Immunocytochemical detection of 5′-bromodeoxyuridine incorporation in the central nervous system of the mouse

doi:10.1016/0165-3806(89)90033-3

Developmental Brain Research

Volume 49, Issue 2, 1 October 1989, Pages 311-317

https://doi.org/10.1016/0165-3806(89)90033-3 Get rights and content

Abstract

In order to label postmitotic neuroblasts, pregnant mice received a single pulse of 5′-bromodeoxyuridine (BrdU) between embryonic days E11 and E17. Young adult offspring were stained for BrdU using a monoclonal antibody and the peroxidase-antiperoxidase method. The distribution of immunoreactive nuclei was very similar to the [³H]thymidine data reported in the literature. The pattern of labelled nuclei after injection at different embryonic ages followed the spatiotemporal gradients described for cortical and hippocampal neurogenesis. Lightly and heavily labelled nuclei, probably representing subsequent postmitotic generations, were distinguished in BrdU-immunoreacted preparations. These data indicate that BrdU can be used to map neuronal birthdates.

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Calabash chalk (CaC) is an aluminium silicate hydroxide compound with heavy metal constituents, making it a potential neurotoxicant. Pregnant women often consume CaC as an antiemetic, which may interfere with the normal development of the foetal brain. Here, we evaluated the effects of CaC administration in pregnant rats on the brain of the offspring. Wistar rat dams were assigned to one of three groups: control, 200 mg/kg and 800 mg/kg of a CaC suspension. Administrations lasted 14 days (gestation days 7–20). On day 14, 5-bromo-2′-deoxyuridine (BrdU) was administered and dams were allowed to term. Behavioural tests were performed on different days as the pups matured, and they were sacrificed on post-natal days 30 and 60. Brains were processed for histology and Western blotting. Results showed no significant differences in surface righting reflex, cliff avoidance, negative geotaxis and open-field activity. No hippocampal and somatosensory cortical cytoarchitectonic alterations and no significant signs of glial fibrillary acidic protein (GFAP) activation were observed. Neuronal nuclei counts showed variability in the somatosensory cortex and hippocampus of the CaC group. BrdU-positive cells were significantly lower in the 200 mg/kg group and higher in the 800 mg/kg group. Doublecortin-X-positive cells were not different in all the CaC groups. Astrocytes and microglia Western blotting quantification confirmed no significant increase in pup glial cells in adulthood. Prenatal consumption of CaC at indicated dosages may not be deleterious to the developing brain, especially after cessation of exposure and during maturation of the animal. However, the differences in neuronal and glial populations may be due to their ability to cope with CaC.
Decreased use of spatial pattern separation in contemporary lifestyles may contribute to hippocampal atrophy and diminish mental health
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Citation Excerpt :
Reduction in hippocampal and DG/CA3 dendritic spine size, branching, and density in hibernating ground squirrels [129,130] and hamsters [131] compared to post-arousal measures may also suggest effects of disuse of the spatial pattern separation process on hippocampal and DG/CA3 volume. In addition, environmental enrichment leads to significant increases in hippocampal thickness [132,133], dentate gyrus volume [86], dendritic arborization [134], glial cells [133], size of granule cell layer (the type of cell produced through neurogenesis), and 15% more adult-born neurons [86] in rodents, implying enhanced hippocampal size in enriched rodents compared to standard-housed controls. In a study that exposed rodents to four different levels of enrichment ranging from a non-enriched cage to a semi-natural outdoor condition, exposure to the semi-natural outdoor condition exhibited the greatest effect on cerebral measures including weight [135].
The spatial pattern separation process has not yet been proposed as a pivotal neural activity affecting hippocampal volume, a metric of mental health. The dentate gyrus/CA3 region is increasingly implicated in hippocampal atrophy, its putative role in spatial pattern separation appears to be impaired in mental health disorders, and performance on a task indicative of pattern separation correlates with dentate gyrus/CA3 volume. Spatial pattern separation is thought to utilize the heightened neural plasticity of newborn neurons in the dentate gyrus to distinguish highly similar aspects of scene so that these remain distinct in memory rather than lost. The level of such activity associates with BDNF secretion, which may affect neuroplasticity and therefore hippocampal volume. Distinguishing fine-grained aspects of surroundings was likely of great importance during hunting and gathering for survival in nature. However the need to make