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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.
doi:10.1159/000322550
PMCID: PMC3202948  PMID: 21266803
Medial septum; Metamorphosis; Midbrain; Multisensory convergence; Nucleus isthmi; Optic tectum; Tadpole; Thalamus
2.  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.
doi:10.1159/000095027
PMCID: PMC3257804  PMID: 16912473
Tadpoles; Anurans; Vestibular nucleus complex; Dorsal medullary nucleus; Superior olivary complex; Torus semicircularis; Lipophilic dyes; PHA-L; Cholera toxin; Metamorphosis
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.
doi:10.1016/j.brainres.2007.05.020
PMCID: PMC2040256  PMID: 17573049
amphibian; BrdU; cell proliferation; cell migration; ventricular zone
4.  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.
doi:10.1196/annals.1327.054
PMCID: PMC2967251  PMID: 15891054
androgen receptor; amphibian; brain; PG-21
5.  Birth of neural progenitors during the embryonic period of sexual differentiation in the Japanese quail brain 
The Journal of comparative neurology  2012;520(18):4226-4253.
Several brain areas in the diencephalon are involved in the activation and expression of sexual behavior, including in quail, the medial preoptic nucleus (POM). However, the ontogeny of these diencephalic brain nuclei has not to this date been examined in detail. We investigated the ontogeny of POM and other steroid-sensitive brain regions by injecting quail eggs with 5-bromo-2-deoxyuridine (BrdU) at various stages between E3 and E16 and killing animals at postnatal (PN) days 3 or 56. In the POM, large numbers of BrdU-positive cells were observed in subjects injected from E3 to E10, the numbers of these cells was intermediate in birds injected on E12, and most cells were post-mitotic in both sexes on E14-E16. Injections on E3-E4 labeled large numbers of Hu positive cells in POM. In contrast, injections performed at a later stage labeled cells that do not express aromatase nor neuronal markers such as Hu or NeuN in the POM and other steroid-sensitive nuclei and thus do not have a neuronal phenotype in these locations contrary to what is observed in the telencephalon and cerebellum. No evidence could also be collected to demonstrate that these cells have a glial nature. Converging data, including the facts that these cells divide in the brain mantle and express PCNA, a cell cycling marker, indicate that cells labeled by BrdU during the second half of embryonic life are slow cycling progenitors born and residing in the brain mantle. Future research should now identify their functional significance.
doi:10.1002/cne.23153
PMCID: PMC3522871  PMID: 22628012
preoptic area; sex steroids; neurogenesis; neural progenitors; glial cell; sexual behavior
6.  Neural Proliferation and Restoration of Neurochemical Phenotypes and Compromised Functions Following Capsaicin-Induced Neuronal Damage in the Nodose Ganglion of the Adult Rat 
We previously reported that neuronal numbers within adult nodose ganglia (NG) were restored to normal levels 60 days following the capsaicin-induced destruction of nearly half of the neuronal population. However, the nature of this neuronal replacement is not known. Therefore, we aimed to characterize neural proliferation, neurochemical phenotypes, and functional recovery within adult rat NG neurons following capsaicin-induced damage. Sprague-Dawley rats received intraperitoneal injections of capsaicin or vehicle solution, followed by 5-bromo-2-deoxyuridine (BrdU) injections to reveal cellular proliferation. NG were collected at multiple times post-treatment (up to 300 days) and processed for immunofluorescence, RT-PCR, and dispersed cell cultures. Capsaicin-induced cellular proliferation, indicated by BrdU/Ki-67-labeled cells, suggests that lost neurons were replaced through cell division. NG cells expressed the stem cell marker, nestin, indicating that these ganglia have the capacity to generate new neurons. BrdU-incorporation within β-III tubulin-positive neuronal profiles following capsaicin suggests that proliferating cells matured to become neurons. NG neurons displayed decreased NMDAR expression up to 180-days post-capsaicin. However, both NMDAR expression within the NG and synaptophysin expression within the central target of NG neurons, the NTS, were restored to pre-injury levels by 300 days. NG cultures from capsaicin-treated rats contained bipolar neurons, normally found only during development. To test the functional recovery of NG neurons, we injected the satiety molecule, CCK. The effect of CCK on food intake was restored by 300-days post-capsaicin. This restoration may be due to the regeneration of damaged NG neurons or generation of functional neurons that replaced lost connections.
doi:10.3389/fnins.2011.00012
PMCID: PMC3034227  PMID: 21344007
neurogenesis; injury; BrdU; sensory; TRPV1; vagus
7.  Detection of BrdU-label Retaining Cells in the Lacrimal Gland: Implications for Tissue Repair 
Cell and tissue research  2011;346(3):317-326.
