Regulation of protein synthesis during postnatal maturation of mouse brain.

On a DNA basis, there is higher concentration of polysomes in the brain of newborn than in the brain of adult mice, but there is no maturation-dependent decrease in tRNA content during postnatal development. The amino acid incorporating activity of cell-free systems with polysomes or mitochondria from newborn brain exceeds that of adult controls significantly in contrast to a smaller incorporating rate of labelled amino acids into synaptosomal protein. Addition of polysomes isolated from newborn brain increases the amino acid incorporation by cell-free systems with adult brain tRNA and enzymes, whereas the polysomes from adult brain decrease the incorporating activity of newborn brain systems. The loading capacity of newborn brain tRNA exceeds that of the adult controls and the velocities of its aminoacylation are four times faster. Uncharged as well as precharged newborn brain tRNA increases the amino acid incorporating activity of tRNA-dependent cell-free systems with adult brain polysomes and enzymes. In contrast to polysomes and tRNA, the newborn brain enzymes involved in protein synthesis seem to be less active in cell-free amino acid incorporation than the enzyme fractions from adult brain. These data indicate that the different protein synthesizing activity in developing and adult mouse brain is the result not only of higher amino acid incorporating activities of the newborn polysomes, but also of a stimulated acceptance and transfer function of the newborn brain tRNA.     

There is no loss of motor neurons in the rat spinal cord during postnatal maturation

Motor neurons are lost during embryonic development, but it remains controversial whether motor neuron cell death occurs during postnatal life. In this study we investigated the effect of postnatal maturation on the number of intact spinal motor neurons in the rat using retrograde labelling with model-based counting, and an unbiased stereological counting technique. To determine the number of motor neurons innervating a specific forelimb muscle in rats of different postnatal ages FluoroGold was injected into the flexor carpi radialis. Before postnatal day 21 there were higher numbers of retrogradely labelled motor neurons than in adult rats, suggesting a 'loss' with postnatal maturation. This loss may be attributed to tracer diffusion to adjacent muscles and to the permeability of the muscle spindle capsule in younger animals. To obtain an unbiased estimate of the number of motor neurons in the C7 and C8 segments of the postnatal rat cervical spinal cord the fractionator/optical disector counting technique was used. This method did not show a loss of spinal motor neurons between birth and adulthood. The main conclusion from this study is that there is no loss of spinal motor neurons during postnatal maturation.     

Projections from the brain to the spinal cord in the mouse

The cells that project from the brain to the spinal cord have previously been mapped in a wide range of mammalian species, but have not been comprehensively studied in the mouse. We have mapped these cells in the mouse using retrograde tracing after large unilateral Fluoro-Gold (FG) and horseradish peroxidase (HRP) injections in the C1 and C2 spinal cord segments. We have identified over 30 cell groups that project to the spinal cord, and have confirmed that the pattern of major projections from the cortex, diencephalon, midbrain, and hindbrain in the mouse is typically mammalian, and very similar to that found in the rat. However, we report two novel findings: we found labeled neurons in the precuneiform area (an area which has been associated with the midbrain locomotor center in other species), and the epirubrospinal nucleus. We also found labeled cells in the medial division of central nucleus of the amygdala in a small number of cases. Our findings should be of value to researchers engaged in evaluating the impact of spinal cord injury and other spinal cord pathologies on the centers which give rise to descending pathways.     

