Neuronal differentiation and long‐term survival of newly generated cells in the olfactory midbrain of the adult spiny lobster,Panulirus argus

The fate of continuously generated cells in the soma clusters of the olfactory midbrain of adult spiny lobsters, Panulirus argus, was investigated by in vivo pulse‐chase experiments with the proliferation marker 5‐bromo‐2′‐deoxyuridine (BrdU) combined with immunostainings for neuropeptides of mature neurons. A BrdU injection after a survival time (ST) of 14 h labeled about 100 nuclei in the lateral soma clusters (LC), comprised of projection neurons, and about 30 nuclei in the medial soma clusters (MC), comprised of local interneurons. The BrdU‐positive nuclei were confined to small regions at the inside of these clusters, which also contain nuclei in different phases of mitosis and thus represent proliferative zones. After STs of 2 weeks or 3 months, the number of BrdU‐positive nuclei was doubled, indicating a mitosis of all originally labeled cells. Dependent on ST, the BrdU‐positive nuclei were translocated from the proliferative zones towards the outside of the clusters, where somata of mature neurons reside. Immunostainings with antibodies to the neuropeptides FMRFamide and substance P, both of which label a large portion of somata in the MC and a pair of giant neurons projecting into the LC, revealed that in both clusters the proliferative zones are surrounded by, but are themselves devoid of, labeling. In the MC, some BrdU‐positive somata were double‐labeled by the FMRFamide antibody after an ST of 3 months, and by the substance P antibody after STs of 6 and 11/14 months, but not after 3 months. In the LC, BrdU‐positive somata after an ST of 3 months partially and after 6 and 11/14 months widely overlapped with the arborizations of the giant neurons, indicating the establishment of synaptic input. The experiments show that cells generated in proliferative zones in the LC and MC of adult spiny lobsters after a final mitosis differentiate into neurons within months, survive for at least 1 year, and are integrated into the circuitry of the olfactory midbrain. A new hypothesis about the mechanism of adult neurogenesis in the central olfactory pathway of decapod crustaceans is developed, linking it to neurogenesis during embryonic and larval development. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 181–203, 2001     

Newborn cells in the adult crayfish brain differentiate into distinct neuronal types

Mitotically active regions persist in the brains of decapod crustaceans throughout their lifetimes, as they do in many vertebrates. The most well-studied of these regions in decapods occurs within a soma cluster, known as cluster 10, located in the deutocerebrum. Cluster 10 in crayfish and lobsters is composed of the somata of two anatomically and functionally distinct classes of projection neurons: olfactory lobe (OL) projection neurons and accessory lobe (AL) projection neurons. While adult-generated cells in cluster 10 survive for at least a year, their final phenotypes remain unknown. To address this question, we combined BrdU labeling of proliferating cells with specific neuronal and glial markers and tracers to examine the differentiation of newborn cells in cluster 10 of the crayfish, Cherax destructor. Our results show that large numbers of adult-generated cells in cluster 10 differentiate into neurons expressing the neuropeptide crustacean-SIFamide. No evidence was obtained suggesting that cells differentiate into glia. The functional phenotypes of newborn neurons in cluster 10 were examined by combining BrdU immunocytochemistry with the application of dextran dyes to different brain neuropils. These studies showed that while the majority of cells born during the early postembryonic development of C. destructor differentiate in AL projection neurons, neurogenesis in adult crayfish is characterized by the addition of both OL and AL projection neurons. In addition to our examination of neurogenesis in the olfactory pathway, we provide the first evidence that adult neurogenesis is also a characteristic feature of the optic neuropils of decapod crustaceans.     

