label in target nuclei. Combined injections of cholera toxin subunit β and Phaseolus vulgaris leucoagglutinin consistently label cell bodies, puncta, or fiber segments bilaterally in midbrain targets including the pretectal gray, laminar nucleus of the torus semicircularis, and the nucleus of the medial longitudinal fasciculus. Developmentally stable label was observed bilaterally in medullary targets including the medial vestibular nucleus, lateral vestibular nucleus, and reticular gray, and in forebrain targets including the posterior and ventromedial nuclei of the thalamus. The nucleus isthmi, cerebellum, lateral line nuclei, medial septum, ventral striatum, and medial pallium show more developmentally variable patterns of connectivity. Our results suggest that even during larval development, the optic tectum contains substrates for integration of visual with auditory, vestibular, and somatosensory cues, as well as for guidance of motivated behaviors.
PMCID: PMC3202948  PMID: 21266803
Medial septum; Metamorphosis; Midbrain; Multisensory convergence; Nucleus isthmi; Optic tectum; Tadpole; Thalamus
2.  Spatial distribution and cellular composition of adult brain proliferative zones in the teleost, Gymnotus omarorum 
Proliferation of stem/progenitor cells during development provides for the generation of mature cell types in the CNS. While adult brain proliferation is highly restricted in the mammals, it is widespread in teleosts. The extent of adult neural proliferation in the weakly electric fish, Gymnotus omarorum has not yet been described. To address this, we used double thymidine analog pulse-chase labeling of proliferating cells to identify brain proliferation zones, characterize their cellular composition, and analyze the fate of newborn cells in adult G. omarorum. Short thymidine analog chase periods revealed the ubiquitous distribution of adult brain proliferation, similar to other teleosts, particularly Apteronotus leptorhynchus. Proliferating cells were abundant at the ventricular-subventricular lining of the ventricular-cisternal system, adjacent to the telencephalic subpallium, the diencephalic preoptic region and hypothalamus, and the mesencephalic tectum opticum and torus semicircularis. Extraventricular proliferation zones, located distant from the ventricular-cisternal system surface, were found in all divisions of the rombencephalic cerebellum. We also report a new adult proliferation zone at the caudal-lateral border of the electrosensory lateral line lobe. All proliferation zones showed a heterogeneous cellular composition. The use of short (24 h) and long (30 day) chase periods revealed abundant fast cycling cells (potentially intermediate amplifiers), sparse slow cycling (potentially stem) cells, cells that appear to have entered a quiescent state, and cells that might correspond to migrating newborn neural cells. Their abundance and migration distance differed among proliferation zones: greater numbers and longer range and/or pace of migrating cells were associated with subpallial and cerebellar proliferation zones.
PMCID: PMC4157608  PMID: 25249943
thymidine analogs; adult cell proliferation; weakly electric fish; electrosensory; teleosts
3.  Regional distribution and migration of proliferating cell populations in the adult brain of Hyla cinerea (Anura, Amphibia) 
Brain research  2007;1159:112-118.
We examined the distribution of adult cell proliferation throughout the brain of an anuran amphibian using 5-bromo-2′-deoxyuridine (BrdU). BrdU, a thymidine analog, is a commonly used cellular marker that is incorporated into actively dividing progenitor cells. Adult green treefrogs, Hyla cinerea, received injections of BrdU and were sacrificed 2 hours, 2 days, 2 weeks, or 30 days later. Immunohistochemistry revealed BrdU-immunopositive (BrdU+) cells to be distributed in ventricular zones throughout the brain. The heaviest concentrations of cells were located in the telencephalon, primarily in the ventrolateral region of the lateral ventricles, and the ventricles of olfactory bulbs. Numerous BrdU+ cells were located around the preoptic and hypothalamic recesses and few around the third ventricle in the diencephalon. Proceeding caudally towards the midbrain, there was a marked decrease in BrdU-labeling and few BrdU+ cells were found in the hindbrain. Consistent with previous studies in ectothermic vertebrates, BrdU+ cells were found predominantly in the ventricular zone (VZ) and immediately adjacent to the VZ; at later time points (i.e., 30 days), the cells appeared to have migrated into parenchymal regions. The extent of cellular proliferation in anurans is similar to that of fishes and reptiles and thus is more widespread compared to mammals.
PMCID: PMC2040256  PMID: 17573049
amphibian; BrdU; cell proliferation; cell migration; ventricular zone
4.  Plasticity of Auditory Medullary-Midbrain Connectivity across Metamorphic Development in the Bullfrog, Rana catesbeiana 
Brain, Behavior and Evolution  2006;69(1):1-19.