Early development of the brain and spinal cord in dysraphic mice

Summary The development and closure of the neural folds was studied in C57BL/6J and loop-tail (Lp) mutant mice by means of scanning electron microscopy on a series of embryos ranging in age from 7.5 to 9.0 days of gestation. The normal embryos (C57BL/6J; +/+; Lp/+) showed a transitional zone of flattened cells lying between the surface ectoderm and neuroepithelial cells at the apices of the neural folds in the presumptive hindbrain and spinal cord, and ruffles occurred at the boundary between the flattened cells and surface ectoderm in regions of the folds which were about to fuse. In the abnormal loop-tail homozygotes (Lp/Lp) which exhibit dysraphism, the ruffles were arranged erratically along the zone of flattened cells. Moreover, at the stage when the folds became apposed and fused in the normal embryos, the abnormals showed ruffles extending the entire length of the unfused folds, thereby distinguishing the abnormals from retarded n normal embryos. Within the neural groove of the hindbrain region, the lateral neuroepithelial cells of the abnormal dysraphic embryos exhibited more flattened surfaces and fewer villous projections than in the normal embryos. The abnormal embryos also lagged behind their normal littermates in converting the body axis from the initial V-shape to the C-shaped configuration.This research was supported by NIH grant no. HD09562 from the National Institute of Child Health and Human Development, USPHS     

Activity-dependent development of cortical axon terminations in the spinal cord and brain stem

 Corticospinal (CS) axon terminations in several species are widespread early in development but are subsequently refined into a spatially more restricted distribution. We studied the role of neural activity in sensorimotor cortex in shaping postnatal development of CS terminations in cats. We continuously infused muscimol unilaterally into sensorimotor cortex to silence neurons during the postnatal CS refinement period (weeks 3–7). Using anterograde transport of WGA-HRP, we examined the laterality of terminations from the muscimol-infused (i.e., silenced) and active sides in the spinal cord, as well as in the cuneate nucleus and red nucleus. We found that CS terminations from the muscimol-infused cortex were very sparse and limited to the contralateral side, while those from the active cortex maintained an immature bilateral topography. Controls (saline infusion, noninfusion) had dense, predominantly contralateral, CS terminations. There was a substantial decrease in the spinal gray matter area occupied by terminations from the side receiving the blockade and a concomitant increase in the area occupied by ipsilateral terminations from the active cortex. Optical density measurements of HRP reaction product from the active cortex in muscimol-infused animals showed substantial increases over controls in the ratio of ipsilateral to contralateral CS terminations for all laminae examined (IV–V, VI, VII). Our findings suggest that ipsilateral dorsal horn terminations reflect new axon growth during the refinement period because they are not present there earlier in development. Those in the ventral horn are present earlier in development and thus could reflect maintenance of transient terminations. Increased ipsilateral terminations from active cortex were due to recrossing of CS axons in lamina X and not to an increase in labeled CS axons in the ipsilateral white matter. Examination of brain stem terminations suggested that, between postnatal weeks 3 and 7, development of corticocuneate terminations also is activity-dependent but that development of corticorubral terminations is not. Activity-dependent CS development is a plausible mechanism by which early motor experiences could shape the anatomical and functional organization of the motor systems during a critical postnatal period. Received: 4 August 1998 / Accepted: 2 September 1998     

Fetal development and postnatal maturation of the longus colli muscle

In adults, the predominant expression of a slow phenotype in the m. longus colli corresponds to its important postural function. Morphologically, there is a dispersion in fiber size predominating on the fast type 2 fibers which are significantly smaller than the slow type 1 fibers. We deemed it of interest, therefore, to analyze the metabolic differentiation of the muscle longus colli during its development. This study has been carried out on six anatomical samples, in foetuses aged between 16 and 40 weeks of pregnancy and in an 18 month-old child. The histological study combined H&E staining and immunohistochemical techniques (using antibodies specific for the slow and the fast isoforms of the myosin heavy chains). Our results indicate that the m. longus colli differentiates during the foetal period in a way which is quite comparable to that of other skeletal muscles, such as the quadriceps. In this series, a major slow predominance with a significant dispersion in fiber size was first observed in the 18 month-old child. Thus, it can be concluded that the establishment of the adult phenotype of this muscle starts during postnatal life, following the development of the mechanisms holding up the head and neck and leading to the appearance of the cervical lordosis.     