“Impoverished” and “enriched” living conditions influence the proliferation and survival of neurons in crayfish brain

New neurons are added to two bilateral clusters of neurons in crayfish brain throughout their lives. These interneurons are associated with the olfactory and accessory lobes, areas of the brain that receive primary olfactory information and higher order inputs from the visual and tactile receptor systems. The rate of cell proliferation in these four clusters, revealed by BrdU labeling, is sensitive to the living conditions of the animals: individuals isolated in small spaces (impoverished condition) exhibit a lower rate of cell proliferation in comparison to their siblings living together in larger areas (enriched condition), although both groups were fed to satiation. Reduction in the rate of proliferation can be measured 1 to 2 weeks after the animals are subjected to the impoverished condition. Counts of the labeled neurons that survive after 4 weeks of subjection to the two conditions show that fewer new neurons survive in the brains of animals that have lived for 2 weeks in the impoverished condition in comparison to their siblings living in the enriched conditions. Factors such as surface area, depth of water, and social interaction can all play a role in determining both the rate of new neuron production and the incorporation of the new neurons into the brain of freshwater crayfish. The results indicate a high degree of neuronal plasticity in the crayfish brain that is highly sensitive to the conditions under which the animals are kept. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 215–226, 2000     

"Impoverished" and "enriched" living conditions influence the proliferation and survival of neurons in crayfish brain

New neurons are added to two bilateral clusters of neurons in crayfish brain throughout their lives. These interneurons are associated with the olfactory and accessory lobes, areas of the brain that receive primary olfactory information and higher order inputs from the visual and tactile receptor systems. The rate of cell proliferation in these four clusters, revealed by BrdU labeling, is sensitive to the living conditions of the animals: individuals isolated in small spaces (impoverished condition) exhibit a lower rate of cell proliferation in comparison to their siblings living together in larger areas (enriched condition), although both groups were fed to satiation. Reduction in the rate of proliferation can be measured 1 to 2 weeks after the animals are subjected to the impoverished condition. Counts of the labeled neurons that survive after 4 weeks of subjection to the two conditions show that fewer new neurons survive in the brains of animals that have lived for 2 weeks in the impoverished condition in comparison to their siblings living in the enriched conditions. Factors such as surface area, depth of water, and social interaction can all play a role in determining both the rate of new neuron production and the incorporation of the new neurons into the brain of freshwater crayfish. The results indicate a high degree of neuronal plasticity in the crayfish brain that is highly sensitive to the conditions under which the animals are kept.     

BrdU Birth Dating Can Produce Errors in Cell Fate Specification in Chick Brain Development

Birth dating neurons with bromodeoxyuridine (BrdU) labeling is an established method widely employed by neurobiologists to study cell proliferation in embryonic, postnatal, and adult brain. Birth dating studies in the chick dorsal telencephalon and the mammalian striatum have suggested that these structures develop in a strikingly similar manner, in which neurons with the same birth date aggregate to form “isochronic clusters.” Here we show that isochronic cluster formation in the chick dorsal telencephalon is an artifact. In embryos given standardly employed doses of BrdU, we observed isochronic clusters but found that clusters were absent with BrdU doses close to the limits of detection. In addition, in situ hybridization experiments established that neurons in the clusters display errors in cell type specification: BrdU cell clusters in nidopallium adopted a mesopallial neuronal fate, mesopallial clusters were misspecified as nidopallial cells, and in some instances, the BrdU clusters failed to express neuronal differentiation markers characteristic of the dorsal telencephalon. These results demonstrate that the chick dorsal telencephalon does not develop by isochronic cluster formation and highlight the need to test the integrity of BrdU-treated tissue with gene expression markers of regional and cell type identity.     

Neuronal Ras activation inhibits adult hippocampal progenitor cell division and impairs spatial short‐term memory

A large number of endogenous and exogenous factors have been identified to upregulate and downregulate proliferation, differentiation and/or survival of newborn cells in the adult hippocampus. For studying neuronal mechanisms mediating the impact of those factors, we used a transgenic synRas mouse model expressing constitutively activated Valin12‐Harvey Ras selectively in differentiated neurons. BrdU injections showed significantly reduced proliferation of new cells within the adult hippocampus of transgenic animals compared with their wild‐type siblings. In contrast, the relative survival of newborn cells was increased in synRas mice, although this effect did not fully compensate for diminished proliferation. Inhibition of progenitor cell proliferation and enhancement of cellular survival were more pronounced in males compared with females. Double labelling and doublecortin immunostaining verified that specifically newborn neurons were decreased in synRas mice. Reduced cell generation was observed already 2 h after BrdU pulse injections, identifying an early precursor cell population as target of the inhibitory transgene effect. Differences in proliferation remained stable after 24 h and were specific for the subgranular zone of the dentate gyrus, as subventricular cell generation was not affected supporting a non‐cell autonomous effect on neural hippocampal progenitors. Transgene expression only starts with synaptic differentiation and therefore reduced proliferation must represent an indirect secondary consequence of synRas activity in differentiated neurons. This was associated with impaired spatial short‐term memory capacities as observed in a radial maze paradigm. Our data suggest that constantly high Ras activity in differentiated neurons downregulates hippocampal precursor cell generation in the neuronal lineage, but is modulated by sex‐dependent factors.     

