大鼠皮质神经元的体外培养和纯化

目的：探讨大鼠皮质神经元分离纯化的有效方法。方法：取胎鼠大脑皮质细胞，分别在不同的培养液中进行原代培养，利用相差显微镜对培养的细胞进行动态观察；并以免疫细胞化学技术对神经元进行染色。结果：在相差显微镜下可见加用N2添加剂和胎牛血清培养的神经元生长良好，胞体形态多样，突起数目多，长度长，与邻近神经元的胞体或突起形成接触，而胶质细胞则大量死亡，神经元纯化率达94％以上，未加用N2添加剂培养的神经元胶质细胞大量存在，神经元纯化率达50％左右。结论：N2添加剂和胎牛血清联合应用可使大鼠皮质神经元得到良好的生长和有效纯化。     

pp60c-src encoded by the proto-oncogene c-src is a product of sensory neurons

The advent of recombinant DNA technology has led to the identification in the DNA of normal animal cells of over 30 proto-oncogenes that are homologous to retroviral transforming genes. One of these encodes a protein kinase (pp60c-src) of unknown function, that is preferentially synthesized in brain and neural retina. Here the expression of pp60c-src in the peripheral nervous system was examined in sensory neurons from chick dorsal root ganglia with antisera raised against the transforming protein of Rous sarcoma virus (pp60v-src) expressed in Escherichia coli carrying the cloned v-src gene. This antiserum recognizes pp60c-src specifically in normal chicken cells. Western immunoblotting showed that dorsal root ganglia of stage 30 (day 6.5) chick embryos contained elevated levels of pp60c-src. Immunoperoxidase staining of neuron-enriched cultures prepared from chick dorsal root ganglia showed pp60c-src immunoreactivity in cells with neuronal morphology; flat, fibroblastic cells contained no detectable immunoreactivity. Indirect double immunofluorescence with pp60src antibodies and monoclonal antibodies against the 200-kD subunit of neurofilament protein confirmed that the cells expressing pp60c-src were neurons. Ninety-six percent of the neurofilament-positive cells were immunoreactive with pp60src antibodies, and conversely, all pp60c-src-positive cells were immunoreactive with neurofilament antibodies. pp60c-src immunofluorescence appeared to be distributed over the cell body, processes, and growth cones. These results clearly demonstrate that pp60c-src is a product of neurons and is expressed in sensory neurons in culture.     

In vivo Intracellular Recording of Neurons in the Supraoptic Nucleus of the Rat Hypothalamus

Intracellular recordings were made from cells in the hypothalamic supraoptic nucleus in the urethane-anaesthetized male rat using the ventral surgical approach. Impalements lasted from 5 min to 1 h and recorded cells had an input resistance of 55 to 170 megohms. Spikes of over 50 mV were recorded from 14 cells which could be antidromically activated by stimulation of the neural stalk. The spikes showed a hyperpolarizing afterpotential and the broadening characteristic of rapidly firing magnocellular neurons, which recovered rapidly (<200 ms). When depolarized, the cells showed evidence of a transient potassium current. Recurrent synaptic coupling between the recorded cell and adjacent cells would be expected to alter the hyperpolarizing afterpotential of an antidromic spike as compared with a spontaneous spike; no perceptible difference in the waveforms of the different types of spike could be detected in 11 spontaneously active cells. Application of just subthreshold stimuli to the neural stalk did not evoke depolarizing or hyperpolarizing potentials. Suprathreshold shocks to the neural stalk, when the antidromic spike was prevented by collision, also had no discernible effect on membrane potential. Thus intracellular recordings from magnocellular neurons in vivo revealed electrophysiological properties similar to those seen in vitro. No evidence for synaptic interconnection between magnocellular neurons was found in male rats.     

Chemically distinct rat olivocochlear neurons.

