Evidence that late-generated granule cells do not simply replace earlier formed neurons in the rat dentate gyrus

Summary Ten-day-old Sprague-Dawley rats were injected intraperitoneally with ³H-thymidine (³HTdR) and allowed to survive until postnatal day (P) 40, P120, P200, P300 or P450. Following the preparation of the tissue for autoradiography, the location of the labeled neurons within the granule cell layer of the dentate gyrus was determined at two different septo-temporal levels. At P40 the labeled cells in the suprapyramidal blade of the dentate gyrus were found only in the deep part of the granule cell layer, where it borders on the hilus, and more than four cell diameters from the molecular layer. The latter relationship remained constant at each of the five ages studied, but between P40 and P120 the average distance from the labeled granule cells to the hilus almost doubled. At the middle of the temporal portion of the dentate gyrus this trend continues with age, so that by P450 the labeled neurons, though no nearer to the molecular layer than at earlier stages, were found, on average, in the middle of the granule cell layer, more-or-less halfway between the hilus and the molecular layer. Near the middle of the septal half of the dentate gyrus a similar pattern was seen at P40 and P120 but thereafter the labeled cells within the granule cell layer remained at about the same distance from the hilus. These observations permit the following three conclusions: (i) there is a gradual accumulation of dentate granule cells throughout the life of Sprague-Dawley rats; (ii) the newly-generated cells are preferentially added to the deep aspect of the granule cell layer from which they progressively displace the earlier-formed cells; and (iii) there is no concomitant loss of a corresponding number of earlier-generated granule cells: in other words, there is no substantial turnover of granule cells in adult animals.     

出生后大鼠海马结构的发育

目的：定量观察出生后大鼠海结构锥体细胞层和颗粒细胞层的发育。方法：用体视学原理定量分析。结果：阿蒙角锥体层,尤其是ＣＡ１区,其细胞层数逐渐减少。数密度、面数密度和体密度逐渐下降,平均体积逐渐增大。齿状回内臂逐渐延长,至Ｐ１４天接近成年水平。内外臂的细胞层数逐渐增加,至Ｐ９０天仍有增加趋势。在ＣＡ１、ＣＡ３、ＣＡ４区的锥体层和齿状回颗粒层中,数密度,面数密度和体密度按大小排序为：颗粒层〉ＣＡ１〉ＣＡ     

The persistent expression of a highly polysialylated NCAM in the dentate gyrus of the adult rat.

The expression of a highly polysialylated form of the neural cell adhesion molecule (NCAM-H), often termed 'embryonic NCAM', has been investigated in the hippocampal formation of developing and adult rats. To determine the immunohistochemical localization of NCAM-H, a monoclonal antibody that reacts with the polysialic acid portion of NCAM-H was used. In the late embryonic and early postnatal periods, immunoreactivity for NCAM-H was found throughout the hippocampal formation, except for the ventricular layer. Thereafter, the immunoreactivity gradually decreased and almost vanished in most parts in the adult. However, a strong immunoreactivity remained on a number of cells in the dentate gyrus of adult rats, particularly in the deepest part of the granular layer. The immunoreactive arborized dendrites, mostly arising from the primary apical pole of the granule cells, were found to enter the molecular layer. The mossy fibers also were positive. Electron-microscopic examination of the hilus portion showed that the immunoreactivity was detected on the plasma membrane of some axons in the mossy fiber bundles. Since postnatal neurogenesis is known to continue into adulthood in the deepest part of the granule cell layer of the dentate gyrus, these results suggest that, in the adult dentate gyrus, NCAM-H is expressed by newly generated granule cells, and that the NCAM-H-expressing new cells may participate in the formation of new neural circuits.     