Species‐specific injury‐induced cell proliferation in the hippocampus and subventricular zone of food‐storing and nonstoring wild birds

Cells are continuously born and incorporated into the adult hippocampus (HP). Adult neurogenesis might act to increase the total number of cells or replace dead cells. Thus, neurogenesis might be a primary factor in augmenting, maintaining, or even recovering functions. In zebra finches, HP injury increases cell proliferation in the HP and stem cell rich subventricular zone (SVZ). It is unknown what effect injury has on a species dependent upon the HP for survival in the wild. In food‐storing birds, recovery of caches is seasonal, necessary for survival, dependent upon the HP and is concomitant with a peak in HP neurogenesis. During the fall, food‐storing black‐capped chickadees (BCCs) and nonstoring dark‐eyed juncos (DEJs) were captured and given a unilateral penetrating lesion to the HP one day later. On day 3, birds were injected with the mitotic marker 5‐bromo‐2′‐deoxyuridine (BrdU) and perfused on day 10. If unlesioned, more BrdU‐labeled cells were observed in the HP and SVZ of BCCs compared to DEJs, indicating higher innate cell proliferation or incorporation in BCCs. If lesioned, BrdU‐labeled cells increased in the injured HP of both species; however, lesions caused larger increases in DEJs. DEJs also showed increases in BrdU‐labeled cells in the SVZ and contralateral HP. BCCs showed no such increases on day 10. Thus, during the fall food‐storing season, storers showed suppressed injury‐induced cell proliferation and/or reduced survival rates of these new cells compared to nonstorers. These species differences may provide a useful model for isolating factors involved in cellular responses following injury. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010     

Neurogenesis in subclasses of vomeronasal sensory neurons in adult mice

The vomeronasal sensory epithelium contains two distinct populations of vomeronasal sensory neurons. Apical neurons express G_i2α‐linked V1R vomeronasal receptors and project to the anterior portion of the accessory olfactory bulb, while basal neurons express G_oα‐linked V2R receptors and project to the posterior portion. Sensory neurons expressing V1R and V2R vomeronasal receptors are sensitive to different stimuli. Neurons in the vomeronasal system undergo continuous cell turnover during adulthood. To analyze over time neurogenesis of the different sensory cell populations, adult mice were injected with bromodeoxyuridine (BrdU) and sacrificed at postinjection days 1, 3, 5, 7, and 11. Newborn vomeronasal neurons were revealed by antibodies against BrdU while subclasses of vomeronasal neurons were identified using antibodies against G_oα or G_i2α proteins. To ascertain whether G proteins are early expressed during neurogenesis, multiple labeling experiments using PSA‐NCAM and doublecortin were performed. Distribution of BrdU‐labeled cells was analyzed in angular segments from the margin of the sensory epithelium. No sexual differences were found. Within survival groups, BrdU‐G_oα labeled cells were found more marginally when compared with BrdU‐G_i2α labeled cells. The number of BrdU‐positive cells decreased from day 1 to day 3 to remain constant afterwards. The relative proportions of BrdU‐G_i2α and BrdU‐G_oα labeled cells remained similar and constant from postinjection day 1 onwards. This rate was also comparable with BrdU‐positive cells starting day 3. These results indicate an early, constant, and similar rate of neurogenesis in the two major subclasses of vomeronasal neurons, which suggests that both cell populations maturate independently. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 961–970, 2010     

Neuronal differentiation and long-term survival of newly generated cells in the olfactory midbrain of the adult spiny lobster, Panulirus argus

