THE NISSL SUBSTANCE OF LIVING AND FIXED SPINAL GANGLION CELLS : II. AN ULTRAVIOLET ABSORPTION STUDY

Living chick spinal ganglion neurons grown for 19 to 25 days in vitro were photographed with a color-translating ultraviolet microscope (UV-91) at 265, 287, and 310 mµ. This instrument was unique in permitting rapid accumulation of ultraviolet information with minimal damage to the cell. In the photographs taken at 265 mµ of the living neurons, discrete ultraviolet-absorbing cytoplasmic masses were observed which were found to be virtually unchanged in appearance after formalin fixation. These were identical with the Nissl bodies of the same cells seen after staining with basic dyes. The correlation of ultraviolet absorption, ribonuclease extraction, and staining experiments with acid and basic dyes confirmed the ribonucleoprotein nature of these Nissl bodies in the living and fixed cells. No change in distribution or concentration of ultraviolet-absorbing substance was observed in the first 12 ultraviolet photographs of a neuron, and it is concluded that the cells had not been subjected to significant ultraviolet damage during the period of photography. On the basis of these observations, as well as previous findings with phase contrast microscopy, it is concluded that Nissl bodies preexist in the living neuron as discrete aggregates containing high concentrations of nucleoprotein.     

The Nissl substance of living and fixed spinal ganglion cells. I. A phase contrast study

Living chick embryo spinal ganglion neurons grown from 1 to 4 weeks in vitro were studied under the phase contrast microscope. In the peripheral cytoplasm of the earliest stages studied, a homogeneous, phase-dense material is seen which corresponds in location to the cytoplasmic basophil material of the same stages. As maturation proceeds, this material increases in extent, and becomes separated by lighter channels into discrete bodies. Short fixation by 1 per cent buffered osmium tetroxide followed by post-fixation with neutral buffered formalin does not significantly alter the size, shape, or distribution of any of the cytoplasmic components, and the fixed, hydrated cell is almost indistinguishable from the living cell. Dehydration causes some shrinkage of the fixed preparations, but if the photographs of the stained preparations are enlarged to correspond with those of the living cell, excellent correspondence can be made between at least the larger basophil masses and the larger dark masses seen with phase contrast. Fixation by a formalin-mercuric chloride procedure also results in satisfactory correspondence between the stained Nissl bodies and the phase-dark homogeneous areas. It is concluded that discrete Nissl bodies preexist in the living neuron and are essentially unchanged after good cytological fixation. Evidence is also presented of the presence of neurofibrils in the living state.     

THE NISSL SUBSTANCE OF LIVING AND FIXED SPINAL GANGLION CELLS : I. A PHASE CONTRAST STUDY

Living chick embryo spinal ganglion neurons grown from 1 to 4 weeks in vitro were studied under the phase contrast microscope. In the peripheral cytoplasm of the earliest stages studied, a homogeneous, phase-dense material is seen which corresponds in location to the cytoplasmic basophil material of the same stages. As maturation proceeds, this material increases in extent, and becomes separated by lighter channels into discrete bodies. Short fixation by 1 per cent buffered osmium tetroxide followed by post-fixation with neutral buffered formalin does not significantly alter the size, shape, or distribution of any of the cytoplasmic components, and the fixed, hydrated cell is almost indistinguishable from the living cell. Dehydration causes some shrinkage of the fixed preparations, but if the photographs of the stained preparations are enlarged to correspond with those of the living cell, excellent correspondence can be made between at least the larger basophil masses and the larger dark masses seen with phase contrast. Fixation by a formalin-mercuric chloride procedure also results in satisfactory correspondence between the stained Nissl bodies and the phase-dark homogeneous areas. It is concluded that discrete Nissl bodies preexist in the living neuron and are essentially unchanged after good cytological fixation. Evidence is also presented of the presence of neurofibrils in the living state.     

胎脑提取液对衰老小鼠大脑皮质神经元的影响

目的 观察胎脑提取液对衰老小鼠大脑皮质神经元酸性磷酸酶(ACP)活性和尼氏体含量的影响,探讨胎脑提取液的抗衰老作用。方法 选用健康昆明种小白鼠30只,随机分为3组;采用D-半乳糖制备亚急性衰老模型;组织化学方法显示大脑皮质神经元内的酸性磷酸酶(ACP)和尼氏体;显微图像分析仪进行定量分析。结果 衰老模型组与正常对照组相比,小鼠大脑皮质神经元ACP活性明显升高,尼氏体含量明显减少;给药组与衰老模型组相比,小鼠大脑皮质神经元ACP活性明显降低,尼氏体含量明显增加。结论 胎脑提取液可以稳定大脑皮质神经元的内环境,减少神经元的损伤,促进神经元蛋白质的合成,具有一定的抗衰老作用。     