We have produced a neurochemical map of the cell bodies of origin of the cochlear efferent terminals in rat by combining glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), or calcitonin gene-related peptide (CGRP) immunocytochemistry with retrograde transport of horseradish peroxidase. The locations of cochlear efferent cell bodies are in general agreement with the medial and lateral systems described by White and Warr (J. Comp. Neurol. 219:203-214, 1983) with some minor modifications. The lateral system consists of at least two pools of chemically distinct neurons located within the lateral superior olive (LSO) ipsilateral to the injected cochlea. One pool immunostains with an antibody to GAD while the other immunostains with antibodies to ChAT and to CGRP. The medial efferent system consists of periolivary neurons that are almost exclusively large and ChAT-positive but CGRP-negative. They are located both ipsilateral and contralateral to the cochlea they project to. There are a few GAD-positive small neurons in the medioventral and rostral periolivary regions that project ipsilaterally, but these may prove tobe ectopic neurons. The ipsilateral lateroventral periolivary region (LVPO) contains some efferent neurons, all of which are ChAT-positive but CGRP-negative. Additional cochlear efferent neurons, some of which are ChAT-positive and others GAD-positive, are present within and immediately dorsal to the fiber capsule surrounding the medial limb, and to a lesser extent the lateral limb, of the ipsilateral LSO. Not all GAD-positive or ChAT-positive olivary cells project to the cochlea. We have complemented the results in the brainstem by demonstrating two immunocytochemically distinct populations of efferent terminals in the cochlea simultaneously, one CGRP-positive and the other GAD-positive. Approximately equal numbers of boutons immunoreactive for both markers are present beneath inner hair cells throughout the entire length of the cochlea. Surprisingly high numbers of GAD-positive and CGRP-positive boutons are also present on outer hair cells, with each class having its spatially and morphologically distinct features. The lack of CGRP-positive periolivary cells that are retrogradely labeled by cochlear injections of HRP suggests that the lateral olivocochlear system sends projections to outer hair cells. Our results raise questions about species differences in the organization of targets of the lateral and medial olivocochlear systems.     

Cat-301 immunoreactivity in the lateral geniculate nucleus and visual cortex of the strabismic amblyopic cat

Purpose: It was investigated whether alterations in neuronal structure and function occasioned by strabismic amblyopia also may be reflected in alterations in the expression on Y type neurons of a Cat-301 antibody sensitive antigen in the lateral geniculate nucleus (LGN) and cortex of our cat model of strabismic amblyopia. Methods/Results: The percentage of positively labelled cells was reduced in LGN laminae that received input from the deviated eye in strabismic amblyopic cats compared with normal cats. In the strabismic cortex, the density of immunopositive neurons was significantly reduced compared with normal, the effect being most pronounced in layer IV Conclusions: Despite previous physiological recordings indicating a decrease in X-cell associated acuity in strabismic amblyopia, the present findings imply that the changes in the early visual experience occasioned by strabismus also produce specific molecular changes in theY neuronal class.     

谷氨酸诱导体外培养的鸡胚脊髓神经细胞释放NO

用鸡胚脊髓神经细胞的原代培养,测定细胞中亚硝酸盐的含量,研究了谷氨酸(Glu)对原代培养神经细胞中NO的影响。结果表明,谷氨酸作用于原代培养的鸡胚脊髓神经细胞,在诱导神经细胞释放乳酸脱氢酶(LDH)的同时,还可诱导细胞释放一氧化氮(NO)。如先用NO合成酶抑制剂L-NOARG作用细胞,再加入Glu,则发现L-NOARG能降低Glu导致的培养液中LDH活性升高。提示NO可能参与介导Glu的神经毒性作用。     

Electrophysiological Characterization of a Novel Conotoxin That Blocks Molluscan Sodium Channels