The fate of continuously generated cells in the soma clusters of the olfactory midbrain of adult spiny lobsters, Panulirus argus, was investigated by in vivo pulse-chase experiments with the proliferation marker 5-bromo-2'-deoxyuridine (BrdU) combined with immunostainings for neuropeptides of mature neurons. A BrdU injection after a survival time (ST) of 14 h labeled about 100 nuclei in the lateral soma clusters (LC), comprised of projection neurons, and about 30 nuclei in the medial soma clusters (MC), comprised of local interneurons. The BrdU-positive nuclei were confined to small regions at the inside of these clusters, which also contain nuclei in different phases of mitosis and thus represent proliferative zones. After STs of 2 weeks or 3 months, the number of BrdU-positive nuclei was doubled, indicating a mitosis of all originally labeled cells. Dependent on ST, the BrdU-positive nuclei were translocated from the proliferative zones towards the outside of the clusters, where somata of mature neurons reside. Immunostainings with antibodies to the neuropeptides FMRFamide and substance P, both of which label a large portion of somata in the MC and a pair of giant neurons projecting into the LC, revealed that in both clusters the proliferative zones are surrounded by, but are themselves devoid of, labeling. In the MC, some BrdU-positive somata were double-labeled by the FMRFamide antibody after an ST of 3 months, and by the substance P antibody after STs of 6 and 11/14 months, but not after 3 months. In the LC, BrdU-positive somata after an ST of 3 months partially and after 6 and 11/14 months widely overlapped with the arborizations of the giant neurons, indicating the establishment of synaptic input. The experiments show that cells generated in proliferative zones in the LC and MC of adult spiny lobsters after a final mitosis differentiate into neurons within months, survive for at least 1 year, and are integrated into the circuitry of the olfactory midbrain. A new hypothesis about the mechanism of adult neurogenesis in the central olfactory pathway of decapod crustaceans is developed, linking it to neurogenesis during embryonic and larval development.     

雌百灵端脑新生神经前体细胞的产生和分布并与白腰文鸟的比较

用BrdU标记DNA的ABC免疫细胞化学方法,观察雌性蒙古百灵端脑神经前体细胞的产生和分布特点,并与白腰文鸟作比较。结果如下:1.在百灵和白腰文鸟胸肌注射BrdU短时程组(存活1天),在端脑室带区外侧壁(LVZ)有大量的标记细胞,新生神经细胞起源于端脑室带区(VZ)中的增殖细胞层,并在纹状体腹侧的VZ形成标记细胞增殖热点,如在百灵和白腰文鸟靠近中缝线处的外侧纹状体(LSt)与内侧纹状体(MSt)腹侧的LVZ形成标记最多的‘第1增殖热点’区;在靠近中缝线处LVZ的头端形成密集的新生标记细胞,形成‘第2增殖热点’区;在百灵LSt尾端的LVZ标记细胞形成‘第3增殖热点’,但白腰文鸟此脑区的标记细胞较少。2.在百灵胸肌注射BrdU长时程组中5天起,大量的LVZ的标记细胞开始迁移,存活5-30天期间在高级发声中枢(HVc)和高位发声运动中枢-古纹状体栎核(RA)有新生标记细胞,在端脑靠近LVZ的区域有较多的标记细胞。但在雌性白腰文鸟胸肌注射BrdU存活30天期间,在HVC、RA内未见到标记细胞。结果提示雌百灵端脑HVc和RA不断地产生新生神经细胞,这可能与雌性需要不断地感知、识别雄百灵鸣唱的新语句有关,而白腰文鸟不需要这种功能。     

Functional restoration of acoustic units and adult‐generated neurons after hypothalamic lesion