The purpose of the present study was to determine if the lacrimal gland contains 5-bromo-2’-deoxyuridine (BrdU)-label retaining cells and if they are involved in tissue repair. Animals were pulsed daily with BrdU injections for 7 consecutive days. After a chase period of 2, 4, or 12 weeks, the animals were sacrificed and the lacrimal glands were removed and processed for BrdU immunostaining. In another series of experiments, the lacrimal glands of 12-week chased animals were either left untreated or were injected with interleukin 1 (IL-1) to induce injury. Two and half day post-injection, the lacrimal glands were removed and processed for BrdU immunostaining. After 2 and 4 week of chase period, a substantial number of lacrimal gland cells were BrdU+ (11.98 ± 1.84 and 7.95 ± 1.83 BrdU+ cells/mm2, respectively). After 12 weeks of chase, there was a 97% decline in the number of BrdU+ cells (0.38 ± 0.06 BrdU+ cells/mm2), suggesting that these BrdU-label retaining cells may represent slow-cycling adult stem/progenitor cells. In support of this hypothesis, the number of BrdU labeled cells increased over 7-fold during repair of the lacrimal gland (control: 0.41 ± 0.09 BrdU+ cells/mm2, injured: 2.91 ± 0.62 BrdU+ cells/mm2). Furthermore, during repair, among BrdU+ cells 58.2 ± 3.6 % were acinar cells, 26.4 ± 4.1% were myoepithelial cells, 0.4 ± 0.4% were ductal cells, and 15.0 ± 3.0% were stromal cells. We conclude that the murine lacrimal gland contains BrdU-label retaining cells that are mobilized following injury to generate acinar, myoepithelial and ductal cells.
doi:10.1007/s00441-011-1271-x
PMCID: PMC3245820  PMID: 22101331
Progenitor cells; BrdU-label retaining cells; Tissue repair; Lacrimal gland
8.  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.
doi:10.1002/cne.20638
PMCID: PMC3962047  PMID: 15984004
neurogenesis; ERα; amygdala; hypothalamus; progenitor
9.  Intracerebral estrogen provision increases cytogenesis and neurogenesis in the injured zebra finch brain 
Developmental neurobiology  2011;71(2):170-181.
To determine whether or not local, injury-induced aromatization and/orestrogen provision can affect cyto-or neuro-genesis following mechanical brain damage, two groups of adult male zebra finches sustained bilateral penetrating brain injuries. The first received contralateral injections of vehicle or the aromatase inhibitor fadrozole. The second group received contalateral injections of fadrozole, or fadrozole with 17β-estradiol. Subsequent to injury, birds were injected with the thymidine analog 5-Bromo-2′-deoxyuridine (BrdU). Two weeks following injury, the birds were perfused, and coronal sections were labeled using antibodies against BrdU and the neuronal proteins HuC/HuD. In a double blind fashion, BrdU positive cells and BrdU/Hu double-labeled cells in the subventricular zone (SVZ) and at the injury site (INJ) were imaged and sampled. The average numbers of cells per image were compared across brain regions and treatments using repeated measures ANOVAs and, where applicable, post-hoc, pairwise comparisons. Fadrozole administration had no detectable effect on cytogenesis or neurogenesis, however, fadrozole coupled with estradiol significantly increased both measures. The dorsal SVZ had the greatest proportion of new cells that differentiated into neurons, though the highest numbers of BrdU labeled and BrdU, Hu double-labeled cells were detected at the injury site. In the adult zebra finch brain, local estradiol provision can increase cytogenesis and neurogenesis, however, whether or not endogenous glial aromatization is sufficient to similarly affect these processes remains to be seen.
doi:10.1002/dneu.20839
PMCID: PMC3019250  PMID: 20878945
neurogenesis; estrogens; neurosteroids; neuroplasticity; songbirds
10.  Cell Proliferation, Movement and Differentiation during Maintenance of the Adult Mouse Adrenal Cortex 
PLoS ONE  2013;8(12):e81865.