Synapsin-dependent development of glutamatergic synaptic vesicles and presynaptic plasticity in postnatal mouse brain

Inactivation of the genes encoding the neuronal, synaptic vesicle-associated proteins synapsin I and II leads to severe reductions in the number of synaptic vesicles in the CNS. We here define the postnatal developmental period during which the synapsin I and/or II proteins modulate synaptic vesicle number and function in excitatory glutamatergic synapses in mouse brain. In wild-type mice, brain levels of both synapsin I and synapsin IIb showed developmental increases during synaptogenesis from postnatal days 5-20, while synapsin IIa showed a protracted increase during postnatal days 20-30. The vesicular glutamate transporters (VGLUT) 1 and VGLUT2 showed synapsin-independent development during postnatal days 5-10, following which significant reductions were seen when synapsin-deficient brains were compared with wild-type brains following postnatal day 20. A similar, synapsin-dependent developmental profile of vesicular glutamate uptake occurred during the same age periods. Physiological analysis of the development of excitatory glutamatergic synapses, performed in the CA1 stratum radiatum of the hippocampus from the two genotypes, showed that both the synapsin-dependent part of the frequency facilitation and the synapsin-dependent delayed response enhancement were restricted to the period after postnatal day 10. Our data demonstrate that while both synaptic vesicle number and presynaptic short-term plasticity are essentially independent of synapsin I and II prior to postnatal day 10, maturation and function of excitatory synapses appear to be strongly dependent on synapsin I and II from postnatal day 20.     

Innervation of the thymus gland by brain stem and spinal cord in mouse and rat

Central nervous system (CNS) projections to the thymus were studied in the mouse and rat using the horseradish peroxidase (HRP)-retrograde transport method. With discrete HRP injections localized to the thymus, labeled neurons are evident in both medulla and spinal cord. In the medulla the largest population of labeled neurons is present in the retrofacial nucleus. Within this cytoarchitectonically distinct nucleus, the majority of neurons are labeled with large HRP injections in the thymus. In addition to retrofacial nucleus, scattered labeled neurons are found throughout the rostrocaudal extent of the nucleus ambiguus and in the dorsal medullary tegmentum adjacent to the dorsal motor vagus nucleus. With HRP injections restricted to thymus parenchyma, no labeled neurons are evident in the dorsal motor vagus nucleus. Three groups of spinal cord neurons are labeled. In segments C2–C4, neurons localized to the ventral horn are labeled in two distinct columns, one located lying laterally in the ventral horn and the other located medially. Labeling of neurons in these segments is distinct from that of large motor neurons located medially in the ventral horn extending from the level of the decussation of the pyramids through the C1 segment. The location and sizes of neurons labeled in these areas following HRP injection in the thymus are identical in the mouse and rat. These observations provide evidence for previously unknown projections from spinal cord and brain stem to the thymus which may play an important role in the regulation of thymic function.     

小鼠脊髓发育与神经细胞凋亡

目的通过观察小鼠脊髓发育过程中神经元的增殖、分化与凋亡,探讨小鼠脊髓的发育过程及其调控机制。方法取妊娠第18天(E18)至生后第90天(P90)小鼠173只,用5’-溴脱氧尿嘧啶核苷(BrdU)技术标记增殖的神经干细胞,免疫荧光法(DCX,NeuN和Caspase8)标记脊髓中的新生神经元,成熟神经元和凋亡细胞。结果在胚胎与出生早期,BrdU阳性细胞均匀分布于小鼠脊髓各部位。随着小鼠日龄的增加,脊髓神经干细胞可以分化为神经胶质细胞与神经元。其后,位于神经管侧脑室的新生神经元向中间层(未来的灰质)迁移,逐渐分化为成熟神经元。最后,神经元向脊髓中央聚集,灰质呈现典型的"H"型。随着神经元的分化,一些凋亡神经细胞出现在新生神经元与成熟神经元中。荧光双标显示,大部分的凋亡神经细胞都是新生神经元,说明神经元凋亡常常发生在新生神经元中。统计学分析表明,DCX、NeuN、Caspase-8标记的阳性细胞数均随小鼠日龄的增加而减少,说明神经元的分化和凋亡在脊髓的发育过程中逐渐减少。结论在脊髓的发育过程中存在着神经元的增殖、分化与凋亡。三者相互协同,共同调节脊髓的形成与发育。     