On the basis of patterns of anterograde, retrograde, and bi-directional transport of tracers from both the superior olivary nucleus (SON) and the torus semicircularis (TS), we report anatomical changes in brainstem connectivity across metamorphic development in the bullfrog, Rana catesbeiana. In early and late stages of larval development (Gosner stages 25–37), anterograde or bi-directional tracers injected into the SON produce terminal/fiber label in the contralateral SON and in the ipsilateral TS. Between stages 38–41 (deaf period), only sparse or no terminal/fiber label is visible in these target nuclei. During metamorphic climax (stages 42–46), terminal/fiber label reappears in both the contralateral SON and in the ipsilateral TS, and now also in the contralateral TS. Injections of retrograde tracers into the SON fail to label cell bodies in the ipsilateral TS in deaf period animals, mirroring the previously-reported failure of retrograde transport from the TS to the ipsilateral SON during this developmental time. Bilateral cell body label emerges in the dorsal medullary nucleus and the lateral vestibular nucleus bilaterally as a result of SON transport during the late larval period, while cell body label in the contralateral TS emerges during climax. At all larval stages, injections into the SON produce anterograde and retrograde label in the medial vestibular nucleus bilaterally. These data show anatomical stability in some pathways and plasticity in others during larval development, with the most dramatic changes occurring during the deaf period and metamorphic climax. Animals in metamorphic climax show patterns of connectivity similar to that of froglets and adults, indicating the maturation during climax of central anatomical substrates for hearing in air.
PMCID: PMC3257804  PMID: 16912473
Tadpoles; Anurans; Vestibular nucleus complex; Dorsal medullary nucleus; Superior olivary complex; Torus semicircularis; Lipophilic dyes; PHA-L; Cholera toxin; Metamorphosis
5.  Estrogen Regulation of Cell Proliferation and Distribution of Estrogen Receptor-α in the Brains of Adult Female Prairie and Meadow Voles 
Adult female prairie (Microtus ochrogaster) and meadow (M. pennsylvanicus) voles were compared to examine neural cell proliferation and the effects of estrogen manipulation on cell proliferation in the amygdala, ventromedial hypothalamus (VMH), and dentate gyrus of the hippocampus (DG). Unlike prior studies, our study focused on the amygdala and VMH, because they are involved in social behaviors and may underlie behavioral differences between the species. Meadow voles had a higher density of cells labeled with the cell proliferation marker 5-bromo-2′-deoxyuridine (BrdU) in the amygdala and DG than did prairie voles. Treatment with estradiol benzoate (EB) for 3 days increased the density of BrdU-labeled cells in the amygdala, particularly in the posterior cortical (pCorA) and medial (pMeA) nuclei, in meadow, but not prairie, voles. Furthermore, the majority of the BrdU-labeled cells in the pCorA and pMeA displayed either a neuronal or a glial progenitor phenotype, but no species or treatment differences were found in the percentage of neuronal or glial progenitor cells. To understand better estrogen’s effects on adult neurogenesis, we also examined estrogen receptor-α (ERα) distribution. Meadow voles had more ERα-labeled cells in the pCorA and VMH, but not in the pMeA or DG, than did prairie voles. In addition, more than one-half of the BrdU-labeled cells in the amygdala of both species coexpressed ERα labeling. Together, these data indicate that estrogen alters cell proliferation in a species- and region-specific manner, and some of these effects may lie in the specific localization of estrogen receptors in the adult vole brain.
PMCID: PMC3962047  PMID: 15984004
neurogenesis; ERα; amygdala; hypothalamus; progenitor
6.  HDAC1 Regulates the Proliferation of Radial Glial Cells in the Developing Xenopus Tectum 
PLoS ONE  2015;10(3):e0120118.
In the developing central nervous system (CNS), progenitor cells differentiate into progeny to form functional neural circuits. Radial glial cells (RGs) are a transient progenitor cell type that is present during neurogenesis. It is thought that a combination of neural trophic factors, neurotransmitters and electrical activity regulates the proliferation and differentiation of RGs. However, it is less clear how epigenetic modulation changes RG proliferation. We sought to explore the effect of histone deacetylase (HDAC) activity on the proliferation of RGs in the visual optic tectum of Xenopus laevis. We found that the number of BrdU-labeled precursor cells along the ventricular layer of the tectum decrease developmentally from stage 46 to stage 49. The co-labeling of BrdU-positive cells with brain lipid-binding protein (BLBP), a radial glia marker, showed that the majority of BrdU-labeled cells along the tectal midline are RGs. BLBP-positive cells are also developmentally decreased with the maturation of the brain. Furthermore, HDAC1 expression is developmentally down-regulated in tectal cells, especially in the ventricular layer of the tectum. Pharmacological blockade of HDACs using Trichostatin A (TSA) or Valproic acid (VPA) decreased the number of BrdU-positive, BLBP-positive and co-labeling cells. Specific knockdown of HDAC1 by a morpholino (HDAC1-MO) decreased the number of BrdU- and BLBP-labeled cells and increased the acetylation level of histone H4 at lysine 12 (H4K12). The visual deprivation-induced increase in BrdU- and BLBP-positive cells was blocked by HDAC1 knockdown at stage 49 tadpoles. These data demonstrate that HDAC1 regulates radial glia cell proliferation in the developing optical tectum of Xenopus laevis.
PMCID: PMC4366096  PMID: 25789466
7.  Determination of key aspects of precursor cell proliferation, cell cycle length and kinetics in the adult mouse subgranular zone 
Neuroscience  2007;146(1):108-122.