Cell formation in the human hippocampal formation from mid-gestation to the late postnatal period

In the present study cell formation was studied in the human hippocampal formation from the 24th gestational week until the end of the first postnatal year. Proliferating cells were detected with the monoclonal antibody MIB-1.The cytoarchitectonic layers of Ammon's horn are formed before the 24th gestational week. In harmony with this observation, cell proliferation in the hippocampal ventricular zone is minimal after the 24th week. In addition, local cell multiplication in Ammon's horn is occasional and the proliferating cells are glial or endothelial cells. In contrast, cell formation continues in the hilar region of the dentate gyrus even after birth. Immature cells accumulate in the hilus, and at the border between the hilus and the granule cell layer throughout the first eight postnatal months. The subgranular zone of the dentate gyrus becomes a cell sparse area at about the 11th postnatal month, indicating that immature cells from the hilus have already migrated to the granule cell layer and differentiated into granule cells. There is an increase in glial cell proliferation both in Ammon's horn and the dentate gyrus at the 11.5th postnatal month suggesting the onset of myelination by the end of the first year.Our findings indicate that most pyramidal neurons of Ammon's horn are generated in the first half of pregnancy and no pyramidal neurons are formed after the 24th gestational week. In contrast, granule cells of the dentate gyrus proliferate in a decreasing rate during the second half of pregnancy and after birth. Proliferating neuronal precursors occur in a low percentage in the dentate gyrus of 3-, 5- and 11.5-month-old children.     

胚胎时期大鼠海马结构的发育

为研究胚胎时期大鼠海马结构的发育状况,用浸泡固定、石蜡切片、混合尼氏染色方法,观察了Ｅ１４、Ｅ１６、Ｅ１８、Ｅ２２天大鼠胚胎海马结构的发育过程。结果：Ｅ１４天基本上能区分出海马原基。Ｅ１６天海马原基背侧与下托部神经上皮延续,腹侧与隔区神经上皮及脉络丛基质相连续;此时海马原基可区分出３部分,即背侧的阿蒙角神经上皮,其下的原始齿状回神经上皮,腹侧的伞部胶质上皮。Ｅ１８天海马裂形成,将阿蒙角神经上皮与齿状回生发基质区分开来;ＣＡ１区锥体层和海马伞开始出现,齿状回外臂和门也开始形成并第一次见向齿状回迁移的细胞。Ｅ２２天海马裂消失,阿蒙角分子层与齿状回分子层贴在一起;ＣＡ１区锥体层增厚,ＣＡ３区锥体层出现并穿透门区;阿蒙角神经上皮变成单层结构,中间层变薄,仍见大量逗留的锥体细胞,海马槽形成;齿状回外臂轮廓出现,颗粒细胞呈单层排列,内臂起始部开始形成;ＣＡ３区中间层周围及个部附近见大量的细胞正朝门区迁移。研究表明：出生前阿蒙角ＣＡ１、ＣＡ３区锥体层已经形成,齿状回外臂的轮廓已形成,内臂刚开始出现,其轮廓将于出生后形成。     

Synaptic formation in subsets of glutamatergic terminals in the mouse hippocampal formation is affected by a deficiency in the neural cell recognition molecule NB-3

The neural cell recognition molecule NB-3, which is also referred to as contactin-6, is a member of the contactin subgroup molecules that are expressed prominently in the developing nervous system after birth. In mice, an NB-3 deficiency impairs motor coordination and reduces the synaptic density between parallel fibers and Purkinje cells in the cerebellum. Here, we studied the role of NB-3 in the formation of glutamatergic synapses in the hippocampal formation. At postnatal day 5, NB-3 immunoreactivity was detected in the subiculum, the stratum lacunosum–moleculare of the CA1 region and the hilus of the dentate gyrus. NB-3 expression in the strata radiatum and oriens was weak, and it was very weak in the granule cell layer of the dentate gyrus, the pyramidal cell layer of regions CA3 to CA1 and the stratum lucidum. NB-3-positive puncta partially overlapped with vesicular glutamate transporter 1 (VGLUT1) and 2 (VGLUT2), excitatory presynaptic markers, but not with vesicular GABA transporter (VGAT), an inhibitory presynaptic marker. The density of VGLUT1 and VGLUT2 puncta in the regions where NB-3 was strongly expressed in wild-type mice was reduced by ∼20–30% in NB-3 knockout mice relative to wild-type mice, whereas that of VGAT puncta was not affected by NB-3 deficiency. Thus, NB-3 has key roles in the formation of glutamatergic, but not GABAergic, synapses during postnatal development of the hippocampal formation as well as the cerebellum.     

Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections

Summary Fully mature rats were injected intraperitoneally with ³H-TdR on postnatal day (P) 100. After an additional 28–32 days, a retrograde fluorescent tracer, either FB or DY, was injected into the regio inferior of the hippocampal formation to label granule cells of the dentate gyrus through their mossy fiber axons. Examination of autoradiographs from these brains reveals that ³H-TdR labeled cells within the granule cell layer of the dentate gyrus are often labeled with the retrograde tracer as well. This indicates that within the mature hippocampal formation, newly generated dentate granule cells are capable of extending axonal projections for considerable distances.Abbreviations FB fast blue - DY diamidino yellow dihydrochloride - ³H-TdR tritiated thymidine - UV ultraviolet     

Arrival of afferents and the differentiation of target neurons: studies of developing cholinergic projections to the dentate gyrus

This study examined the relationship between the development of cholinergic axons originating from the septum and a group of their target cells, the granule cells of the dentate gyrus of the rat. Acetylcholinesterase histochemistry was used to identify septal cholinergic afferents to the dentate gyrus; parallel studies used anterograde movement of a carbocyanine dye to label the septal projections. Septal cholinergic axons are present in the molecular layer of the internal blade of the dentate gyrus shortly after birth, but these axons do not reach the external blade until several days later. Results demonstrate that acetylcholinesterase positive septal axons grow into the external blade of the dentate gyrus only after the recently generated granule cells have coalesced to form a clearly defined layer. Results from studies using in situ hybridization techniques demonstrate that dentate gyrus granule cells express messenger RNAs for brain derived neurotrophic factor and for neurotrophic factor 3 shortly after formation of the granule cell layer. Ingrowth of septal cholinergic axons follows two days after the formation of the external blade of the dentate gyrus and the expression of neurotrophin messenger RNAs by the dentate granule cells.These data support the hypothesis that target cell development is a prerequisite for attracting the ingrowth of septal afferent axons.     

切割穹窿海马伞大鼠海马内Lhx8 mRNA表达的变化

目的:探讨切割穹窿海马伞大鼠切割侧与正常侧海马内Lhx8 mRNA表达的差异。方法:切割SD大鼠右侧穹窿海马伞。切割后7d制备海马冰冻切片,用体外转录法制备地高辛标记的Lhx8 RNA探针进行原位杂交,分析切割侧和正常侧海马锥体细胞层和齿状回颗粒层中及齿状回门区和颗粒下层中的Lhx8 mRNA阳性细胞的数量和平均光密度值。结果:切割侧和正常侧海马锥体细胞层和齿状回颗粒层Lhx8 mRNA阳性细胞数量无明显差异,但切割侧平均光密度值较正常侧明显增加;在齿状回的门区和颗粒下层,切割侧Lhx8 mRNA阳性细胞数和平均光密度值均较正常侧升高。结论:切割穹窿海马伞后海马中Lhx8 mRNA表达上调,可能与其中的神经干细胞向胆碱能神经元分化的神经再生机制有关。     