The hypothalamus of the adult ring dove contains acoustic units that respond to species‐specific coo vocalization. Loss of nest coo leads to unsuccessful breeding. However, the recovery of nest coo in some doves suggests that these units are capable of self‐renewal. We have previously shown that lesioning the hypothalamus generates the addition of new neurons at the lesioned area. In this study, we sought to determine whether lesion‐induced new neurons are involved in the recovery of coo‐responsive units. We systematically recorded electrical activity in the ventromedial nucleus (VMN) of the hypothalamus, before and after lesion, for varying periods up to 3 months. Recordings were made when the birds were at rest (spontaneous discharge) and when the birds were exposed to acoustic stimulations (evoked discharge). Concurrently, the lesioned area was monitored for changes in cell types by using bromodeoxyuridine (BrdU) to label newly divided cells and NeuN to identify mature neurons. For 1 month after lesion, there was no sign of electrical activity, and only BrdU‐labeled cells were present. When the first electrical activity occurred, it displayed abnormal spontaneous bursting patterns. The mature discharge patterns (both spontaneous and evoked) occurred after detection of BrdU⁺/NeuN⁺ double‐labeled cells 2–3 months postlesion and were similar to those found in intact and sham‐lesioned birds. Double‐labeled cells bore morphologic characteristics of a neuron and were confirmed with z‐stack analysis using confocal laser scanning microscopy. Moreover, double‐labeled cells were not stained for glial fibrillary acidic protein (GFAP), suggesting that they were neurons. The number of coo‐responsive units was significantly correlated with that of BrdU⁺/NeuN⁺ cells. Furthermore, the marker for recording sites revealed that coo‐responsive units were colocalized with BrdU⁺/NeuN⁺ cells. Taken together, the evidence strongly suggests that lesion‐induced addition of new neurons promotes the functional recovery of the adult hypothalamus. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 197–213, 2004     

Intracerebral estrogen provision increases cytogenesis and neurogenesis in the injured zebra finch brain

To determine whether or not local, injury‐induced aromatization and/or estrogen 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 INJ. 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. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 170‐181, 2011     

Stress-induced decrease of granule cell proliferation in adult rat hippocampus: assessment of granule cell proliferation using high doses of bromodeoxyuridine before and after restraint stress

Stress is known to inhibit granule cell proliferation in the hippocampus. However, recent studies suggest that the commonly used dose of bromodeoxyuridine (BrdU) is insufficient to label all fractions of granule cells. Furthermore, stress-induced changes in BrdU availability may influence the labeling of newly born cells. To investigate whether changes in BrdU availability affect measurements of stress-induced granule cell proliferation, granule cell proliferation was assessed using injection of high doses of BrdU before and after restraint stress lasting 1 h. In addition, to determine whether stress-induced changes in plasma corticosterone levels were influenced by the BrdU, time-dependent changes in plasma corticosterone levels over 2 h after BrdU injection were compared with total accumulated plasma corticosterone levels [as determined by areas under the curve (AUC)]. Restraint stress significantly reduced the numbers of BrdU-labeled cells and clusters in the granule cell layer (GCL) of rats that received BrdU after stress, and decreases of similar magnitude were observed when the rats were given BrdU before stress. BrdU injection enhanced the stress-induced plasma corticosterone response, but there was no difference between the mean AUCs of plasma corticosterone levels of animals injected with BrdU before or after stress. These observations suggest that restraint stress decreases granule cell proliferation, and that this may be influenced by the extent and duration of plasma corticosterone increases rather than by changes in the availability of BrdU.     

Evidence that brain-derived neurotrophic factor is required for basal neurogenesis and mediates,in part,the enhancement of neurogenesis by dietary restriction in the hippocampus of adult mice

To determine the role of brain-derived neurotrophic factor (BDNF) in the enhancement of hippocampal neurogenesis resulting from dietary restriction (DR), heterozygous BDNF knockout (BDNF +/-) mice and wild-type mice were maintained for 3 months on DR or ad libitum (AL) diets. Mice were then injected with bromodeoxyuridine (BrdU) and killed either 1 day or 4 weeks later. Levels of BDNF protein in neurons throughout the hippocampus were decreased in BDNF +/- mice, but were increased by DR in wild-type mice and to a lesser amount in BDNF +/- mice. One day after BrdU injection the number of BrdU-labeled cells in the dentate gyrus of the hippocampus was significantly decreased in BDNF +/- mice maintained on the AL diet, suggesting that BDNF signaling is important for proliferation of neural stem cells. DR had no effect on the proliferation of neural stem cells in wild-type or BDNF +/- mice. Four weeks after BrdU injection, numbers of surviving labeled cells were decreased in BDNF +/- mice maintained on either AL or DR diets. DR significantly improved survival of newly generated cells in wild-type mice, and also improved their survival in BDNF +/- mice, albeit to a lesser extent. The majority of BrdU-labeled cells in the dentate gyrus exhibited a neuronal phenotype at the 4-week time point. The reduced neurogenesis in BDNF +/- mice was associated with a significant reduction in the volume of the dentate gyrus. These findings suggest that BDNF plays an important role in the regulation of the basal level of neurogenesis in dentate gyrus of adult mice, and that by promoting the survival of newly generated neurons BDNF contributes to the enhancement of neurogenesis induced by DR.     