Neurogenesis studies on the adult mouse hippocampal subgranular zone (SGZ) typically report increases or decreases in proliferation. However, key information is lacking about these proliferating SGZ precursors, from the fundamental - what dose of bromodeoxyuridine (BrdU) is appropriate for labeling all S phase cells? - to the detailed - what are the kinetics of BrdU-labeled cells and their progeny? To address these questions, adult C57BL/6J mice were injected with BrdU and BrdU-immunoreactive (IR) cells were quantified. Initial experiments with a range of BrdU doses (25-500 mg/kg) suggested that 150 mg/kg labels all actively dividing precursors in the mouse SGZ. Experiments using a saturating dose of BrdU suggested BrdU bioavailability is less than 15 minutes, notably shorter than in the developing mouse brain. We next explored precursor division and maturation by tracking the number of BrdU-IR cells and colabeling of BrdU with other cell cycle proteins from 15 min to 30 days after BrdU. We found that BrdU and the G2/M phase protein pHisH3 maximally colocalized 8 hr after BrdU, indicating that the mouse SGZ precursor cell cycle length is 14 hr. In addition, triple labeling with BrdU and PCNA and Ki-67 showed that BrdU-IR precursors and/or their progeny express these endogenous cell cycle proteins up to 4 days after BrdU injection. However, the proportion of BrdU/Ki-67-IR cells declined at a greater rate than the proportion of BrdU/PCNA-IR cells. This suggests that PCNA protein is detectable long after cell cycle exit, and that reliance on PCNA may overestimate the length of time a cell remains in the cell cycle. These findings will be critical for future studies examining the regulation of SGZ precursor kinetics in adult mice, and hopefully will encourage the field to move beyond counting BrdU-IR cells to a more mechanistic analysis of adult neurogenesis.
PMCID: PMC2230096  PMID: 17307295
BrdU; PCNA; pHisH3; Ki-67; mitosis; neurogenesis
8.  Thymidine analog methods for studies of adult neurogenesis are not equally sensitive 
Adult neurogenesis is often studied by labeling new cells with the thymidine analog bromodeoxyuridine (BrdU) and using immunohistochemical methods for their visualization. Using this approach, considerable variability has been reported in the number of new cells produced in the dentate gyrus of adult rodents. We examined whether immunohistochemical methods, including BrdU antibodies from different vendors (Vector, BD, Roche, Dako, Novocastra, Accurate) and DNA denaturation pretreatments, alter the quantitative and qualitative patterns of BrdU labeling. We also compared the sensitivity and specificity of BrdU with two other thymidine analogs, iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU). We found that the number of BrdU-labeled cells in the dentate gyrus of adult rats was dependent on the BrdU antibody used but was unrelated to differences in antibody penetration. Even at a higher concentration, some antibodies stained fewer cells (Vector, Novocastra). A sensitive BrdU antibody (BD) was specific for dividing cells; all BrdU-labeled cells stained for Ki67, an endogenous marker of cell proliferation. We also observed that DNA denaturation pretreatments affected the number of BrdU-labeled cells and staining intensity for a marker of neuronal differentiation, NeuN. Finally, we found that IdU and CldU, when used at molarities comparable to those that label the maximal number of cells with BrdU, are less sensitive. These data suggest that antibody and thymidine analog selection, as well as the staining procedure employed, can affect the number of newly generated neurons detected in the adult brain thus providing a potential explanation for some of the variability in the adult neurogenesis literature.
PMCID: PMC2749999  PMID: 19731267
BrdU; IdU; CldU; hippocampus; dentate gyrus; subventricular zone
9.  Identification of Adult Stem Cells in Schwalbe's Line Region of the Primate Eye 
To identify stem cells in the chamber angle of the monkey eye by detection of 5-bromo-2′-deoxyuridine (BrdU) long-term retention.
Four cynomolgus monkeys were treated with BrdU via subcutaneous pumps for 4 weeks. The eyes of two animals were processed immediately thereafter (group 1) while in the other animals, BrdU treatment was discontinued for 4 weeks to allow identification of cells with long-term BrdU retention (group 2). The number of BrdU-positive nuclei was quantified, and the cells were characterized by immunohistochemistry and transmission electron microscopy (TEM).
The number of BrdU-positive cells was higher at Schwalbe's line covering the peripheral end of Descemet's membrane than in Schlemm's canal (SC) endothelium, trabecular meshwork (TM), and scleral spur (SS). Labeling with BrdU in SC, TM, and SS was less intense and the number of labeled cells was smaller in group 2 than in group 1. In contrast, in cells of Schwalbe's line the intensity of BrdU staining and the number of BrdU-positive cells was similar when group 1 and 2 monkeys were compared with each other, indicating long-term BrdU retention. Cells that were BrdU-positive in Schwalbe's line region stained for the stem cell marker OCT4. Details of a stem cell niche in Schwalbe's line region were identified by TEM.
We provide evidence for a niche in the Schwalbe's line region harboring cells with long-term BrdU retention and OCT4 immunoreactivity. The cells likely constitute a population of adult stem cells with the capability to compensate for the loss of TM and/or corneal endothelial cells.
Schwalbe's line region in the monkey eye harbors cells with long-term BrdU retention and OCT4 immunoreactivity. The cells likely constitute a population of adult stem cells with the capability to compensate for the loss of TM and/or corneal endothelial cells.