Tbr1影响大脑皮质发育及细胞分化与迁移

目的 探究Tbr1基因在大脑新皮质与海马发育过程中的功能。 方法 分别取胚胎165d、185d,出生后0d、3d、7d、14d昆明小鼠的脑组织,每个年龄点取材8～10 (共52) 只,常规固定脱水后石蜡切片,用免疫荧光检测小鼠大脑皮质、海马及齿状回神经细胞迁移与片层化发育过程中Tbr1的表达与分布情况。结果 1.在大脑皮质,Tbr1最早在皮质板广泛表达,随日龄的增加其表达逐渐向皮质板下层移动且最终定位于皮质的第Ⅵ层;2.相似地,齿状回处Tbr1在颗粒细胞层表达,P7之后其表达定位于颗粒细胞下层;3.根据其表达位置及组织发生规律,推测Tbr1阳性细胞即是皮质板内迁移中的新生神经元。 结论 Tbr1是影响小鼠大脑皮质发育及神经细胞迁移与分化的关键分子。作为新生神经元的标记物,Tbr1参与细胞分化与迁移以及大脑皮质片层化的形成过程。     

钙/钙调蛋白依赖性蛋白激酶Ⅱ在大鼠生后海马发育中的表达

目的 探讨Wistar大鼠生后海马发育过程中钙/钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）的表达。 方法 应用免疫荧光方法检测CaMKⅡ在生后不同时期大鼠海马CA1、CA3区和齿状回（DG）中的表达情况（n =48）。结果 CaMKⅡ于生后各期海马CA1区和DG的表达逐渐增强，生后第10天（P10）达高峰期，此后逐渐减弱；于CA3区的表达在P4和P10时均较高。其中，CaMKⅡ在CA3区的表达高于在CA1区和DG的表达，在多形层和分子层的表达高于在锥体细胞层或颗粒细胞层的表达。结论 CaMKⅡ在CA1、CA3区和DG中的表达具有特异性的时空分布模式，这可能与其在生后发育过程中的突触发生，树突、轴突形成，海马的成熟以及学习记忆功能相关。     

切割穹窿海马伞大鼠海马内Brn-4 mRNA的表达变化——原位杂交法

目的 探讨大鼠穹隆海马伞切割侧与正常侧海马内Brn-4 mRNA表达的差异.方法 切割大鼠右侧穹窿海马伞,切割后14d制备海马冰冻切片,用体外转录法制备地高辛标记的Brn-4 RNA探针进行原位杂交.每只动物随机计数3张切片切割侧和正常侧Brn-4 mRNA的阳性细胞,测定其吸光度(A)值,进行配对t检验分析.结果 切割侧和正常侧海马锥体细胞层和齿状回颗粒层均见Brn-4 mRNA阳性细胞,两侧细胞数无明显差异,但切割侧阳性细胞平均吸光度值较正常侧明显增加(P＜0.01);而在齿状回门区和颗粒下层,切割侧Brn-4 mRNA阳性细胞数和平均吸光度值均较正常侧升高(均P＜0.01).结论 穹窿海马伞切割侧海马锥体细胞层和齿状回颗粒层细胞中Brn-4 mRNA的表达量明显增强,而在齿状回门区和颗粒下层中,其阳性细胞数和表达量均较正常侧明显升高.结合本课题组以往的工作,提示切割穹窿海马伞后,海马中Brn-4 mRNA表达的增高可能与促进其中的神经干细胞向神经元分化有关.     

The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus

Summary One of the most distinctive and common cell types in Golgi preparations of the hilus of the rat dentate gyrus is the mossy cell. We have used a variety of techniques including the Golgi method, the combined Golgi and electron microscopic (EM) method and the retrograde transport of horseradish peroxidase (HRP) to study the development, ultrastructure and synaptic connections of this cell type. The mossy cells identified in our light microscopic preparations are characterized by: (1) triangular or multipolar shaped somata; (2) three to four primary dendrites that arise from the soma and bifurcate once or more to produce an extensive dendritic arborization restricted, for the most part, to the hilus; (3) numerous thorny excrescences on their somata and proximal dendrites with typical spines on distal dendrites; and (4) axons that bifurcate and are directed toward the fimbria and the molecular layer of the dentate gyrus.The mossy cells have an immature appearance at birth and on subsequent days their maturation appears to lag somewhat behind that of the hippocampal pyramidal cells. On postnatal day 1, many of the dendrites bear growth cones primarily at their termini and have long, thin filipodia emanating from various points along their lengths. Many of the dendrites enter the molecular layer of the dentate gyrus, though this is rarely seen in the mature brain. Typical pedunculate spines are first commonly seen on the distal dendrites around postnatal day 7 while thorny excrescences are first commonly seen between postnatal days 11 and 14. By postnatal day 21, the dendrites have attained a mature appearance although the density of both typical spines and thorny excrescences is less than that found in adults.Two different retrograde transport methods were used to confirm that mossy cells give rise to the commissural projection to the contralateral dentate gyrus. The first method combined HRP histochemistry with a silver intensification procedure and the second method combined HRP histochemistry with Golgi staining. While the majority of commissurally projecting hilar neurons had the appearance of mossy cells, there were others that were smaller and either ovoid or fusiform.     

