Enrichment of spinal cord cell cultures with motoneurons

Spinal cord cell cultures contain several types of neurons. Two methods are described for enriching such cultures with motoneurons (defined here simply as cholinergic cells that are capable of innervating muscle). In the first method, 7-day embryonic chick spinal cord neurons were separated according to size by 1 g velocity sedimentation. It is assumed that cholinergic motoneurons are among the largest cells present at this stage. The spinal cords were dissociated vigorously so that 95-98% of the cells in the initial suspension were isolated from one another. Cells in leading fractions (large cell fractions: LCFs) contain about seven times as much choline acetyltransferase (CAT) activity per unit cytoplasm as do cells in trailing fractions (small cell fractions: SCFs). Muscle cultures seeded with LCFs develop 10-70 times as much CAT as cultures seeded with SCFs and six times as much CAT as cultures seeded with control (unfractionated) spinal cord cells. More than 20% of the large neurons in LCF-muscle cultures innervate nearby myotubes. In the second method, neurons were gently dissociated from 4-day embryonic spinal cords and maintained in vitro. This approach is based on earlier observations that cholinergic neurons are among the first cells to withdraw form the mitotic cycle in the developing chick embryo (Hamburger, V. 1948. J. Comp. Neurol. 88:221-283; and Levi-Montalcini, R. 1950. J. Morphol. 86:253-283). 4-Day spinal cord-muscle cultures develop three times as much CAT as do 7-day spinal cord-muscle plates, prepared in the same (gentle) manner. More than 50% of the relatively large 4-day neurons innervate nearby myotubes. Thus, both methods are useful first steps toward the complete isolation of motoneurons. Both methods should facilitate study of the development of cholinergic neurons and of nerve-muscle synapse formation.     

鸡脊髓运动神经元中钙离子通道α2亚基和钠离子通道α亚基的免疫学表达

目的观察电压门控钙离子通道α2（votage—gated calcium channel,VGCC）亚基和电压门控钠离子通道α（voltage—gated sodium channel,VGSC）亚基在鸡脊髓运动神经元中的表达,并探讨相互表达的关系。方法应用免疫组织化学（ABC法）观察鸡脊髓前角神经元中cCα2亚基和sCα亚基的表达,并应用免疫荧光双标记法观察鸡脊髓运动神经元中CCα2亚基和SCα亚基表达的关系。结果CCα2亚基主要表达于脊髓IX层的大型神经元中,Ⅷ层的部分小型神经元亦呈CCα2亚基免疫阳性,大约83％的脊髓运动神经元呈CCα2阳性。CCα2亚基免疫反应物位于神经元胞浆和近位树突中。SCα亚基免疫反应物主要表达于脊髓运动神经元细胞核中,或同时表达在神经元的细胞核及胞浆中。一些有髓轴突和神经元近位树突亦呈SCα亚基免疫强阳性。大约46％的运动神经元呈SCα亚基阳性,并所有SCα亚基免疫阳性运动神经元均为CCα2亚基阳性。结论脊髓运动神经元中CCα2亚基和SCα亚基有多样性表达,可能表明运动神经元的不同运动活性。     

Transneuronal tracing of neural pathways influencing both diaphragm and genioglossal muscle activity in the ferret.

In prior experiments that employed the transneuronal transport of isogenic recombinants of pseudorabies virus (PRV), we demonstrated that neurons located ventrally in the medial medullary reticular formation (MRF) of the ferret provide collateralized projections to both diaphragm and abdominal muscle motoneurons as well as to multiple abdominal muscle motoneuron pools. The goal of the present study was to determine whether single MRF neurons also furnish inputs to diaphragm motoneurons and those innervating an airway muscle with inspiratory-related activity: the tongue protruder genioglossus. For this purpose, PRV recombinants expressing unique reporters (beta-galactosidase or enhanced green fluorescent protein) were injected into either the diaphragm or the genioglossal muscle. The virus injections produced transneuronal infection of overlapping populations of MRF neurons. A small proportion of these neurons (<15%) was infected by both PRV recombinants, which indicated that they provide collateralized inputs to genioglossal and diaphragm motoneurons. These findings show that, whereas some MRF neurons simultaneously influence the activity of upper airway and respiratory pump muscles, other cells in this brain stem region independently contribute to diaphragm and genioglossal muscle contraction regulation.     