Neurogenesis response of middle-aged hippocampus to acute seizure activity

Acute Seizure (AS) activity in young adult age conspicuously modifies hippocampal neurogenesis. This is epitomized by both increased addition of new neurons to the granule cell layer (GCL) by neural stem/progenitor cells (NSCs) in the dentate subgranular zone (SGZ), and greatly enhanced numbers of newly born neurons located abnormally in the dentate hilus (DH). Interestingly, AS activity in old age does not induce such changes in hippocampal neurogenesis. However, the effect of AS activity on neurogenesis in the middle-aged hippocampus is yet to be elucidated. We examined hippocampal neurogenesis in middle-aged F344 rats after a continuous AS activity for >4 hrs, induced through graded intraperitoneal injections of the kainic acid. We labeled newly born cells via daily intraperitoneal injections of the 5'-bromodeoxyuridine (BrdU) for 12 days, commencing from the day of induction of AS activity. AS activity enhanced the addition of newly born BrdU+ cells by 5.6 fold and newly born neurons (expressing both BrdU and doublecortin [DCX]) by 2.2 fold to the SGZ-GCL. Measurement of the total number of DCX+ newly born neurons also revealed a similar trend. Furthermore, AS activity increased DCX+ newly born neurons located ectopically in the DH (2.7 fold increase and 17% of total newly born neurons). This rate of ectopic migration is however considerably less than what was observed earlier for the young adult hippocampus after similar AS activity. Thus, the plasticity of hippocampal neurogenesis to AS activity in middle age is closer to its response observed in the young adult age. However, the extent of abnormal migration of newly born neurons into the DH is less than that of the young adult hippocampus after similar AS activity. These results also point out a highly divergent response of neurogenesis to AS activity between middle age and old age.     

A decrease in the addition of new cells in the nucleus accumbens and prefrontal cortex between puberty and adulthood in male rats

Adolescence involves shifts in social behaviors, behavioral flexibility, and adaptive risk‐taking that coincide with structural remodeling of the brain. We previously showed that new cells are added to brain regions associated with sexual behaviors, suggesting that cytogenesis may be a mechanism for acquiring adult‐typical behaviors during adolescence. Whether pubertal cell addition occurs in brain regions associated with behavioral flexibility or motivation and whether these patterns differ between pubertal and adult animals had not been determined. Therefore, we assessed patterns of cell proliferation or survival in the prefrontal cortex and nucleus accumbens. Pubertal and adult male rats were given injections of bromo‐deoxyuridine (BrdU). To assess cell proliferation, half of the animals from each group were sacrificed 24 h following the last injection. The remaining animals were sacrificed at Day 30 following the last injection to evaluate cell survival. Adult animals had significantly lower densities of BrdU‐immunoreactive (ir) cells in the prefrontal cortex, irrespective of post‐BrdU survival time, whereas in the nucleus accumbens, adult animals had a lower density of BrdU‐ir cells at the short survival time; however, the density of BrdU‐ir cells was equivalent in pubertal and adult animals at the longer survival time. These data provide evidence that cell addition during puberty may contribute to the remodeling of brain regions associated with behavioral flexibility and motivation, and this cell addition continues into adulthood, albeit at lower levels. Higher levels of cell proliferation or survival in younger animals may reflect a higher level of plasticity, possibly contributing to the dynamic remodeling of the pubertal brain. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 633–642, 2014     