Caspase-3在大鼠齿状回生后发育过程中的作用研究

为探讨在大鼠海马齿状回(DG)生后发育过程中,活化的caspase-3在细胞增殖中的作用,本研究采用双重免疫荧光染色方法检测活化的caspase-3与Ki-67的表达情况及其相互关系。结果显示:在DG生后发育过程中,活化的caspase-3与Ki-67的表达无相关性,但明显存在小部分共表达的颗粒细胞。研究结果提示,在终生都有自我更新能力的DG中,活化的caspase-3在颗粒细胞增殖中可能存在重要的非凋亡作用。     

Tetanus-induced re-activation of evoked spiking in the post-ischemic dentate gyrus

This study aimed at investigating and influencing the basic electrophysiological functions and neuronal plasticity in the dentate gyrus in freely moving rats at several time-points after global ischemia. Although neuronal death was induced selectively in the cornu ammonis, subfield 1 (CA1)-region of the hippocampus, we found an additional loss of the population spike in the dentate gyrus after stimulation of the perforant path. Input/output-measurements revealed that as early as 1 day post-ischemia population spike generation in the granular cell layer is greatly decreased when compared with pre-ischemic values and to sham-operated animals, despite an apparently intact morphology of granular cells as evidenced by Nissl-staining. In contrast, the synaptic transmission (excitatory postsynaptic field potential) shows no significant difference when comparing values before and after ischemia and ischemic and sham-operated animals. Despite reduced output function, indicated by very small population spike amplitudes, long lasting potentiation can be induced 10 days after ischemia. Surprisingly, even "silent" populations of neurons, which appear selectively post-ischemia and do not show any evoked population spike, can be re-activated by tetanisation which is followed by a normal appearing long-term potentiation. However, this functional recovery seems to be partial and transient under current conditions: population spike-values do not reach pre-ischemic values and return to the low pre-tetanic baseline values the next day. Electrophysiological measurements ex vivo after ischemia indicate that the neuronal dysfunction in the dentate gyrus is not due to locally destroyed structures but that the activity of granular cells is merely suppressed only under in vivo conditions. In summary, global ischemia leaves a neighboring morphologically intact input area, functionally impaired. However, neuronal function can be partially regenerated by electrophysiological tetanic stimulation.     

Thyroid hormone and development of the rat hippocampus: cell acquisition in the dentate gyrus

A quantitative autoradiographic histological study was carried out to examine mechanisms underlying the reduction in the rates of growth and of cell acquisition, including that of granule cells, in the dentate gyrus of hypothyroid rats. Thyroid deficiency in early life had no effect on the replication of intrinsic cells present in the polymorph and granular layers. The pyknotic index was also normal in the "proliferative zone", polymorph layer and granule cell layer, indicating that thyroid hormone had no effect on the survival of replicating, migrating or maturing granule cells. By contrast, the arrival of migrating cells from the "proliferative zone" to the granular layer was severely retarded in thyroid deficiency. This deficit was rapidly restored after a physiological dose of thyroxine given to hypothyroid rats. The present findings are consistent with our previous proposal that the role of thyroid hormone in the formation and/or the maintenance of nerve cells is related to changes in either cell migration or maturation, rather than to alterations in the replication of germinal cells.     