Reactivation of nuclear RNA polymerase activity in growth stimulated epithelial cells

The activation of endogenous RNA polymerases has been studied in mouse kidney epithelial cells which have been induced to grow in culture. Slide cultures were assayed for RNA polymerase, in situ, after brief fixation. Enzyme activity was detected by autoradiography. Polymerases I (nucleolar) and II (nucleoplasmic) were distinguished by localization and by the activities in the presence of different ions as well as sensitivity to α-amanitin.Kidney epithelial cells resuming their growth activity when inoculated in vitro show an early and rapid increase in nuclear size (nuclear protein content) which precedes cell multiplication. Both nucleolar and nucleoplasmic polymerase activities appear at an early phase of growth activation and increase during the whole period of growth activation in proportion to increase in nuclear size. RNA polymerases may also be activated in cells which have not undergone an increase in nuclear size if assayed in the presence of a high salt concentration (0.4 M ammonium sulphate). The role of modification of chromatin structure for genetic reactivation is discussed.     

不同固定方法对植物细胞扫描电镜观察用冷冻断裂样品的影响

用冷冻断裂法在扫描电镜下研究了洋葱(Allium cepa)根端分生组织细胞内部的三维结构。采用了两种固定方法。冷冻断裂前只用1％锇酸固定的材料容易在细胞质和核之间断开,而用卡诺固定液(无水乙醇:冰醋酸3:1)前固定,然后再用1％锇酸固定的材料容易使细胞核断裂。前一固定方法适于研究细胞质的内部结构(细胞骨架的纤维、线粒体、内质网等及其三维分布关系):后一固定方法适于研究核内结构(染色质、核仁、核基质纤维)的三维形象,特别是核仁纤维中心染色质的三维结构。     

Cloning of the cDNA and mRNA expression of CLRP,a complex leucine repeat protein of the Golgi apparatus expressed by specific neurons of the rat brain

We report the molecular cloning of one novel cDNA isolated from the rat brain. We have named the putative protein CLRP, for complex leucine-repeat protein. The predicted CLRP amino acid sequence shares homology in the amino acid composition with the Galactose, N-Acetylglucosamine, and Sialic acid transporters, and shows 91% identity with the sequence of one human chromosome 5 BAC clone. Expression of the CLRP cDNA tagged with GFP in COS-7 cells was found in cell organelles that resemble the Golgi apparatus of the cytoplasm. In Northern blot, the CLRP probe labels a single band of 2.4 kb in the brain, kidney, lung, testis, and prostate. In the brain, CLRP mRNA is expressed by limited sets of neurons, such as the pyramidal cells of the cortex, the Purkinje cells of the cerebellum, and the motoneurons of the brainstem. In the brain, the CLRP mRNA is expressed at embryonic day 15; levels of expression are maintained until postnatal day 10 and decrease in adults. The results suggest that CLRP codes a novel member of the nucleotide-sugar family of proteins of the Golgi apparatus.     

Morphologic and cytophotometric indices of the state of the neurons of the spinal cord and spinal cord ganglia following exposure to gravitational stress]

The work presents data on the state of the neurons of the spinal ganglia and the spinal cord of the lumbosacral part in 96 cats subjected to single stresses (10g) and repeated ones (6g). The material was stained with thionin after Nissl. RNA was detected after Einarson. Photometry of the sections was made in MUP.5. Morphological changes were shown to be more pronounced in sensory cells. The method of cytophotometry established the increase by 25% amount of RNA in their cytoplasm after single stresses. Repeated stresses resulted in depletion of RNA. The amount of RNA returned to the initial level within 2 days after single stresses and within 3 days after repeated rotations. The shifts were much less pronounced in motoneurons.     

免疫磁珠法分离纯化胚胎大鼠脊髓源运动神经元的方法探讨

目的探讨应用免疫磁珠法分选脊髓源性运动神经元的实验条件,为研究运动神经元的特性,培养和移植创造有利条件。方法取孕16天胚胎大鼠的脊髓腹侧组织,制备成单细胞悬液,应用免疫磁珠分选系统,在无血清限定性培养基条件下获得相对纯化的运动神经元。应用运动神经元特异的胆碱乙酰转移酶（ChAT）抗体对培养细胞进行免疫细胞化学鉴定。结果分离纯化的细胞在体外可以存活,经免疫荧光检测,ChAT阳性率可达95%。结论本实验提供了一种有效的纯化脊髓运动神经元的方法,纯化所得的运动神经元可用于进一步的运动神经元的后续实验研究。     

A comparative analysis of the morphochemical changes in the neurons of the visual and sensorimotor cortices and the nucleus accumbens of rats administered tuftsin

White rats were treated with a single administration of tetrapeptide tuftsin (Thr-Lys-Pro-Arg) in the dose 300 mcg/kg (b.w). Using interferometry, the protein content and concentration were assessed 15, 30 and 60 minutes after injection. The area of the neuron cytoplasm and nucleus were measured too. The nucleoplasmic balance and dispersion were calculated. Significant alterations in the protein contents and cellular area, nucleoplasmic balance and dispersion were detected in neurons of visual, sensomotor cortex and of n. accumbens. A possible interrelation is discussed between tuftsin action and the functional activity of neurons, between the level of their protein metabolism and establishment of emotional and motor response.     