subtle environmental discriminations is much reduced in modern survival activity. Ancestrally, exploration and utilization of the spatial pattern separation process may have resulted in detection of potential food items, an activity that was likely followed by intensive effort-based reward (EBR) activity to obtain the food. EBR activity and restorative walking in nature demonstrate positive mental health benefits, while their lack indicates negative effects on mental health. Data support the hypothesis that spatial pattern separation activity and neural circuitry are separate from, and may precede, those of EBR as well as restorative walking in nature. Spatial pattern separation therefore represents an additional nature-related neural process whose modern decrease in use may negatively affect mental health. Interventions that increase spatial pattern separation experiences may enhance BDNF secretion, neuroplasticity, hippocampal volume and mental health.
Systematic differences in time of cerebellar-neuron origin derived from bromodeoxyuridine immunoperoxidase staining protocols and tritiated thymidine autoradiography: A comparative study
2015, International Journal of Developmental Neuroscience
Citation Excerpt :
Given that the present work uses peroxidase staining and brightfield microscopy to infer the times of origin profiles of newly produced neurons, additional experiments are needed to assess the extent to which these findings apply for fluorescence methods. Finally, earlier research on the development of the rodent central nervous system has shown that results obtained with BrdU and [3H]TdR are close (Miller and Nowakoswi, 1988; del Rio and Soriano, 1989). However, there is evidence indicating quantitative differences in the number and placement of tagged neurons in the cerebral cortex of macaque monkeys exposed to either BrdU or [3H]TdR as embryos (Duque and Rakic, 2011, 2015).
As exogenous markers of DNA synthesis, 5-bromo-2′-deoxyuridine (BrdU) and tritiated thymidine ([³H]TdR) have revolutionized our ability to identify proliferating neuroblasts and follow their fate during the development of the central nervous system. The effect of the incorporation of these molecules into DNA on cell proliferation, migration and differentiation is frequently neglected (Duque and Rakic, 2011. J. Neurosci. 31, 15205–15217). By a progressively delayed cumulative labeling method, the current paper analyzes the development of the cerebellum in mice exposed to either BrdU or [³H]TdR as embryos and collected at postnatal day 90. We observed that, in comparison to the saline group, several parameters of the cerebellum such as length of the cerebellar cortex, the area of the molecular layer, Purkinje cell (PCs) number, the areas of the cerebellar nuclei, and the number of the deep cerebellar nuclei (DCN) neurons were lower in the BrdU injected group. No consequence of [³H]TdR administration was observed. On the other hand, we also studied whether immunohistochemical methods, including BrdU antibodies from different vendors (Sigma and Dako), partial DNA denaturation procedures and trypsin pretreatments, alter the neurogenetic timetables of PC and DCN neurons that resulted from analysis of these tissue specimens. Our analysis revealed that the generative programs of these macroneurons were unrelated to differences in the sensibility of BrdU antibodies but were dependent on the partial denaturation of DNA and trypsin digestion protocols. Finally, we also compare the generation and spatial distribution of PC and DCN neurons in mice exposed to either BrdU or [³H]TdR to assess whether the results obtained by these two markers are quantitatively similar. The data presented here show that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar neurons between mice injected with BrdU or [³H]TdR. These findings have implications for the interpretation of results obtained by both exogenous makers as an index of the production, migration and settling of neurons in the developing central nervous system.
Targeting of microglial K<inf>Ca</inf>3.1 channels by TRAM-34 exacerbates hippocampal neurodegeneration and does not affect ictogenesis and epileptogenesis in chronic temporal lobe epilepsy models
2014, European Journal of Pharmacology