PMCID: PMC4575086  PMID: 25324280
Schwalbe's line; stem cells; BrdU retention; monkey eye
10.  Proliferation dynamics of germinative zone cells in the intact and excitotoxically lesioned postnatal rat brain 
BMC Neuroscience  2005;6:26.
The forebrain subventricular zone (SVZ)-olfactory bulb pathway and hippocampal subgranular zone (SGZ) generate neurons into adulthood in the mammalian brain. Neurogenesis increases after injury to the adult brain, but few studies examine the effect of injury on neural and glial precursors in the postnatal brain. To characterize the spatio-temporal dynamics of cell proliferation in the germinative zones, this study utilized a model of postnatal damage induced by NMDA injection in the right sensorimotor cortex at postnatal day 9.
Dividing cell populations were labeled with 5-Bromodeoxyuridine (BrdU) in the intact and damaged postnatal brain. Identity of proliferating cells was determined by double immunolabeling with nestin, GFAP, NeuN and tomato lectin (TL).
In the control brain, grouped BrdU+ cells were observed in the Rostral Migratory Stream (RMS), SVZ and SGZ. Maximal proliferation was seen at P12, persisted until P23 and diminished by P49. After injury, a striking reduction in the number of BrdU+ cells was observed in the ipsilateral SVZ from 10 hours (58% decrease) until 14 days post-lesion (88% decrease). In contrast, an increase in grouped BrdU+ cells was seen in the striatum adjacent to the depleted SVZ. Significantly reduced numbers of BrdU+ cells were also seen in the RMS until 3 days post-lesion. No changes were noted in the SGZ. Both in controls and lesioned hemispheres, BrdU+ cells located in the germinal zones were mostly nestin positive and negative for GFAP, NeuN, and TL. In the SVZ area lining the ventricle, BrdU+/nestin+ cells were mainly located between TL+ ependyma and parenchymal GFAP+ astrocytes. After excitotoxicity, a decrease in the number and orientation of GFAP/nestin+ prolongations leaving the SVZ to the cortex, corpus callosum and striatum was noted until 5 days post-lesion.
Postnatal excitotoxic injury differentially affects proliferating cells in the germinative zones: no change is observed in the dentate gyrus whereas excitotoxicity causes a significant decrease in proliferating cells in the SVZ and RMS. Depletion of BrdU+ cells in the postnatal SVZ and RMS differs from previous studies after adult brain injury and may affect the SVZ-RMS migration and is suggestive of progenitor recruitment to injured areas.
PMCID: PMC1087489  PMID: 15826306
11.  Immunochemical Detection and Isolation of DNA from Metabolically Active Bacteria 
Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community.
PMCID: PMC91166  PMID: 10049885
12.  The cell cycle–apoptosis connection revisited in the adult brain 
The Journal of Cell Biology  2005;171(4):641-650.
Adult neurogenesis is studied in vivo using thymidine analogues such as bromodeoxyuridine (BrdU) to label DNA synthesis during the S phase of the cell cycle. However, BrdU may also label DNA synthesis events not directly related to cell proliferation, such as DNA repair and/or abortive reentry into the cell cycle, which can occur as part of an apoptotic process in postmitotic neurons. In this study, we used three well-characterized models of injury-induced neuronal apoptosis and the combined visualization of cell birth (BrdU labeling) and death (Tdt-mediated dUTP-biotin nick end labeling) to investigate the specificity of BrdU incorporation in the adult mouse brain in vivo. We present evidence that BrdU is not significantly incorporated during DNA repair and that labeling is not detected in vulnerable or dying postmitotic neurons, even when a high dose of BrdU is directly infused into the brain. These findings have important implications for a controversy surrounding adult neurogenesis: the connection between cell cycle reactivation and apoptosis of terminally differentiated neurons.
PMCID: PMC2171559  PMID: 16291699
13.  Neurodistribution of Androgen Receptor Immunoreactivity in the Male Frog, Rana esculenta 
Sexual behavior in vertebrates depends on the cyclic release of steroids and their binding to the brain receptors. Previously, we demonstrated the presence of specific binding of 3H-testosterone and staining with PG-21 in the brain of the adult male frog, Rana esculenta. Here, we report our further receptor characterization using an anti–androgen receptor antiserum, PG-21, and the androgen site of action in frog brain. Nuclei, which contained cells labeled for the androgen receptor (AR), were mainly identified in the olfactory bulbs, preoptic-septal region, infundibulum, amygdala, thalamus, tectum, torus semicircularis, and medulla. The neuroanatomical AR staining appears similar to that in other lower vertebrates.
PMCID: PMC2967251  PMID: 15891054
androgen receptor; amphibian; brain; PG-21
14.  Reduced Proliferation in the Adult Mouse Subventricular Zone Increases Survival of Olfactory Bulb Interneurons 
PLoS ONE  2012;7(2):e31549.