Cytoarchitectonic and neurochemical properties of spinal cord in teleost fishes

Traditional neuromorphological and NADPH-diaphorase methods were used to study the topography, morphology and neurochemical organization properties of spinal cord in teleosts fishes. The heterogeneous population of NO-producing motoneurons was revealed in the motor column of spinal cords from studied species. Dendrites of primary motoneurons formed rich plexus at the spinal segment periphery. This morphological pattern is determined by translational motion of the fishes in the water (trunk-tail movement), and has no connection with the origin of upper and lower extremities. The NO-producing capacity of spinal motoneurons shows their connection with premotor NO-ergic brain system, including over situated motor centers of reticular formation and descending projections of giant steam neurons (Mauthner and Muller cells). The NO-producing Rohon-Berd neurons were found in the dorso-medial part of spinal cord from studied fishes. These cells with the ascending propriospinal targets form spinal nociceptive system. Thus, the sense Rohon-Berd cells and most motor neurons of studied bony fishes are nitric oxide synthesizing ones. Spinal cord NO-synthesizing territories are situated in concordance with dorso-ventral histochemical gradient. Spinal cord interneurons of these fishes produce nitric oxide selectively. The quantity of NO-synthesizing reticular cells is determined by two main factors: the connection with the specialized neurochemical complexes, where NO is a specific neuromodulator, and individual properties of spinal cord structure directed by conditions of morphoadaptation.     

Cloning of the cDNA and mRNA expression of CLRP,a complex leucine repeat protein of the golgi apparatus expressed by specific neurons of the rat brain

We report the molecular cloning of one novel cDNA isolated from the rat brain. We have named the putative protein CLRP, for complex leucine‐repeat protein. The predicted CLRP amino acid sequence shares homology in the amino acid composition with the Galactose, N‐Acetylglucosamine, and Sialic acid transporters, and shows 91% identity with the sequence of one human chromosome 5 BAC clone. Expression of the CLRP cDNA tagged with GFP in COS‐7 cells was found in cell organelles that resemble the Golgi apparatus of the cytoplasm. In Northern blot, the CLRP probe labels a single band of 2.4 kb in the brain, kidney, lung, testis, and prostate. In the brain, CLRP mRNA is expressed by limited sets of neurons, such as the pyramidal cells of the cortex, the Purkinje cells of the cerebellum, and the motoneurons of the brainstem. In the brain, the CLRP mRNA is expressed at embryonic day 15; levels of expression are maintained until postnatal day 10 and decrease in adults. The results suggest that CLRP codes a novel member of the nucleotide–sugar family of proteins of the Golgi apparatus. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 166–173, 2002     

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.     

Cellular characteristics of immunolabeled luteinizing hormone releasing hormone (LHRH) neurons

This study utilized the preembedding immunocytochemical technique in order to identify LHRH-containing neurons in rat brain and define their ultrastructural characteristics. LHRH-containing neurons in the vertical limb of the diagonal band of Broca, medial septum, triangular nucleus of the septum and other regions were studied by taking ultrathin serial sections. These neurons had scant cytoplasm surrounding a centrally-located, spheroid, euchromatic nucleus. Neurosecretory granules were evenly distributed throughout the cell, but many tended to lie directly under the plasmalemma. The cytoplasm was organized in such a way that the most extensive portion of the rough endoplasmic reticulum was polar opposite to areas having high concentrations of Golgi complex, lysosome-like bodies, smooth endoplasmic reticulum and ribosomes. The perikarya had no axosomatic synapses but functional interaction via unspecialized appositions to the plasmalemma cannot be discounted. Many of the perikarya bore at least one cilium. Processes from immunonegative cells were occasionally observed to penetrate the cytoplasm of the LHRH perikaryon or its processes. At their points of origin, dendrites were found to be broadened processes containing many elements common to the cytoplasm: ribosomes, smooth endoplasmic reticulum, cristal and lamellar mitochondria, neurotubules, and an occasional alveolate caveola. Infrequently, some of the LHRH axons were partially myelinated. This method of studying serial-sectioned immunocytochemically-identified cells is suggested as a means of describing the cellular and subcellular characteristics of other specific peptide-containing cells.     

Breeding status affects motoneuron number and muscle size in naked mole-rats: recruitment of perineal motoneurons?