An ultrastructural study of the development of the leptomeninx of the mouse spinal cord

The ultrastructure of the mouse spinal cord leptomeninx was studied from 11 days post-conception to 5 days post natum. At all ages the cells investing the developing cord resembled immature fibroblasts. The most characteristic feature was the rough endoplasmic reticulum, filled with an amorphous ground substance and frequently branched. At first other cytoplasmic organelles were sparse apart from numerous free ribosomes. During development mitochondria became more numerous. From E11 presumptive leptomeningeal processes were joined by tight junctions and the endothelial cells of leptomeningeal vessels were united by zonulae occludentes. Collagen fibrils were found from E11 and in prenatal mice were of small diameter, although the quantity of collagen increased with age. In the postnatal mice collagen was of larger diameter with the characteristic banded appearance.     

Urotensin-II expression in the mouse spinal cord

Urotensin-II (UII), a 12 amino acid peptide, was discovered in the teleost fish neurosecretory cells located in the caudal portion of the spinal cord and which project to a neurohemal gland called the urophysis. The distribution of UII and of its prepro-UII mRNA is not limited to fish and was found for example in the rat spinal cord. In view of the potential interest of obtaining transgenic mice, we have therefore characterized the distribution of mouse pro-UII mRNA and UII immunoreactivity, by in situ hybridization and immunohistochemistry, respectively, in the mouse spinal cord. A population of UII-like immunoreactive cell bodies was located in the ventral horn of the different segments. These cells displayed all the features of motoneurons, as confirmed by a double immunohistochemical labelling showing the co-occurrence of UII and vesicular acetylcholine transporter, and by electron microscope immunocytochemistry. Retrograde labelling of motoneurons innervating the bulbocavernosus penile muscle showed that some of them contained UII. In situ hybridization histochemistry revealed that pro-UII mRNA was located in some ventral horn neuronal perikarya. The pro-UII mRNA-containing cell bodies possessed the same motoneuron characteristics, confirming the results of the immunohistochemical studies and showing that the gene of mouse UII is expressed in a subpopulation of motoneurons in the spinal cord. Our results support the assumption that UII peptide characterized as endocrine in fish is also expressed within mammalian motoneurons.     

Effect of tetanus toxin on protein composition of synaptic structures of the rat brain and spinal cord

It was shown by electrophoresis on polyacrylamide gel that the content of proteins with low electrophoretic mobility rises in a Triton extract of the fractions of synaptic structures from the spinal cord tissue of rats with local tetanus, whereas no change was found in the protein spectrum in the dodecyl sulfate extract. In experiments in vitro tetanus toxin stimulated the incorporation of lysine-H³ into total proteins of cortical synaptosomes.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 79, No. 4, pp. 19–22, April, 1975.     

大鼠脊髓发育及损伤后NIDD mRNA表达的实验研究

目的：探讨在大鼠发育过程中及急性脊髓损伤后脊髓组织中NIDD （nNOS-interacting DHHC domain-containing protein with dendritic mRNA）mRNA的表达变化及意义。方法：采用改良Allen＇s打击法，咬除T8-10椎板后，造成大鼠脊髓损伤模型，致伤量为10×10g·cm；借助实时荧光定量PCR、原位杂交与免疫荧光结合的方法，定量、定位研究发育过程中及脊髓损伤后早期大鼠脊髓组织中NIDD mRNA与nNOS mRNA表达的时间和空间分布特征。结果：大鼠发育过程中，胚胎16d的大鼠脊髓中可见NIDD mRNA的高表达，在生后1d，与nNOS共表达于尚未分化成熟的前角，在白质也见NIDD的阳性信号。成年后呈低表达；nNOS mRNA于生后1～3d出现表达高峰；脊髓损伤后NIDD mRNA表达明显增多，在8h到达高峰，分布于脊髓前角、中间带、中央管周围及后角nNOS阳性的神经元，7d恢复至正常水平；nNOS mRNA在损伤后8h达到高峰，1d降低至正常水平。而且，在脊髓损伤后NIDD mRNA与nNOS mRNA二者表达呈正相关。结论：胎鼠脊髓中，NIDD高表达于nNOS阳性细胞，提示其在脊髓组织的发育成熟过程中的作用可能与nNOS相关。脊髓损伤后脊髓组织中NIDD与nNOS表达增多，提示在脊髓损伤的病理过程中，NIDD可能通过调节nNOS的细胞亚定位及活性而发挥一定的生物学作用。     