Neurogenesis in the adult brain is largely restricted to the subependymal zone (SVZ) of the lateral ventricle, olfactory bulb (OB) and the dentate subgranular zone, and survival of adult-born cells in the OB is influenced by factors including sensory experience. We examined, in mice, whether survival of adult-born cells is also regulated by the rate of precursor proliferation in the SVZ. Precursor proliferation was decreased by depleting the SVZ of dopamine after lesioning dopamine neurons in the substantia nigra compacta with 6-hydroxydopamine. Subsequently, we examined the effect of reduced SVZ proliferation on the generation, migration and survival of neuroblasts and mature adult-born cells in the SVZ, rostral migratory stream (RMS) and OB. Proliferating cells in the SVZ, measured by 5-bromo-2-deoxyuridine (BrdU) injected 2 hours prior to death or by immunoreactivity against Ki67, were reduced by 47% or 36%, respectively, 7 days after dopamine depletion, and by 29% or 31% 42 days after dopamine depletion, compared to sham-treated animals. Neuroblast generation in the SVZ and their migration along the RMS were not affected, neither 7 nor 42 days after the 6-hydroxydopamine injection, since the number of doublecortin-immunoreactive neuroblasts in the SVZ and RMS, as well as the number of neuronal nuclei-immunoreactive cells in the OB, were stable compared to control. However, survival analysis 15 days after 6-hydroxydopamine and 6 days after BrdU injections showed that the number of BrdU+ cells in the SVZ was 70% higher. Also, 42 days after 6-hydroxydopamine and 30 days after BrdU injections, we found an 82% increase in co-labeled BrdU+/γ-aminobutyric acid-immunoreactive cell bodies in the granular cell layer, while double-labeled BrdU+/tyrosine hydroxylase-immunoreactive cell bodies in the glomerular layer increased by 148%. We conclude that the number of OB interneurons following reduced SVZ proliferation is maintained through an increased survival of adult-born precursor cells, neuroblasts and interneurons.
PMCID: PMC3283653  PMID: 22363671
15.  Immortalized, premalignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA 
During selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents, simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy.
The glands of 3-week-old immune-competent Balb/C female mice were used intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-Bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells that retain their template DNA. Nulliparous mice were then either injected with [3H]-thymidine (3H-TdR) to distinguish 5-BrdU label-retaining cells that enter the cell cycle and euthanized, or mated, injected with 3H-TdR, and euthanized at various days after coitus. Sections were stained for estrogen receptor-α (ER-α) or progesterone receptor (PR) with immunohistochemistry. Cells labeled with both 5-BrdU and 3H-TdR were indicative of label-retaining epithelial cells (LRECs).
Cells that retained a 5-BrdU label and cells labeled with [3H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labeled nuclei (LRECs) were found in the intact mammary glands of both pregnant and nulliparous mice, and in mammary glands implanted with premalignant cells. Double-labeled cells (3H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-α or PR positive. LRECs distributed their second label (3H-TdR) to daughter cells, and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary-implant groups.
The results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-α and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.
PMCID: PMC3096979  PMID: 20964820
Neuroscience  2012;222:417-428.
Exposure to ethanol during the prenatal period contributes to increased alcohol consumption and preference in rodents and increased risk for alcoholism in humans. With studies in adult animals showing the orexigenic peptides, enkephalin (ENK), galanin (GAL) and orexin (OX), to stimulate ethanol consumption, the question addressed here is whether prenatal ethanol alters the development in utero of specific neurons that express these peptides. With reports describing suppressive effects of high doses of ethanol, we examined the offspring of dams gavaged from embryonic day 9 to parturition with a control solution or lower ethanol doses, 1 and 3 g/kg/day, known to promote ethanol consumption in the offspring. To understand underlying mechanisms, measurements were taken in postnatal offspring of the expression of ENK in the hypothalamic paraventricular nucleus (PVN) and nucleus accumbens (NAc), GAL in the PVN, and OX in the perifornical lateral hypothalamus (PFLH) using real-time qPCR and in situ hybridization, and also of the cell proliferation marker, 5-bromo-2-deoxyuridine (BrdU), and its double-labeling with either neuronal nuclei (NeuN), a marker of mature neurons, or the peptides. On postnatal day 15 (P15), after two weeks without ethanol, the offspring showed increased expression of ENK in the PVN and NAc core but not shell, GAL in the PVN, and OX in the PFLH. In these same areas, prenatal ethanol compared to control increased the density at birth (P0) of neurons expressing these peptides and at P0 and P15 of neurons double-labeling BrdU and NeuN, indicating increased neurogenesis. These BrdU-positive neurons were found to express ENK, GAL and OX, indicating that prenatal ethanol promotes neurogenesis in these specific peptide systems. There were no changes in gliogenesis or apoptosis. This increase in neurogenesis and density of peptide-expressing neurons suggests the involvement of these hypothalamic and accumbal peptide systems in mediating the increased alcohol consumption observed in prenatal ethanol-exposed offspring.
PMCID: PMC3605889  PMID: 22742906
prenatal ethanol; hypothalamus; nucleus accumbens; orexigenic peptides; neurogenesis
17.  Housing in Environmental Complexity Following Wheel Running Augments Survival of Newly-Generated Hippocampal Neurons in a Rat Model of Binge Alcohol Exposure during the Third Trimester Equivalent 
Binge-like alcohol exposure in neonatal rats during the brain growth spurt causes deficits in adult neurogenesis in the hippocampal dentate gyrus (DG). Previous data from our lab demonstrated that twelve days of voluntary wheel-running (WR) beginning on postnatal day (PD) 30 significantly increased the number of newly-generated cells evident in the DG on PD42 in both alcohol-exposed and control rats, but 30 days later a sustained beneficial effect of WR was evident only in control rats. This study tested the hypothesis that housing rats in environmental complexity (EC) following WR would promote survival of the newly-generated cells stimulated by WR, particularly in alcohol-exposed rats.