Cell degeneration in early development of the forebrain and cerebellum

The hippocampus, dentate gyrus, cerebellum, amygdala and caudate were examined for degenerating cells in normal neonatal hamsters. Each structure was studied from postnatal day 5 (P5) to P 10. The cerebellum was also studied on P 12. This time span was chosen to coincide with cessation of migration and establishment of connectivity in these structures. Substantial numbers of pycnotic cells were found in all structures except the dentate gyrus. The timing and amount of cell death varied between structures. Both the caudate nucleus and the amygdala showed greater cell losses in their peripheral margins than in their centers. The deep half of the internal granule cell layer of the cerebellum showed greater cell loss than the superficial half on all postnatal days studied.     

出生后大鼠海马结构兴奋性氨基酸神经元的发育

用免疫组化方法结合体视学研究,对出生后雄性大鼠海马结构中谷氨酸和门冬氨酸神经元的发育状况进行了定性与定量观察。结果证明：谷氨酸和门冬氨酸神经元主要分布于锥体层和颗粒层,其他各层相对稀少。颗粒层中两者的平均灰度值都高于锥体层（Ｐ＜ ０．０５）。锥体层中两种神经元的数密度、面数密度和体密度在发育过程中逐渐下降,平均体积逐渐增大,至生后２１ｄ 趋于稳定。颗粒层中上述参数变化不大,但随着细胞层次的增多,两种神经元的总数不断增长。谷氨酸和门冬氨酸神经元的体视学参数排列顺序如下：密度参数为颗粒层＞ ＣＡ１＞ ＣＡ３＞ ＣＡ４ 锥体层,平均体积为ＣＡ３＞ ＣＡ４＞ ＣＡ１ 锥体层＞ 颗粒层。在细胞总数中（尼氏染色标本计算）,ＣＡ１、ＣＡ３、ＣＡ４ 锥体层和颗粒层中谷氨酸神经元分别占７２．７９％ 、７４．５６％ 、６９．８３％ 、７３．５７％ ;门冬氨酸神经元分别占６９．２３％ 、７１．４４％ 、６８．１３％ 、６７．９６％ ,两者相比存在显著性差异（Ｐ＜ ０．０５）,即各亚区中谷氨酸神经元均多于门冬氨酸神经元。     

Bromodeoxyuridine immunohistochemical determination of the lengths of the cell cycle and the DNA-synthetic phase for an anatomically defined population

Summary A cumulative labelling protocol using 5-bromo-2-deoxyuridine (BUdR) was followed to determine: (1) the growth fraction (i.e., the proportion of cells that comprise the proliferating population), (2) the length of the cell cycle, and (3) the length of the DNA-synthetic phase (S-phase) for proliferative cells in the dentate gyrus of the mouse. On postnatal day 20 (P20), C57BL/6J mice were injected with BUdR at two hour intervals for a total period of 12 hours. Animals were sacrificed at selected intervals, and the brains were processed for immunohistochemistry using a monoclonal antibody directed against single-stranded DNA containing BUdR. The numbers of BUdR-labelled and unlabelled cells in sections through the hilus of the dentate gyrus were counted. The number of BUdR-labelled cells increased linearly from an initial value of about 12% of the total number of cells to a maximum value of just over 24% of the total. These findings indicate that, at P20, a maximum of 24.2 ± 1.2% of the cells in the dentate hilus are part of the proliferating population. The calculated length of the cell cycle of the cells comprising the intrahilar proliferative zone was estimated to be 16.1 ± 0.8h. The length of the S-phase was estimated at 8.0 ± 0.4 h. In addition, mathematical analysis, using one and two population models, indicates that over 90% of the proliferating cells in the dentate hilus at this age comprise a single population at least in terms of the lengths of the cell cycle and the S-phase. This protocol provides a convenient method for thein situ analysis of the cell cycle for anatomically defined proliferative populations.