地高辛原位杂交显示锌转运体 3 mRNA 在脑、脊髓和脊神经节中的分布

目的:为研究锌转运体 3(ZnT3) 在神经系统功能中的作用提供形态学依据.方法:制备带有地高辛标记物的 ZnT3反义 RNA 探针,取雄性 SD 大鼠脑、脊髓和脊神经节行冷冻切片,作 ZnT3 mRNA 原位杂交组织化学显色,杂交后用 AKP标记的抗地高辛抗体显色.结果:脑内 ZnT3 mRNA 主要分布于海马结构,包括街状回颗粒细胞和 CAl～CA4 区的锥体细胞,在梨状皮质、扣带后皮质中也有分布,显示的细胞较小;腩干中也有 ZnT3 mRNA 分布,面神经核处分布较多,网状结构中有少量分布;脊髓灰质前角内有少数胞体较大的细胞呈 ZnT3 mRNA 阳性;脊神经节内的多数神经元呈 ZnT3 mRNA 强阳性.结论:ZnT3 mRNA 主要分布于海马、大脑皮质、杏仁核、脑干网状结构等可塑性较强结构和周围神经神经元中,提示ZnT3 可能在学习、记忆、初级感觉传入和肌运动调控等方面发挥重要作用.     

Genetic variation in the development of mouse brain and behavior: evidence from the middle postnatal period.

Six inbred strains and 3 F2 hybrid crosses of mice were assessed for developmental status at 32 days after conception (about 13 days after birth). Phenotypes measured included body weight, brain weight, maturity of 14 reflexive behaviors, myelination of 80 fiber tracts, and thickness of the external granular layer of the cerebellum. All measures of brain and behavior showed a similar pattern of results: hybrids were generally more advanced than either of their inbred parent strains; differences among inbred strains were large, but differences among hybrid crosses were quite small. Acceleration of F2 mice compared to their homozygous relatives ranged from .5 to 2.4 days mean difference. Developmental ages of inbred litters ranged from 28.7 to 32.2 days, whereas hybrid litters ranged from 31.5 to 32.7 days.     

Apoptosis in the rat spinal cord during postnatal development; the effect of perinatal asphyxia on programmed cell death

The aim of our study was to investigate the effect of perinatal asphyxia on developmental apoptosis in the cervical and lumbar spinal cord in the neonatal rat. Perinatal asphyxia was induced by keeping pups at term in utero in a water bath at 37 degrees C for 20 min, followed by resuscitation. Effects of this treatment on developmental apoptosis were studied on postnatal days 2, 5 and 8 using terminal deoxynucleotidyl transferase (TdT)-dUTP-biotin nick end labelling (TUNEL) and caspase-3 staining. TUNEL positive cells were identified using double immunostaining. On postnatal day 2 an increase of 215% in TUNEL positive cells was detected (P=0.005) in laminae IV-VII of the lumbar spinal cord of rats which underwent perinatal asphyxia compared to controls. An increase of 55% compared to controls (P=0.03) was seen in laminae I-III of the lumbar spinal cord at postnatal day 8. TUNEL positive cells could be partly identified as microglia cells (ED1 positive) and oligodendrocytes (O4 positive). The effect of perinatal asphyxia on programmed cell death in the neonatal rat spinal cord was mainly observed in the intermediate zone and dorsal horn of the lumbar spinal cord.We conclude that perinatal asphyxia has a pronounced effect on the survival of cells in a specific region of the spinal cord and thus may have a profound effect on the development of motor networks.