Mitotic sympathetic neuroblasts initiate axonal pathfinding in vivo

Neuronal precursor proliferation and axodendritic outgrowth have been traditionally regarded as discrete and sequential developmental stages. However, we recently found that sympathetic neuroblasts in vitro often elaborate long neuritic processes before dividing. Furthermore, these “paramitotic” neurites were maintained during cell division and neuritic morphology was consistently preserved by daughter cells after mitosis. This inheritance of neuritic morphology in vitro raised the possibility that proliferating neuroblasts engage in axodendritic outgrowth. To determine whether mitotic superior cervical ganglion (SCG) neuroblasts are engaged in pathfinding in vivo, we have combined retrograde axonal tracing of efferent nerve trunks with bromodeoxyuridine (BrdU) labeling of cells in S‐phase. In fact, about 13% of BrdU(+) cells were retrogradely labeled, indicating that mitotic neuroblasts often have extraganglionic axonal projections. Moreover, the presence of axons during S‐phase was observed at two developmental ages (E15.5 and E16.5), implicating an ongoing function of paramitotic axons during neuronal ontogeny. Using a calculation to account for experimental limitations, we estimate that virtually all mitotic SCG neuroblasts have direct access to extraganglionic signals during development. We conclude that mitotic neuronal precursors in vivo engage in pathfinding, raising the possibility that interaction of proliferating populations with distant signals actively coordinates cell division and neural connectivity. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 366–374, 1999     

Supporting cell proliferation after hair cell injury in mature guinea pig cochlea in vivo

In cold-blooded animals, lost sensory hair cells can be replaced via a process of regenerative cell proliferation of epithelial supporting cells. In contrast, in mammalian cochlea, receptor (hair) cells are believed to be produced only during embryogenesis; after maturity, sensory or supporting cell proliferation or regeneration are thought to occur neither under normal conditions nor after trauma. Using bromodeoxyuridine (BrdU) as a proliferation marker, we have assessed cell proliferation activity in the mature organ of Corti in the cochlea of young guinea pigs following severe damage to the outer hair cells induced by kanamycin sulfate and ethacrynic acid. Although limited, we have found BrdU-labeled nuclei in the regions of Deiters cells when BrdU is given for 3 days or longer. When BrdU is given for 10 days, at least one labeled nucleus can be observed in the organ of Corti in approximately half of the ears; proliferating cells typically appear as paired daughters, with one nucleus being displaced away from the basement membrane to the position expected of the hair cells. Double-staining with antibodies to cytokeratin, vimentin, and p27 have shown that the BrdU-labeled nuclei are located in cells phenotypically similar to Deiters cells. Most of the uptake of BrdU occurs 3–5 days following ototoxic insult, and the number of BrdU-labeled cells does not decrease until 30 days following insult. These findings indicate that Deiters cells in the mature mammalian cochlea maintain a limited competence to re-enter the cell cycle and proliferate after hair cell injury, and that they can survive at least for 1 month.This work was supported by the Ministry of Health, Labour, and Welfare, Japan (grants 12120101, 15110201) and by the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant 13470357) to T.Y.     

Reduced proliferation in the adult mouse subventricular zone increases survival of olfactory bulb interneurons

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.     

Morphogenesis and pattern formation in the retina of the crayfish Procambarus clarkii

Pattern formation and ommatidial differentiation in the crayfish retina were analyzed using confocal, light and electron microscopy. Optic primordia first appear in the embryo as round elevations covered by a surface epithelial layer. Retinal differentiation begins with a wave of mitotic activity that moves across this epithelium from lateral to medial. Ommatidial cell clusters are visible at the surface along a transition zone, which lies at the interface of the medial undifferentiated retina and the lateral patterned retina. This zone is 8–10 cells wide and composed of small uniform cell profiles. Lateral to the transition zone the initial ommatidial cell clusters form staggered rows across the surface. Each first row cluster contains eight retinula cells surrounded by four cone, two corneagenous and two distal pigment cells. Ommatidial clusters in the first nine rows show significant changes in their organization, which are visible at the surface of the retina. In row 10 the retinula cells recede from the surface and the cone cells close in above them creating a constant cell pattern at the surface. Rhabdome development begins distally and extends downward as the retinula cluster recedes from the surface. Movement of the retinula cells inward and enlargement of the cone and corneagenous cells at the surface creates a two-tiered organization characteristic of each ommatidium. Comparison of retinal pattern formation and differentiation in the crayfish with retinal morphogenesis in Drosophila and other insects show several similarities between the two arthropod groups.