Blockade of K_Ca3.1 channels has been suggested as a novel strategy to reduce microglia activation. The concept has been confirmed by neuroprotective effects in a rat brain ischemia-reperfusion model and reduced microglia activation surrounding glioblastomas. Cumulating evidence exists that microglia activation significantly contributes to epileptogenesis as well as intrinsic severity in the chronic epileptic brain. Taken together these data raised the question whether the K_Ca3.1 channel blocker triarylmethane-34 (TRAM-34) might also exert beneficial effects in chronic epilepsy models. In a rat post-status epilepticus model TRAM-34 treatment following the insult did not result in neuroprotective effects. Whereas status epilepticus-associated neurodegeneration remained unaffected in the piriform cortex, loss of pyramidal cells in the hippocampal CA1 and CA3a region and of neuropeptide Y-positive interneurons in the hilus proved to be exacerbated by pharmacological K_Ca3.1 blockade. The development of spontaneous seizures and of behavioral and cognitive alterations was comparable in animals receiving TRAM-34 treatment or the respective vehicle. The kindling model of temporal lobe epilepsy with a massive stimulation paradigm with frequent seizure elicitation in fully kindled rats was used to assess a putative disease-modifying effect. However, sub-chronic TRAM-34 treatment failed to exert relevant effects on seizure generation and thresholds. In conclusion, the data obtained in two different chronic epilepsy models argue against using KCa3.1 blockers as disease-modifying or antiepileptogenic agents. Exacerbation of neuronal cell loss in TRAM-34 pre-treated epileptic animals rather indicates that translational development of the compound needs to carefully consider the pathophysiological mechanisms associated with different brain insults.
Status epilepticus alters neurogenesis and decreases the number of GABAergic neurons in the septal dentate gyrus of 9-day-old rats at the early phase of epileptogenesis
2013, Brain Research
Citation Excerpt :
To study the effects of seizures on neurogenesis, BrdU (Sigma, St. Louis, MO, USA) dissolved in 0.9% NaCl was given (i.p.; 50 mg/kg) to rats 3 h after the KA injection. BrdU is a thymidine analog that incorporates within DNA for ~30 min during the DNA-synthetic cell cycle phase (S phase) (del Rio and Soriano, 1989; Packard et al., 1973). Thus, the single BrdU injection 3 h after KA will label only a proportion of dividing cells during the 30 min time period after the injection.
The effects of a prolonged seizure, i.e. status epilepticus (SE), on neurogenesis of dentate granule cells (DGCs) in the immature dentate gyrus (DG) and possible changes in the phenotypes of the newborn neurons have remained incompletely characterized. We have now studied neurogenesis of DGCs in 9-day-old (postnatal, P9) rats 1 week after kainate (KA)-induced SE using 5-bromo-2-deoxyuridine (BrdU) immunostaining. The phenotype characterization of the newborn cells was carried out by immunofluorescence double labeling using doublecortin (DCX) and nestin as markers for immature cells, and glial fibrillary acid protein (GFAP) as a marker for glial cells. Newborn GABAergic neurons were further identified with antibodies for parvalbumin, glutamate decarboxylase 67 (GAD67), and the GABA_A receptor α1 subunit, and mRNA expression of GABAergic and immature neurons was measured with quantitative real-time PCR (qPCR) in the DG. Our results show that the number of newborn as well as GABAergic neurons was significantly decreased after SE in the superior blade of the septal DG. The majority of the newborn BrdU-stained neurons co-expressed DCX, but neither nestin nor GFAP. In both experimental groups, newborn neurons were frequently localized in close contact, but not co-localized, with the cells positively stained for the GABAergic cell markers. Nestin and calretinin mRNA expression were significantly increased after SE. Our results suggest that SE-induced disruption of DGC neurogenesis and decreased number of GABAergic neurons could modify the connectivity between these cells and disturb the maturation of the GABAergic neurotransmission in the immature DG at the early epileptogenic phase.
Pax6 limits the competence of developing cerebral cortical cells to respond to inductive intercellular signals
2022, PLoS Biology

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Short communicationImmunocytochemical detection of 5′-bromodeoxyuridine incorporation in the central nervous system of the mouse

Abstract

Dev. Brain Res.

Dev. Brain Res.

Brain Res.

Dev. Brain Res.

Autoradiographic study of cell migration during histogenesis in the cerebral cortex in the mouse

Nature (Lond.)

Time of neuron origin in the hippocampal region

The effects of 5′-bromodeoxyuridine on fusion of the cranial neural folds in the mouse embryo

Teratology

Time of origin of corresponding cell classes in the cerebral cortex of normal and mutant reeler mice: an autoradiographic analysis

J. Comp. Neurol.

Time of neuron origin of the hippocampus and dentate gyrus of normal and reeler mutant mice: an autoradiographic analysis

J. Comp. Neurol.

Rapid fluorometric detection of drug resistant tumour cells

Br. J. Cancer

A flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine

Times of generation of glutamic acid decarboxylase immunoreactive neurons in mouse somatosensory cortex

J. Comp. Neurol.

An immunofluorescence method for monitoring DNA synthesis by flow cytometry

Cytometry

Short communication
Immunocytochemical detection of 5′-bromodeoxyuridine incorporation in the central nervous system of the mouse