Appropriate maintenance and regeneration of adult endocrine organs is important in both normal physiology and disease. We investigated cell proliferation, movement and differentiation in the adult mouse adrenal cortex, using different 5-bromo-2'-deoxyuridine (BrdU) labelling regimens and immunostaining for phenotypic steroidogenic cell markers. Pulse-labelling showed that cell division was largely confined to the outer cortex, with most cells moving inwards towards the medulla at around 13-20 µm per day, though a distinct labelled cell population remained in the outer 10% of the cortex. Pulse-chase-labelling coupled with phenotypic immunostaining showed that, unlike cells in the inner cortex, most BrdU-positive outer cortical cells did not express steroidogenic markers, while co-staining for BrdU and Ki67 revealed that some outer cortical BrdU-positive cells were induced to proliferate following acute adrenocorticotropic hormone (ACTH) treatment. Extended pulse-chase-labelling identified cells in the outer cortex which retained BrdU label for up to 18-23 weeks. Together, these observations are consistent with the location of both slow-cycling stem/progenitor and transiently amplifying cell populations in the outer cortex. Understanding the relationships between these distinct adrenocortical cell populations will be crucial to clarify mechanisms underpinning adrenocortical maintenance and long-term adaptation to pathophysiological states.
doi:10.1371/journal.pone.0081865
PMCID: PMC3852665  PMID: 24324726
11.  Hippocampal Adult Neurogenesis is Enhanced by Chronic Eszopiclone Treatment in Rats 
Journal of sleep research  2010;19(3):384-393.
Summary
The adult hippocampal dentate gyrus (DG) exhibits cell proliferation and neurogenesis throughout life. We examined the effects of daily administration of eszopiclone (Esz), a commonly used hypnotic drug and GABA agonist, compared to vehicle, on DG cell proliferation and neurogenesis, and on sleep-wake patterns. Esz was administered during the usual sleep period of rats, to mimic typical use in humans. Esz treatment for 7 days did not affect the rate of cell proliferation, as measured by 5-bromo-2’-deoxyuridine (BrdU) immunostaining. However, twice daily Esz administration for two weeks increased survival of newborn cells, by 46%. Most surviving cells exhibited a neuronal phenotype, identified BrdU-NeuN double-labeling. NeuN (Neuronal nuclei) is a marker of neurons. NREM sleep was increased on day one, but not on days 7 or 14 of Esz administration. Delta EEG activity was increased on days 1 and 7 of treatment, but not on day 14.
There is evidence that enhancement of DG neurogenesis is a critical component of the effects of antidepressant treatments of major depressive disorder (MDD). Adult born DG cells are responsive to GABAergic stimulation which promotes cell maturation. The present study suggests that Esz, presumably acting as a GABA agonist, has pro-neurogenic effects in the adult DG. This result is consistent with evidence that Esz enhances antidepressant treatment response of MDD patients with insomnia.
doi:10.1111/j.1365-2869.2010.00833.x
PMCID: PMC2920363  PMID: 20408925
sleep; hippocampus; adult neurogenesis; GABA; hypnotic; MDD
12.  Auditory and Lateral Line Inputs to the Midbrain of an Aquatic Anuran; Neuroanatomic Studies in Xenopus laevis 
Computation of rate in auditory signals is essential to call recognition in anurans. This task is ascribed to a group of central nervous sytem nuclei in the dorsal midbrain or torus semicircularis, homologous to the inferior colliculus of mammals. We have mapped the connections of the subnuclei of the torus semicircularis in Xenopus laevis to determine which receive auditory and which receive lateral line information. Relative to terrestrial anurans, the torus of X. laevis is hypertrophied and occupies the entire caudal, dorsal midbrain. Auditory input to the torus, that arising directly from the dorsal medullary nucleus, is present only in the laminar nucleus. The principal and magnocellular nuclei receive their input from the lateral line nucleus of the medulla. All three nuclei of the torus also have reciprocal connections with the superior olive and the nucleus of the lateral lemniscus. Ascending efferents from all three nuclei of the torus innervate central and lateral thalamic nuclei, and all have a weak reciprocal connection with the posterior thalamus. The laminar and magnocellular nuclei have reciprocal connections with the ventral thalamus, and all three nuclei of the torus receive descending input from the anterior entopeduncular nucleus. The laminar and magnocellular nuclei also receive descending input from the preoptic area. Based on our identification of toral nuclei and these results we assign a major function for the detection of water-borne sounds to the laminar nucleus and a major function for the detection of near field disturbances in water pressure to the principal and magnocellular nuclei.