A novel peptide toxin, PnIVB, isolated from the venom of Conus pennaceus blocks voltage-gated sodium current in Aplysia neurons. Complete blockade is obtained at a PnIVB concentration of 80±2.2 nM and 50% blockade at 16±0.86 nM. The potency of PnIVB in blocking Aplysia sodium current is four orders of magnitude larger than that of tetrodotoxin. The toxin has no paralytic activity when injected into fish. The rapid blockade of sodium current by PnIVB is not associated with a change in the activation or inactivation kinetics of the current, or with the reversal potential. Sodium current blockade is reversible after a 30 min wash with 50 times the bath volume. The novel conotoxin PnlVB can be used as a powerful tool for mollusc neurobiological research and as a molecular probe to explore the structure-function relations of voltage-gated sodium channel subtypes.     

Cytoprotective actions of 2,4-dimethoxybenzylidene anabaseine in differentiated PC12 cells and septal cholinergic neurons

The potential cytoprotective actions of a novel nicotinic agent 2,4-dimethoxybenzilidene anabaseine (DMXB) were investigated in differentiated PC12 cells and transected rat septal cholinergic neurons in vivo. In NGF-differentiated PC12 cells, removal of both NGF and serum led to cell loss, a reduced % of cells expressing neurites, the release of lactate dehydrogenase, and a decrease in total cellular protein. Cell loss was apparent within 24 h, and remained constant between 4–8 days post-NGF removal. NGF alone (100 ng/ml), DMXB (10 μM), but not nicotine (10 μM), prevented these cell and neurite losses. DMXB-induced cytoprotection was blocked by 1 μM mecamylamine. DMXB (1 mg/kg, ip) injected twice but not once per day protected cholinesterase-staining septal neurons from retrograde degeneration following unilateral fimbrial transections. The twice per day DMXB injection-protocol also decreased cell roundness among cholinesterase-staining cells in the lesioned septal hemisphere compared to saline-injected animals. These studies suggest that DMXB may exert cytoprotective activity in NGF-sensitive neuronal populations. © 1994 Wiley-Liss, Inc.     

Morphological types of ganglion cells in the dog and wolf retina.

The morphological types of ganglion cells in the dog and wolf retina were studied by intracellular staining with Lucifer Yellow. These retinae contain a range of ganglion cell types that closely correspond to those found in cat retina: alpha cells with large somata and large, relatively densely branched dendritic trees; beta cells with medium-sized somata and small, densely branched dendritic trees; and a variety of other types with smaller somata and varying dendritic branching patterns and dendritic field sizes. The correspondence of canine and cat ganglion cell types strengthens the view that there is a common set of ganglion cell types in carnivores. Alpha and beta cell dendritic trees of dog and wolf are monostratified in either the inner or the outer part of the inner plexiform layer, suggesting an on/off dichotomy in the response to light. Dendritic field sizes of dog alpha and beta cells increase from the central area to peripheral retina: alpha cell fields from 160-200 microns to about 1,100 microns diameter, and beta cell fields from 25 microns to about 360 microns diameter. These sizes are quantitatively very similar to those found in cat retina. The close qualitative and quantitative morphological correspondence of cat and dog ganglion cells suggests that they are also functionally very similar. It is likely that dog alpha cells have brisk-transient (Y), and dog beta cells brisk-sustained (X) concentric receptive fields. From the smallest beta cell sizes it is concluded that the visual acuity of the dog may be as good as that of the cat.     

Localization of proopiomelanocortin mRNA in functional subsets of neurons defined by their axonal projections

In situ cDNA:mRNA hybridization is a technique that has been developed for the visualization of cDNA:mRNA hybrids in individual cells. To use this technique to answer questions of regulation in heterogeneous populations of cells in the brain, it must be combined with other procedures allowing for the identification of functional subgroups of neurons. We report here a procedure by which in situ cDNA:mRNA hybridization may be combined with retrograde axonal tracing using the fluorescent tracer fast blue. Using this technique, it now becomes possible to measure mRNA regulation in functional subsets of cells defined by their axonal projections.