On PD4-9, pups were intubated with alcohol in a binge-like manner (5.25g/kg/day), sham-intubated, or reared normally. In Experiment 1, animals were either assigned to WR during PD30-42 or were socially housed (SH). On PD42, animals were injected with bromodeoxyuridine (BrdU; 200mg/kg) and perfused two hours later to confirm the WR-induced stimulation of proliferation. In Experiment 2, all animals received WR on PD30-42 and were injected with BrdU on the last full day of WR. On PD42, animals were randomly assigned either to EC (WR/EC) or SH (WR/SH) for 30 days and subsequently perfused and brains were processed for immunohistochemical staining to identify BrdU+, Ki67+ and BrdU+/NeuN+ labeled cells in DG.
In Exp. 1, WR exposure significantly increased the number of proliferating cells in all three postnatal conditions. In Exp. 2, the alcohol-exposed rats given WR/SH had significantly fewer BrdU+ cells compared to control rats given WR/SH. However, WR/EC experience significantly increased the number of surviving BrdU+ cells in both the alcohol-exposed and sham-intubated groups compared to WR/SH rats of the same neonatal treatment. Approximately 80% of the surviving BrdU+ cells in the DG across the conditions were co-labeled with NeuN.
WR followed by EC could provide a behavioral model for developing interventions in humans to ameliorate hippocampal-dependent impairments associated with fetal alcohol spectrum disorders.
PMCID: PMC4543282  PMID: 22324755
hippocampal neurogenesis; fetal alcohol spectrum disorders; rehabilitation; environmental enrichment; neuroplasticity
18.  Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens 
Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1-containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.
PMCID: PMC1692227
19.  Second-order projection from the posterior lateral line in the early zebrafish brain 
Neural Development  2006;1:4.
Mechanosensory information gathered by hair cells of the fish lateral-line system is collected by sensory neurons and sent to the ipsilateral hindbrain. The information is then conveyed to other brain structures through a second-order projection. In the adult, part of the second-order projection extends to the contralateral hindbrain, while another part connects to a midbrain structure, the torus semicircularis.
In this paper we examine the second-order projection from the posterior lateral-line system in late embryonic/early larval zebrafish. At four days after fertilization the synaptic field of the sensory neurons can be accurately targeted, allowing a very reproducible labeling of second-order neurons. We show that second-order projections are highly stereotyped, that they vary according to rhombomeric identity, and that they are almost completely lateralized. We also show that the projections extend not only to the contralateral hindbrain and torus semicircularis but to many other brain centers as well, including gaze- and posture-controlling nuclei in the midbrain, and presumptive thalamic nuclei.
We propose that the extensive connectivity observed in early brain development reveals a basic scaffold common to most vertebrates, from which different subsets are later reinforced in various vertebrate groups. The large repertoire of projection targets provides a promising system to study the genetic encoding of this differential projection capacity.
PMCID: PMC1693910  PMID: 17147780
20.  The fate of Müller’s glia following experimental retinal detachment: nuclear migration, cell division, and subretinal glial scar formation 
Molecular Vision  2010;16:1361-1372.
To study the fate of Müller’s glia following experimental retinal detachment, using a “pulse/chase” paradigm of bromodeoxyuridine (BrdU) labeling for the purpose of understanding the role of Müller cell division in subretinal scar formation.
Experimental retinal detachments were created in pigmented rabbit eyes, and 3 days later 10 µg of BrdU was injected intravitreally. The retinas were harvested 4 h after the BrdU was administered (i.e., day 3) or on days 4, 7, and 21 post detachment. The tissue was fixed, embedded in agarose, and sectioned at 100 µm. The sections were labeled with various combinations of probes, including anti-vimentin and anti-S100 (as markers for Müller cells), anti-BrdU, anti-phosphohistone H3 (to identify mitotic cells), and the isolectin B4 (to identify macrophages and microglia). Images were captured using an Olympus Fluoview 500 confocal microscope. To aid in our understanding of how Müller cell nuclei undergo cell division, two additional procedures were used: 1) electron microscopy of normal cat and rabbit retinas and 2) a new method using 5-fluorouracil and subsequent anti-BrdU labeling to detect all Müller cell nuclei, using confocal imaging.
Three days after detachment, anti-vimentin labeled all Müller cells, some of which were also labeled with anti-BrdU. On day 4, many of the anti-BrdU-labeled Müller cell nuclei appeared in columns with one labeled nucleus in the inner nuclear layer and another directly sclerad to it in the outer nuclear layer. By day 7, most anti-BrdU-labeled nuclei were observed in subretinal scars. At 3 weeks, some anti-BrdU-labeled nuclei that remained within the retina did not express vimentin or S100. Anti-phosphohistone H3-labeled (i.e., mitotic) cells, some of which were also labeled with anti-BrdU, were only observed in the outer nuclear layer on day 4, and these nuclei were surrounded by an accumulation of vimentin filaments. Isolectin B4-labeled microglia and macrophages also incorporated BrdU and were observed throughout the retina and in subretinal scars during all times of detachment. Electron microscopy and immunofluorescence labeling of the 5-fluorouracil-injected eyes revealed the presence of a unique structural relationship between Müller cell nuclei and intermediate filament proteins.