PMCID: PMC3493254  PMID: 11536185
torus semicircularis; temporal processing; vocalizations; inferior colliculus
13.  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.
doi:10.1016/j.neuroscience.2006.12.064
PMCID: PMC2230096  PMID: 17307295
BrdU; PCNA; pHisH3; Ki-67; mitosis; neurogenesis
14.  Cell proliferation in the Drosophila adult brain revealed by clonal analysis and bromodeoxyuridine labelling 
Neural Development  2009;4:9.
Background
The production of new neurons during adulthood and their subsequent integration into a mature central nervous system have been shown to occur in all vertebrate species examined to date. However, the situation in insects is less clear and, in particular, it has been reported that there is no proliferation in the Drosophila adult brain.
Results
We report here, using clonal analysis and 5'-bromo-2'-deoxyuridine (BrdU) labelling, that cell proliferation does occur in the Drosophila adult brain. The majority of clones cluster on the ventrolateral side of the antennal lobes, as do the BrdU-positive cells. Of the BrdU-labelled cells, 86% express the glial gene reversed polarity (repo), and 14% are repo negative.
Conclusion
We have observed cell proliferation in the Drosophila adult brain. The dividing cells may be adult stem cells, generating glial and/or non-glial cell types.
doi:10.1186/1749-8104-4-9
PMCID: PMC2662830  PMID: 19254370
15.  α-Aminoadipate Induces Progenitor Cell Properties of Müller Glia in Adult Mice 
PURPOSE
Retinal Müller glia in higher vertebrates have been reported to possess progenitor cell properties and the ability to generate new neurons after injury. This study was conducted to determine the signals that can activate this dormant capacity of Müller glia in adult mice, by studying their behavior during glutamate stimulation.
METHODS
Various concentrations of glutamate and its analogue α-aminoadipate, which specifically binds Müller glia, were injected subretinally in adult mice. Proliferating retinal cells were labeled by subretinal injection of 5′-bromo-2′-deoxyuridine (BrdU) followed by immunohistochemistry. Müller cell fates were analyzed in retinal sections by using double immunolabeling with primary antibodies against Müller and other retinaspecific cell markers. The effects of glutamate and α-aminoadipate were also determined in purified Müller cell cultures.
RESULTS
Although high levels of glutamate induce retinal damage, subtoxic levels of glutamate directly stimulate Müller glia to re-enter the cell cycle and induce neurogenesis in vivo and in purified Müller cell cultures. α-Aminoadipate, which selectively target glial cells, also induced expression of progenitor cell markers by Müller cells in vitro or stimulated Müller cell migration to the outer nuclear layer (ONL) and to differentiate into photoreceptors in vivo.
CONCLUSIONS
Mature Müller glia in adult mice can be induced to dedifferentiate, migrate, and generate new retinal neurons and photoreceptor cells by α-aminoadipate or glutamate signaling. The results of this study suggest a novel potential strategy for treating retinal neurodegeneration, including retinitis pigmentosa and age-related macular degeneration, without transplanting exogenous cells.
doi:10.1167/iovs.07-0434
PMCID: PMC2638994  PMID: 18326742
16.  Prozac during puberty: Distinctive effects on neurogenesis as a function of age and sex 
Neuroscience  2009;163(2):609-617.
Neurogenesis is a possible substrate through which antidepressants alleviate symptoms of depression. In adult male rodents and primates, chronic treatment with fluoxetine increases neurogenesis in the hippocampal formation. Little is known about the effects of the antidepressant on neurogenesis during puberty or in female animals at any age. Therefore we examined the effects of chronic fluoxetine treatment on cell proliferation and survival in male and female rats during puberty and adulthood.
Adult and peri-pubescent male and female rats were treated chronically with fluoxetine (Prozac-5 mg/kg) or saline. Subsequently rats received a single injection of 5-bromo-2′-deoxyuridine (BrdU; 200 mg/kg) to label DNA synthesis. Rats were sacrificed 2 hrs, 24 hrs, or 28 days after BrdU injection to examine cell proliferation, survival and cell fate. Fluoxetine increased cell proliferation in adult male rats but not in peri-pubescent males or female rats of any age or stage of the estrous cycle. Treatment did not alter the number of surviving cells in the male hippocampus but decreased survival in the female hippocampus. Thus, fluoxetine has distinctive effects on neurogenesis as a function of age and sex. Circulating levels of the stress hormone corticosterone were also examined. Treatment of female rats with fluoxetine during puberty decreased circulating levels of corticosterone as adults, even in the absence of the drug suggesting disruption of maturation of the hypothalamic-pituitary-adrenal axis.
doi:10.1016/j.neuroscience.2009.06.057
PMCID: PMC2740329  PMID: 19576966
Corticosterone; adolescence; rats; antidepressants; cell proliferation; cell survival
17.  Moderate Traumatic Brain Injury Promotes Neural Precursor Proliferation Without Increasing Neurogenesis In The Adult Hippocampus 
Experimental neurology  2012;239:38-48.