Naked mole-rats live in large colonies and exhibit a strict reproductive hierarchy. Each colony has one breeding female and one to three breeding males; all other individuals are nonreproductive subordinates. Subordinates show a remarkable lack of sex differences in behavior and anatomy, but can become reproductive if removed from the colony. We recently reported that the striated perineal muscles and their innervating motoneurons, which are sexually dimorphic in all other mammals examined to date, are not dimorphic in subordinate naked mole-rats. Here we asked whether sexual differentiation of this neuromuscular system occurs when a subordinate becomes a breeder. The size and number of cells within Onuf's nucleus (homologue of the rat spinal nucleus of the bulbocavernosus) as well as perineal muscle volume were examined in subordinate and breeding naked mole-rats of both sexes. Sex differences in perineal motoneurons were not observed, regardless of social status. To our surprise, however, counts of motoneurons in Onuf's nucleus were increased approximately 30% in breeders of both sexes. This was accompanied by a reciprocal decrease in cells in Onuf's nucleus that were characterized by small soma size, and lacked a clear nucleus or nucleolus. Although not exhibiting typical motoneuron morphology, some of these small cells were positive for the motoneuron marker, SMI-32. The neuronal changes correlate with increased perineal muscle volumes in breeders. We propose that small, relatively undifferentiated cells are recruited to the pool of large Onuf's nucleus motoneurons when subordinate naked mole-rats become breeders.     

Effects of Different Fixatives on Freeze-Fractured Samples of Plant Cells for SEM Observations

Three-dimensional structures of meristematic cells of Allium cepa were studied using freeze-fracture method under the scanning electron microscope. Two fixation procedures were used. The cells were often fractured between eytoplasm and nucleus when the materials were fixed in 1% OsO4 alone before freeze fracture, whereas the nuclei, were frequently fractured if the materials were fixed first in Carnoy's, solution (ethanol: acetic acid=3:l) and then in 1% OsO4 before freeze fracture. The former fixation procedure is suitable for the study of the interior structures of cytoplasm such as cytoskeleton fibres, mitochondria, endoplasmic reticulum and their three-dimensional topography. The latter fixation method is suitable for the study of interior structures of nucleus such as chromatin, nucleoli, nuclear matrix filaments and their 3-dimensional architectures, especially the 3-dimensional structures of chromatin in fibrillar centre of the nucleolus.     

In situ hybridization technique to localize rRNA and mRNA in mammalian neurons

In situ hybridization provides a method for identifying cells that contain specific nucleic acid sequences. This report outlines an in situ hybridization procedure for mammalian neural tissue. The method maintains morphological quality and produces excellent specificity. Seven tritiated nucleic acid probes were examined: two ribosomal RNA probes, a control pBR322 plasmid probe, two probes encoding portions of the gene for oxytocin, one probe each encoding a portion of vasopressin glycoprotein, and neurophysin. Using cryostat-cut rat brain sections, rRNA probes labeled the cytoplasm of all cells and the nucleoli of larger neurons. The plasmid probe failed to produce a strong signal. Oxytocin and vasopressin probes appropriately labeled the cytoplasm of hypothalamic magnocellular neurons. Vasopressin parvocellular neurons were not identified by the current method, and the shorter length neurophysin probe failed to produce a signal. Methodological variables were examined by counting autoradiographic grains in cells. The longer oxytocin probe produced a stronger signal than the shorter oxytocin and vasopressin probes, and higher probe concentrations resulted in stronger signal. Hybridization could be abolished by tissue pretreatment with RNAse A, and longer exposure time increased signal strength. The outlined fixation steps with fresh-frozen tissue produced a superior signal compared to paraformaldehyde-perfused tissue.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

Breeding status affects motoneuron number and muscle size in naked mole‐rats: Recruitment of perineal motoneurons?

Naked mole‐rats live in large colonies and exhibit a strict reproductive hierarchy. Each colony has one breeding female and one to three breeding males; all other individuals are nonreproductive subordinates. Subordinates show a remarkable lack of sex differences in behavior and anatomy, but can become reproductive if removed from the colony. We recently reported that the striated perineal muscles and their innervating motoneurons, which are sexually dimorphic in all other mammals examined to date, are not dimorphic in subordinate naked mole‐rats. Here we asked whether sexual differentiation of this neuromuscular system occurs when a subordinate becomes a breeder. The size and number of cells within Onuf's nucleus (homologue of the rat spinal nucleus of the bulbocavernosus) as well as perineal muscle volume were examined in subordinate and breeding naked mole‐rats of both sexes. Sex differences in perineal motoneurons were not observed, regardless of social status. To our surprise, however, counts of motoneurons in Onuf's nucleus were increased ～30% in breeders of both sexes. This was accompanied by a reciprocal decrease in cells in Onuf's nucleus that were characterized by small soma size, and lacked a clear nucleus or nucleolus. Although not exhibiting typical motoneuron morphology, some of these small cells were positive for the motoneuron marker, SMI‐32. The neuronal changes correlate with increased perineal muscle volumes in breeders. We propose that small, relatively undifferentiated cells are recruited to the pool of large Onuf's nucleus motoneurons when subordinate naked mole‐rats become breeders. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006