Following retinal detachment, many Müller cell nuclei initially migrate to the outer retina, undergo mitosis, and eventually reside in subretinal glial scars, suggesting a possible link between the early division of Müller cells and the process of subretinal gliosis. In addition, a subpopulation of anti-BrdU-labeled cells, presumably once Müller cells, appears to stop expressing well accepted Müller cell marker proteins, suggesting a potential dedifferentiation of some of these cells over time. Additionally, Müller cell nuclei may use intermediate filaments as a “track” for migration into the outer retina and later as an important component of cell division by the accumulation of vimentin filaments around the mitotic nuclei.
PMCID: PMC2905639  PMID: 20664798
21.  Adult hippocampal neurogenesis and c-Fos induction during escalation of voluntary wheel running in C57BL/6J mice 
Behavioural brain research  2010;213(2):246-252.
Voluntary wheel running activates dentate gyrus granule neurons and increases adult hippocampal neurogenesis. Average daily running distance typically increases over a period of 3 weeks in rodents. Whether neurogenesis and cell activation are greater at the peak of running as compared to the initial escalation period is not known. Therefore, adult C57BL/6J male mice received 5 days of BrdU injections, at the same age, to label dividing cells during the onset of wheel access or after 21 days during peak levels of running or in sedentary conditions. Mice were sampled either 24 hours or 25 days after the last BrdU injection to measure cell proliferation and survival, respectively. Immunohistochemistry was performed on brain sections to identify the numbers of proliferating BrdU labeled cells, and new neurons (BrdU/NeuN co-labeled) in the dentate gyrus. Ki67 was used as an additional mitotic marker. The induction of c-Fos was used to identify neurons activated from running. Mice ran approximately half as far during the first 5 days as compared to after 21 days. Running increased Ki67 cells at the onset but after 21 days levels were similar to sedentary. Numbers of BrdU cells were similar in all groups 24 hours after the final injection. However, after 25 days, running approximately doubled the survival of new neurons born either at the onset or peak of running. These changes co-varied with c-Fos expression. We conclude that sustained running maintains a stable rate of neurogenesis above sedentary via activity-dependent increases in differentiation and survival, not proliferation, of progenitor cells in the C57BL/6J model.
PMCID: PMC2902602  PMID: 20472002
exercise; wheel running; c-Fos; adult hippocampal neurogenesis; granule cell activation; C57BL/6J
22.  Sexual Phenotype Differences in zic2 mRNA Abundance in the Preoptic Area of a Protogynous Teleost, Thalassoma bifasciatum 
PLoS ONE  2011;6(8):e23213.
The highly conserved members of the zic family of zinc-finger transcription factors are primarily known for their roles in embryonic signaling pathways and regulation of cellular proliferation and differentiation. This study describes sexual phenotype differences in abundances of zic2 mRNA in the preoptic area of the hypothalamus, a region strongly implicated in sexual behavior and function, in an adult teleost, Thalassoma bifasciatum. The bluehead wrasse (Thalassoma bifasciatum) is a valuable model for studying neuroendocrine processes because it displays two discrete male phenotypes, initial phase (IP) males and territorial, terminal phase (TP) males, and undergoes socially-controlled protogynous sex change. Previously generated microarray-based comparisons suggested that zic2 was upregulated in the brains of terminal phase males relative to initial phase males. To further explore this difference, we cloned a 727 bp sequence for neural zic2 from field-collected animals. Riboprobe-based in situ hybridization was employed to localize zic2 signal in adult bluehead brains and assess the relative abundance of brain zic2 mRNA across sexual phenotypes. We found zic2 mRNA expression was extremely abundant in the granular cells of the cerebellum and widespread in other brain regions including in the thalamus, hypothalamus, habenula, torus semicircularis, torus longitudinalis, medial longitudinal fascicle and telencephalic areas. Quantitative autoradiography and phosphorimaging showed zic2 mRNA hybridization signal in the preoptic area of the hypothalamus was significantly higher in terminal phase males relative to both initial phase males and females, and silver grain analysis confirmed this relationship between phenotypes. No significant difference in abundance was found in zic2 signal across phenotypes in the habenula, a brain region not implicated in the control of sexual behavior, or cerebellum.