Traumatic brain injury (TBI) promotes neural stem/progenitor cell (NSC) proliferation in the adult hippocampus; however, it remains inconclusive whether proliferation of these cells results in newly generated mature neurons, leading to increased neurogenesis. When we traced the fates of proliferating cells labeled with bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) we found the number of BrdU-positive cells increased in the hippocampus of TBI mice compared to the sham control. However, double immunostaining to distinguish their cell types showed that most of these cells were glia, and that only a small subpopulation is newborn granular neurons. There was no significant difference with respect to neurogenesis in the adult hippocampus between the injured and the control mice. These results indicate that TBI promotes cell proliferation including astrocyte activation and NSC proliferation. Nevertheless, the majority of the BrdU-positive cells are glia. The neurogenesis is not increased by TBI. These data suggest that TBI activates through promotion of NSC proliferation an innate repair and/or plasticity mechanism in the brain. However, additional intervention is required to increase neurogenesis for successfully repairing the damaged brain following TBI.
doi:10.1016/j.expneurol.2012.09.012
PMCID: PMC3755608  PMID: 23022454
Traumatic brain injury; Neurogenesis; Neural stem/progenitor cells; Gliogenesis; Hippocampal dentate gyrus; Subgranular zone
18.  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.
doi:10.1016/j.bbr.2010.05.007
PMCID: PMC2902602  PMID: 20472002
exercise; wheel running; c-Fos; adult hippocampal neurogenesis; granule cell activation; C57BL/6J
19.  Phenotypic transformation and migration of adventitial cells following angioplasty 
The present study was designed to investigate the phenotypic transformation and migration of adventitial fibroblasts using 5-bromo-2′-deoxyuridine (BrdU) labeling following angioplasty and to explore the correlation between adventitial cells and post-angioplasty restenosis. A vascular restenosis model was established in 23 rats by injuring the common carotid artery with a wire. BrDU was used to label the fibroblasts followed by immunohistochemistry for α-actin. Blood vessels were observed under light microscopy and scanning electron microscopy followed by image analysis. The number of BrDU-positive fibroblasts in the intima, media and adventitia of the blood vessels was determined 3, 7, 41 and 28 days after injury. The results demonstrated that at different time points, the number of BrDU-positive cells was significantly different in the intima, media and adventia (P<0.05). Electron microscopy indicated that the fibroblasts were full of cytoplasm. In addition, many secretory granules were noted on the rough endoplasmic reticulum and a large amount of microfilament bundles were noted after angioplasty. The fibroblasts transformed into myofibroblasts. Seven and 14 days after injury, the myofibroblasts formed wide pseudopods stretching to the fenestrae of the external and internal elastic lamina, and cells had a tendency to migrate into the lumen. The fibroblasts in the adventitia underwent transformation after percutaneous transluminal angioplasty and secreted α-actin. In conclusion, the fibroblasts in the adventitia transformed into myofibroblasts, migrated into and proliferated in the intima and became a component of the newly generated intima. Adventitial cells are thus related to vascular restenosis.
doi:10.3892/etm.2012.551
PMCID: PMC3460273  PMID: 23060918
angioplasty; vascular intima; myofibroblast; phenotype; migration
20.  Cell proliferation in the dentate gyrus of the adult rat fluctuates with the light-dark cycle 
Neuroscience letters  2007;422(3):198-201.