The fine structure of neurons

1. Thin sections of representative neurons from intramural, sympathetic and dorsal root ganglia, medulla oblongata, and cerebellar cortex were studied with the aid of the electron microscope. 2. The Nissl substance of these neurons consists of masses of endoplasmic reticulum showing various degrees of orientation; upon and between the cisternae, tubules, and vesicles of the reticulum lie clusters of punctate granules, 10 to 30 mmicro in diameter. 3. A second system of membranes can be distinguished from the endoplasmic reticulum of the Nissl bodies by shallower and more tightly packed cisternae and by absence of granules. Intermediate forms between the two membranous systems have been found. 4. The cytoplasm between Nissl bodies contains numerous mitochondria, rounded lipid inclusions, and fine filaments.     

The chicken trigeminal ganglion. I. An anatomical analysis of the neuron types in the adult

An anatomical analysis of the chicken trigeminal ganglion was made using light microscopy on specimens prepared by usual chemical fixation or freeze-drying methods and by electron microscopy. Two types of neurons were consistently seen, dark and light cells. Dark cells contained a dense cytoplasm with Nissl substance distributed evenly throughout, whereas light cells had a less dense cytoplasm containing clumps of Nissl substance. The Nissl bodies in light cells contained only a few small cisternae of granular endoplasmic reticulum as compared with many stacked cisternae in Nissl bodies of dark cells. The ratio of dark to light cells was approximately 62:38 in all regions of the ganglion. Dark cells were consistently smaller than light cells. In the seven-day old chick, the mean diameters of the dark and light neurons were 21.4 μ and 29.5 μ respectively; in the adult the values were 29.9 μ and 39.7 μ respectively. It is concluded that the dark and light cells belong to two distinct neuronal cell populations.     

Trypan blue in vivo stains nigral dopaminergic neurons killed by 6-hydroxydopamine

Dopaminergic neurons in the substantia nigra killed by 6-hydroxydopamine were stained in vivo by intracerebral injections of trypan blue. Such staining appeared specific for dead neurons, although a proportion of these retained the ability to stain with Nissl dyes for at least 2 days. Neurons retained trypan blue in vivo for periods of up to 9 days. Trypan blue staining of some neurons outside the substantia nigra demonstrated the use of this dye in determining the degree of non-specific toxicity of 6-hydroxydopamine. Twenty-four hours after infusion of trypan blue almost no background staining was present and individually stained neurons were clearly visible. Thus the use of trypan blue may have a general application as a sensitive method for estimating discrete areas of toxin-induced neuronal death, and for estimating the degree of specificity of a toxin.     

Trypan blue in vivo stains nigral dopaminergic neurons killed by 6-hydroxydopamine

Summary Dopaminergic neurons in the substantia nigra killed by 6-hydroxydopamine were stained in vivo by intracerebral injections of trypan blue. Such staining appeared specific for dead neurons, although a proportion of these retained the ability to stain with Nissl dyes for at least 2 days. Neurons retained trypan blue in vivo for periods of up to 9 days. Trypan blue staining of some neurons outside the substantia nigra demonstrated the use of this dye in determining the degree of non-specific toxicity of 6-hydroxydopamine. Twenty-four hours after infusion of trypan blue almost no background staining was present and individually stained neurons were clearly visible. Thus the use of trypan blue may have a general application as a sensitive method for estimating discrete areas of toxin-induced neulonal death, and for estimating the degree of specificity of autoxin.     

神经细胞尼氏体和神经髓鞘的双重组合染色法与应用

探讨了显示实验动物脊髓组织中的神经尼氏体和神经髓鞘等组织成分的双重组合染色法,通过分别选用孔雀石绿(Malachite green)和橙黄G、磷钨酸(Orange G,Phosphotungstic acid)组合染色(简称MG-OP法)。已能够显示狗脊髓神经尼氏体呈绿色,细胞核呈黄色。神经纤维髓鞘轴突呈黄色,神经膜和结缔组织纤维呈绿色,背景呈淡黄色。MG-OP法克服了原法成分单一,色彩效果差,所建立的双重组合染色法,对比清晰,色彩鲜艳,方法简便的较好染色方法。     

睫状神经营养因子对应激引起大鼠海马神经元损伤的保护作用及其机制的研究

本实验用Ｎｉｓｓｌ染色法、Ｂｉｅｌｓｃｈｏｗｓｋｙ－Ｇｒｏｓ－Ｌａｗｒｅｎｔｊｅｗ染色法、常规透射电镜、行为活动测定、双侧海马微量给药、海马神经元原代培养、活细胞连续照相、全细胞膜片钳记录、细胞内游离Ｃａ＾２＋浓度测定及Ｐ５３蛋白免疫组化测定等方法,观察了睫状神经营养因子（ＣＮＴＦ）对应激引起动物行为变化和海马神经元形态学变化的影响,探讨了ＣＮＴＦ的部分作用机制。结果表明,急性应激不引起大鼠海马神     

Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Summary Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance.Nucleolus In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as nucleolar shadow, overlaid by a few small irregular bodies of higher electron density than other nuclear contents.Nucleus The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas.Nissl Substance In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed.The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.This investigation was supported by USPHS Grants NB 03114-04, NB 00690-11 and 5 T 1 GM 495 from the National Institutes of Health, Bethesda, Maryland.Acknowledgement. Mrs. Eleanor W. Morris and Mr. Edwin E. Pitsinger, Jr. gave indispensible aid with the management of the cultures and with photographic procedures.     

Mucopolysaccharides in the dog spinal cord and dorsal root ganglia. A histochemical study.

A histochemical study of mucopolysaccharides in the dog spinal cord and dorsal root ganglia is reported in this paper. The histochemical techniques used were the following: PAS, colloidal iron, toluidine blue (pH 5.4 and 3.5), thionine (pH 5.4 and 3.5) and alcian blue 8GX (pH 1 and 2.5). Some histological stains were used also. Two types of neurons could be observed in spinal cord sections stained with colloidal iron techniques. In some neuron a border line of mucosubstances could be seen. In the dorsal root ganglia, different patterns of Nissl bodies distribution in neurons were described. This different distribution of Nissl bodies is associated with different metachromatic colorations of neurons. By using the colloidal iron method, two types of neurons were also revealed in the dorsal root ganglia: some neurons are of a yellow, small-sized and star-shaped type and others are of a light green, larger and round-shaped type. Mucosubstances in the endoneurium and perineurium of nerve fibers, in the Ranvier nodules and in the Schmidt-Lantermann incisures were observed. The possibility that the functional rhythm in some cases might be responsible for the difference in coloration between the dorsal root ganglia neurons is suggested.     

The fine structure of the neurons in the rat substantia nigra

Three distinct neurons were identified in the substantia nigra of the rat using Golgi, light, and electron microscopic techniques. A large neuron, found in the pars reticulata, is characterized by well-developed RER, a tubular cytoplasmic inclusion, and somatic and dendritec thorns. A medium-sized neuron, found in the pars compacta, has an eccentric nucleus, distinct Nissl bodies, and an inclusion composed of whorls of concentric cisternae. A small neuron, found in both nigral regions, contains a highly invaginated nucleus, fibrous nuclear inclusion, and paucity of cytoplasmic organelles. Its axon synapses around other nigral dendrites. The presence of these neurons was correlated with the efferent projections and function of the substantia nigra.     

A quantitative histological study of Golgi II neurons and pale cells in different cerebellar regions of the adult and ageing mouse brain

Two types of medium to large sized neurons are present in the granular layer of the mouse cerebellum. One type has a large nucleus with a prominent nucleolus and a moderate amount of cytoplasm containing Nissl substance. This type corresponds to the classical Golgi II neuron. The second type has a much smaller nucleus (mean diameter 8.4 microns) with a darkly staining nuclear envelope which is almost invariably deeply indented by cytoplasmic intrusions. The nucleolus is smaller and less conspicuous than in Golgi II neurons. These neurons are identical to the pale cells described by Altman and Bayer (1977). The numbers of both types of neuron were estimated in the spinocerebellum, lobus simplex and nodulus in mice aged 6, 15, 22, 25, 28 and 31 months. There was no significant variation in the number of either Golgi II neurons or pale cells with age in any part of the cerebellum. The number of Golgi II neurons per mm3 was similar in all parts of the cerebellum (mean 3560 mm3). This was identical to the mean number of pale cells per mm3 in the spinocerebellum and pontocerebellum but in the nodulus pale cells were much more numerous (mean 41,170 per mm3). It is postulated that pale cells are small Golgi II neurons.     

新生SD大鼠皮质神经元的体外培养和鉴定

目的：建立高纯度的新生SD大鼠皮质神经元原代培养方法。方法：取24h内的新生SD大鼠皮质,用木瓜酶和DNaseⅠ共同消化,5％胎牛血清终止消化,吹打分离组织获得单细胞悬液,进行细胞计数,用无血清DMEM／F12种植培养,4h后换成用无血清Neurobasal配制的维持培养液继续培养,尼氏小体染色和免疫荧光法鉴定神经元的纯度。结果：培养第10d,神经元胞体饱满,结构清晰完整,光晕明显,折光性强,可见粗长的树突和轴突,相邻细胞形成紧密网状联系,神经元纯度达到96％以上。结论：经改良和优化,无须添加阿糖胞苷抑制胶质细胞的生长即能够获得生长状态良好、高纯度的神经元。     

Calcium: A novel and efficient inducer of differentiation of adipose‐derived stem cells into neuron‐like cells

This study comparatively investigated the effectiveness of calcium and other well‐known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose‐derived stem cells (ADSCs) into neuronal‐like cells. ADSCs were immunophenotyped and differentiated into neuron‐like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron‐specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real‐time polymerase chain reaction (quantitative real‐time polymerase chain reaction (qRT‐PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite‐like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron‐like cells and instantaneous increase in the expression of neuronal markers.     