PMCID: PMC3149650  PMID: 21826240
23.  Hypoxia–Ischemia Induces DNA Synthesis without Cell Proliferation in Dying Neurons in Adult Rodent Brain 
Recent studies suggest that postmitotic neurons can reenter the cell cycle as a prelude to apoptosis after brain injury. However, most dying neurons do not pass the G1/S-phase checkpoint to resume DNA synthesis. The specific factors that trigger abortive DNA synthesis are not characterized. Here we show that the combination of hypoxia and ischemia induces adult rodent neurons to resume DNA synthesis as indicated by incorporation of bromodeoxyuridine (BrdU) and expression of G1/S-phase cell cycle transition markers. After hypoxia–ischemia, the majority of BrdU- and neuronal nuclei (NeuN)-immunoreactive cells are also terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-stained, suggesting that they undergo apoptosis. BrdU+ neurons, labeled shortly after hypoxia–ischemia, persist for >5 d but eventually disappear by 28 d. Before disappearing, these BrdU+/NeuN+/TUNEL+neurons express the proliferating cell marker Ki67, lose the G1-phase cyclin-dependent kinase (CDK) inhibitors p16INK4 and p27Kip1 and show induction of the late G1/S-phase CDK2 activity and phosphorylation of the retinoblastoma protein. This contrasts to kainic acid excitotoxicity and traumatic brain injury, which produce TUNEL-positive neurons without evidence of DNA synthesis or G1/S-phase cell cycle transition. These findings suggest that hypoxia–ischemia triggers neurons to reenter the cell cycle and resume apoptosis-associated DNA synthesis in brain. Our data also suggest that the demonstration of neurogenesis after brain injury requires not only BrdU uptake and mature neuronal markers but also evidence showing absence of apoptotic markers. Manipulating the aberrant apoptosis-associated DNA synthesis that occurs with hypoxia–ischemia and perhaps neurodegenerative diseases could promote neuronal survival and neurogenesis.
PMCID: PMC2590933  PMID: 15564594
neurogenesis; apoptosis; cell cycle; BrdU; ischemia; hypoxia
24.  Association between Toll-Like Receptor 4 Expression and Neural Stem Cell Proliferation in the Hippocampus Following Traumatic Brain Injury in Mice 
Whether or how neural stem cells (NSCs) respond to toll-like receptor 4 (TLR4) in an inflammatory environment caused by traumatic brain injury (TBI) has not been understood. In the present study, association between TLR4 expression and NSCs proliferation in the hippocampus was investigated in a mouse model of TBI using controlled cortical impact (CCI). Hippocampal proliferating cells were labeled with the thymidine analog 5-bromo-2-deoxyuridine (BrdU). In order to identify NSCs, the proliferating cells were further co-labeled with BrdU/sex determination region of Y chromosome related high mobility group box gene 2 (SOX2). Morphological observation on the expression of BrdU, SOX2, and TLR4 in the hippocampus was performed by inmmunofluorescence (IF). Relative quantification of TLR4 expression at the protein and mRNA level was performed using Western blotting and real-time polymerase chain reaction (PCR). It was observed that BrdU+/SOX2+ cells accounted for 95.80% ± 7.91% among BrdU+ cells; several BrdU+ cells and SOX2+ cells in the hippocampus were also TLR4-positive post injury, and that BrdU+ cell numbers, together with TLR4 expression at either protein or mRNA level, increased significantly in TBI mice over 1, 3, 7, 14, and 21 days survivals and changed in a similar temporal pattern with a peak at 3 day post-injury. These results indicate that hippocampal proliferating cells (suggestive of NSCs) expressed TLR4, and that there was a potential association between increased expression of TLR4 and the proliferation of NSCs post TBI. It is concluded that hippocampal TLR4 may play a potential role in endogenous neurogenesis after TBI.
PMCID: PMC4139865  PMID: 25036030
toll-like receptor 4 (TLR4); neural stem cells (NSCs); hippocampus; traumatic brain injury (TBI); mice
25.  Proliferating cell nuclear antigen: a marker for hepatocellular proliferation in rodents. 
Environmental Health Perspectives  1993;101(Suppl 5):211-218.
Two different markers for quantitating cell proliferation were evaluated in livers of control and chemically treated mice and rats. Proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, and bromodeoxyuridine (BrdU), an exogenously administered DNA precursor label, were detected in formalin-fixed, paraffin-embedded tissues using immunohistochemical techniques. The percentage of cells in S phase (labeling indexes, LI) evaluated as PCNA- or BrdU-positive hepatocellular nuclei was compared in recut tissue sections from animals given BrdU by a single IP injection 2 hr before killing the animals. Ten-week-old male B6C3F1 mice and F344 rats were exposed to known mitogenic hepatocarcinogens, Wy-14,643 (WY) in the diet at 0.1% for 2 days or 1,4-dichlorobenzene (DCB) in corn oil by gavage for 2 days (600 mg/kg/day in mice; 300 mg/kg/day in rats). In mice, PCNA and BrdU hepatocyte LI were similar in control, WY-treated, and DCB-treated animals. In rats, PCNA and BrdU gave similar LI in controls and Wy-treated animals. Although PCNA LI was statistically lower than BrdU LI in DCB-treated rats, both PCNA and BrdU LI for DCB-treated rats was increased over LI in control rats. Different patterns of PCNA immunohistochemical staining, interpreted to represent different subpopulations of cells at various phases of the cell cycle, were quantitated using PCNA immunohistochemistry. The proliferating index (PI), defined as the percentage of cells in the cell cycle (G1 + S + G2 + M), was more sensitive than the LI (S phase only) in detecting a chemically induced cell proliferative response.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1519420  PMID: 7912188

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