This study measured cell proliferation in the hippocampal dentate gyrus in the adult rat at different times within a 12:12 h light-dark cycle. The experiments were conducted in animals living in either a complex environment or in standard lab cages. A single dose of the thymidine analog 5-Bromo-2′–deoxyuridine (BrdU) was injected 2 h before animals were sacrificed either 4, 11, 16, or 23 hours after the beginning of the light phase of the light/dark cycle (designated ZT0). In both studies, we found a significant increase in the number of BrdU-positive cells in the subgranular cell layer (SGZ) following BrdU administration at ZT 9 and sacrifice at ZT11, compared to other circadian times examined. BrdU administration at ZT9 was timed to primarily identify proliferating cells that were in the S phase of the cell cycle during the light phase. Our results suggest that cell proliferation is enhanced either by sleep or by other variables coupled to the light phase of the circadian cycle.
doi:10.1016/j.neulet.2007.06.022
PMCID: PMC2052926  PMID: 17611030
Cell proliferation; dentate gyrus; hippocampus; circadian; BrdU
21.  A Single High Dose of Methamphetamine Induces Apoptotic and Necrotic Striatal Cell Loss Lasting Up to Three Months in Mice 
Neuroscience  2011;193:162-169.
Methamphetamine (METH) is an addictive agent that poses a public health problem due to its toxic effects on neural tissue. We have shown that METH induces striatal lesions (cell loss) within 24 hours of administration. Because cell proliferation has been found to follow excitotoxic and other types of lesions in adult brain, we tested the hypothesis that cell proliferation would follow METH-induced striatal cell death. To that end, METH (30 mg/kg ip) was injected into adult male mice followed by a single injection of the proliferation marker 5-bromo-2’-deoxyuridine (BrdU, 100mg/kg ip) at various times post-METH up to 12 weeks. Immunohistochemical analysis of striatal tissue showed that METH-treated animals incorporated BrdU betweem 24–48 hours post-METH. To determine the survival of the newly generated cells, a sub-group of animals received BrdU 36 hours after METH and were sacrificed at various times up to 12 weeks post-METH. Morphological analysis of striatal tissue from these animals showed that by 12 weeks post-METH, approximately 42% and 30% of the newly generated cells showed pyknotic or necrotic morphology, respectively. Thus, approximately 30% of the newly generated cells survive up to 12 weeks post-METH. Striatal volume was increased by METH and normalized to control levels by 12 weeks after METH. The data demonstrate that a single bolus injection of METH induces cellular changes and responses that persist for months after exposure to METH.
doi:10.1016/j.neuroscience.2011.07.020
PMCID: PMC3171578  PMID: 21771641
methamphetamine; striatum; cytogenesis; proliferation; apoptosis; necrosis
22.  High throughput analysis of neural progenitor cell proliferation in adult rodent hippocampus 
Bioscience trends  2009;3(6):233-238.
Summary
Extensive efforts have been made to determine the status on neural progenitor cell proliferation in specific pathological conditions and to evaluate the therapeutic efficacy of drugs for preventing neurogenic deficits in neurodegenerative diseases. However, the most commonly used stereological analysis using 5-bromo-2′-deoxyuridine (BrdU) immuno-positive sections is a time consuming and labor intensive process and is often a bottle neck in neurogenic drug development, particularly when large sample sizes are needed. In addition, BrdU is toxic to new born neurons and also labels DNA damage in old cells. In this study, we established a method that quantitatively measures the number of Ki-67, an endogenous cell proliferation marker, positive cells by flow cytometry which analyzes extracted cell nuclei from rodent hippocampi in suspension. Our results demonstrate that this approach can be applied to a large number of rodent samples, can be accomplished in a short period of time (1-3 days), and can be completed in a more accurately objective manner than by using 3-D cell counting with immunohistochemically processed sections.
PMCID: PMC2830061  PMID: 20103852
Neurogenesis; high throughput screen; hippocampus; flow cytometry; Ki-67
23.  Socially Modulated Cell Proliferation Is Independent of Gonadal Steroid Hormones in the Brain of the Adult Green Treefrog (Hyla cinerea) 
Brain, Behavior and Evolution  2012;79(3):170-180.