Inputs from intrinsic primary afferent neurons to nitric oxide synthase-immunoreactive neurons in the myenteric plexus of guinea pig ileum

Nitric oxide synthase (NOS) immunoreactivity occurs in two groups of neurons in the guinea pig small intestine: descending interneurons that are also immunoreactive for choline acetyltransferase (ChAT), and inhibitory motor neurons that lack ChAT immunoreactivity. Interneurons that are involved in local reflexes would be expected to have inputs from intrinsic primary afferent (sensory) neurons, most of which are calbindin-immunoreactive. We examined this possibility using triple staining for NOS, ChAT and calbindin immunoreactivity and investigated the relationships between calbindin-immunoreactive varicosities and the cell bodies of NOS-immunoreactive neurons, using high-resolution confocal microscopy and electron microscopy. By confocal microscopy, we found that the cell bodies of ChAT/NOS interneurons received 84 +/- 23 (mean +/- SD) direct appositions from calbindin-immunoreactive varicosities and that the cell bodies of NOS-inhibitory motor neurons received 82 +/- 20 appositions. Electron-microscopic examination of the relations of 265-calbindin-immunoreactive varicosities, at distances within the resolution of the confocal microscope (300 nm), to 30 NOS-immunoreactive nerve cells indicated that 84% formed close contacts or synapses and 16% were separated from neurons by thin glial cell processes. Thus, each NOS-immunoreactive nerve cell receives about 70 synaptic inputs or close contacts from the calbindin-immunoreactive varicosities of intrinsic primary afferent neurons. It is concluded that there are monosynaptic reflex connections in which intrinsic primary afferent neurons synapse directly with motor neurons and di- or poly-synaptic reflexes in which ChAT- and NOS-immunoreactive neurons are interneurons, interposed between intrinsic primary afferent neurons and NOS-inhibitory neurons.     

Variations in the axon reaction after different types of nerve lesion. Light and electron microscopic studies on the facial nucleus of the rat

The retrograde nerve cell reaction was studied after evulsion, transection and crush lesion of the facial nerve in rats. Crush lesion caused barely discernible light and electron microscopic changes. The Nissl bodies became slightly smaller than normal and the arrangement of the granular endoplasmic reticulum (rER) somewhat more irregular. The crush lesions were followed by complete functional and morphologic recovery. After nerve evulsion, the cells showed severe chromatolysis, nuclear caps, nuclear eccentricity, and folding of the nuclear membrane. Ultrastructurally there was a dispersion of the rER and formation of laminated dense bodies. Lager, the rER was partly degranulated and some of the polyribosomes dissociated. These neurons ultimately disappeared. Transection of the nerve caused an intermediate axon reaction and a moderate loss of neurons. It is concluded that certain neurons may regenerate after axotomy in spite of minimal light and electron microscopic changes in the nerve cell bodies, and that the same neurons may show the typical axon reaction after more severe nerve injuries. Mechanisms which may be involved in the regulation of the retrograde nerve cell reaction after axotomy are briefly discussed.     

A rapid method combining Golgi and Nissl staining to study neuronal morphology and cytoarchitecture.

The Golgi silver impregnation technique gives detailed information on neuronal morphology of the few neurons it labels, whereas the majority remain unstained. In contrast, the Nissl staining technique allows for consistent labeling of the whole neuronal population but gives very limited information on neuronal morphology. Most studies characterizing neuronal cell types in the context of their distribution within the tissue slice tend to use the Golgi silver impregnation technique for neuronal morphology followed by deimpregnation as a prerequisite for showing that neuron's histological location by subsequent Nissl staining. Here, we describe a rapid method combining Golgi silver impregnation with cresyl violet staining that provides a useful and simple approach to combining cellular morphology with cytoarchitecture without the need for deimpregnating the tissue. Our method allowed us to identify neurons of the facial nucleus and the supratrigeminal nucleus, as well as assessing cellular distribution within layers of the dorsal cochlear nucleus. With this method, we also have been able to directly compare morphological characteristics of neuronal somata at the dorsal cochlear nucleus when labeled with cresyl violet with those obtained with the Golgi method, and we found that cresyl violet-labeled cell bodies appear smaller at high cellular densities. Our observation suggests that cresyl violet staining is inadequate to quantify differences in soma sizes.