Gonadal steroid hormones have been shown to influence adult neurogenesis in addition to their well-defined role in regulating social behavior. Adult neurogenesis consists of several processes including cell proliferation, which can be studied via 5-bromo-2′-deoxyuridine (BrdU) labeling. In a previous study we found that social stimulation altered both cell proliferation and levels of circulating gonadal steroids, leaving the issue of cause/effect unclear. In this study, we sought to determine whether socially modulated BrdU-labeling depends on gonadal hormone changes. We investigated this using a gonadectomy-implant paradigm and by exposing male and female green treefrogs (Hyla cinerea) to their conspecific chorus or control stimuli (i.e. random tones). Our results indicate that socially modulated cell proliferation occurred independently of gonadal hormone levels; furthermore, neither androgens in males nor estrogen in females increased cell proliferation in the preoptic area (POA) and infundibular hypothalamus, brain regions involved in endocrine regulation and acoustic communication. In fact, elevated estrogen levels decreased cell proliferation in those brain regions in the implanted female. In male frogs, evoked calling behavior was positively correlated with BrdU-labeling in the POA; however, statistical analysis showed that this behavior did not mediate socially induced cell proliferation. These results show that the social modulation of cell proliferation can occur without gonadal hormone involvement in either male or female adult anuran amphibians, and confirms that it is independent of a behavioral response in males.
doi:10.1159/000335037
PMCID: PMC3343747  PMID: 22269468
Amphibian; Cell proliferation; BrdU; Neurogenesis; Acoustic communication; Social behavior; Estrogen; Testosterone
24.  Expression of Nestin, Vimentin, and NCAM by Renal Interstitial Cells after Ischemic Tubular Injury 
This work explores the distribution of various markers expressed by interstitial cells in rat kidneys after ischemic injury (35 minutes) during regeneration of S3 tubules of outer stripe of outer medulla (OSOM). Groups of experimental animals (n = 4) were sacrificed every two hours during the first 24 hours post-ischemia as well as 2, 3, 7, 14 days post-ischemia. The occurrence of lineage markers was analyzed on kidney sections by immunohistochemistry and morphometry during the process of tubular regeneration. In postischemic kidneys, interstitial cell proliferation, assessed by 5-bromo-2′-deoxyuridine (BrdU) and Proliferating Cell Nuclear Antigen (PCNA) labeling, was prominent in outer medulla and reach a maximum between 24 and 72 hours after reperfusion. This population was characterized by the coexpression of vimentin and nestin. The density of -Neural Cell Adhesion Molecule (NCAM) positive interstitial cells increased transiently (18–72 hours) in the vicinity of altered tubules. We have also localized a small population of α-Smooth Muscle Actin (SMA)-positive cells confined to chronically altered areas and characterized by a small proliferative index. In conclusion, we observed in the postischemic kidney a marked proliferation of interstitial cells that underwent transient phenotypical modifications. These interstitial cells could be implicated in processes leading to renal fibrosis.
doi:10.1155/2010/193259
PMCID: PMC2896652  PMID: 20617137
25.  Flow Cytometric Analysis of BrdU Incorporation as a High-Throughput Method for Measuring Adult Neurogenesis in the Mouse 
Introduction
The generation of new neurons occurs throughout adulthood in discrete brain regions, and may be regulated by neuropsychiatric diseases and therapeutic drug treatments. Most current methods that study this process measure the labeling of newborn cells by 5-bromo-2-deoxyuridine (BrdU) using immunohistochemical methods followed by the microscopic counting of BrdU positive cells. This method is time consuming and labor intensive, typically taking several weeks to analyze.
Methods
Therefore, we characterized a method to measure BrdU incorporation in the adult mouse hippocampus in vivo by using flow cytometry, which normally allows analysis of data within a single day.
Results
The present study compared multiple BrdU dosing and loading protocols to determine a dosing strategy that produced the best signal to noise ratio. BrdU incorporation was also compared across different brain regions. The method was sensitive to a number of experimental disease manipulations. Induction of type-1 diabetes and depletion of norepinephrine reduced hippocampal cell proliferation. In contrast, chronic administration of electroconvulsive shock, a somatic treatment for depression, as well as chronic treatment with the antidepressant fluoxetine elevated hippocampal cell proliferation. This increase in cell proliferation with fluoxetine was detected as early as 14 days into treatment. Moreover, comparing measures of cell proliferation obtained by immunohistochemical and flow cytometric methods within the same animals were convergent and significantly correlated to each other. Flow cytometry was also sufficiently sensitive to quantify the survival of newly born cells.
Discussion
These experiments validate the utility of flow cytometry in analyzing hippocampal cell proliferation and survival in a reliable and high-throughput fashion. The speedy analysis afforded by flow cytometry lends itself to be utilized in novel drug discovery and physiology.
doi:10.1016/j.vascn.2008.12.002
PMCID: PMC2701659  PMID: 19121403
Antidepressants; Diabetes; DSP-4; Fluoxetine; Flow cytometry; Hippocampus

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