Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

Striatal interneurons in dissociated cell culture

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. It might be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain each of these neuronal types. However, in dissociated rat striatal (caudate/putamen, CPu) cultures arguably the most important interneuron, the giant aspiny cholinergic neuron, is not present. When dissociated striatal neurons from E14.5 Sprague–Dawley rats were mixed with those from E18.5 rats, combined cultures from these two gestational periods yielded surviving cholinergic interneurons and representative populations of the other interneuron types at 5 weeks in vitro. Neurons from E12.5 CD-1 mice were combined with CPu neurons from E14.5 mice and the characteristics of striatal interneurons after 5 weeks in vitro were determined. All four major classes of interneurons were identified in these cultures as well as rare tyrosine hydroxylase positive interneurons. However, E14.5 mouse CPu cultures contained relatively few cholinergic interneurons rather than the nearly total absence seen in the rat. A later dissection day (E16.5) was required to obtain mouse CPu cultures totally lacking the cholinergic interneuron. We show that these cultures generated from two gestational age cells have much more nearly normal proportions of interneurons than the more common organotypic cultures of striatum. Interneurons are generated from both ages of embryos except for the cholinergic interneurons that originate from the medial ganglionic eminence of younger embryos. Study of these cultures should more accurately reflect neuronal processing as it occurs in the striatum in vivo. Furthermore, these results reveal a procedure for parallel culture of striatum and cholinergic depleted striatum that can be used to examine the function of the cholinergic interneuron in striatal networks.     

Long-term cultures of neurons from adult frog brain express GABA and glutamate-activated channels

Cells from adult Xenopus laevis brainstem and spinal cord were dissociated with mild enzymatic treatment and grown in long-term cell culture. These cells had specific attachment/substrate and medium/serum requirements. Cells with bipolar and multipolar morphology were positively identified as neurons using immunohistochemistry with monoclonal antibodies to rat and bovine neurofilament proteins which we show here cross-react with similar amphibian proteins. Patch clamp recordings demonstrated that these neurons have populations of ionic channels which are activated by L-glutamate or gamma-aminobutyric acid (GABA). The characteristics of these channels were similar to those previously described for GABA- and glutamate-activated channels in embryonic mammalian neurons isolated in culture. Cell cultures of neurons isolated from adult Xenopus laevis brain may be a useful and simple preparation with which to examine the modulation of neuronal properties by various agents over longer time intervals then has been previously possible.     

The neuroanatomy of an amphibian embryo spinal cord

Horseradish peroxidase has been used to stain spinal cord neurons in late embryos of the clawed toad (Xenopus laevis). It has shown clearly the soma, dendrites and axonal projections of spinal sensory, motor and interneurons. On the basis of light microscopy we describe nine differentiated spinal cord neuron classes. These include the Rohon-Beard cells and extramedullary cells which are both primary sensory neurons, one class of motoneurons that innervate the segmental myotomes, two classes of interneurons with decussating axons, three classes of interneurons with ipsilateral axons and a previously undescribed class of ciliated ependymal cells with axons projecting ipsilaterally to the brain. We believe that all differentiated neuron classes are described and that this anatomical account is the most complete for any vertebrate spinal cord.     

High affinity choline uptake by spinal cord neurons in dissociated cell culture

Spinal cord-myotube cultures prepared with dissociated embryonic chick spinal cord cells and myoblasts exhibit a high affinity mechanism for accumulating choline. The uptake mechanism has a K_m of 3.4 ± 0.5 μM (7) and a V_m of 40.0 ± 0.1 (7) pmoles/min/mg of protein (mean ± SEM; number of determinations in parentheses). It is inhibited 90–95% by 10 μM hemicholinium-3 or by replacement of Na⁺ in the incubation solution with Li⁺. Part of the choline (10–20%) accumulated by the high affinity system is converted to acetylcholine (ACh). Uptake studies on spinal cord cells and myotubes grown separately demonstrate that the spinal cord cells can account for virtually all of the choline uptake observed in the mixed cultures. Myotubes are unnecessary under these conditions for the expression of the high affinity uptake mechanism by spinal cord cells. Neurons are not the only cell type in culture to exhibit high affinity choline uptake. Chick fibroblasts in both rapidly growing and stationary phase can accumulate choline with kinetics similar to those observed for the high affinity uptake by spinal cord cells. Little if any of the choline accumulated by fibroblasts, however, is converted to ACh. In most uptake studies with spinal cord cells, contributions from fibroblasts were minimized by carrying out the analysis at a time when few non-neuronal cells were present in the spinal cord cultures. These observations suggest that a population of chick central nervous system (CNS) neurons develop a high affinity choline uptake mechanism in cell culture that has many of the properties described for uptake by cholinergic neurons in vivo and that at least part of the choline accumulated by the system can be used for neurotransmitter synthesis.     

An analysis of dorsal root ganglia differentiation using three tissue culture systems

Summary The histogenesis of the dorsal root ganglia of chick embryos (ages 3 to 9 days) was followed in three different tissue culture systems. Organotypic explants included dorsal root ganglia connected to the lumbosacral segment of the spinal cord or isolated explants of the contralateral ganglia. Additionally, dissociated monolayer cultures of ganglia tissue were established. The gradual differentiation of progenitor neuroblasts into distinct populations of large ventrolateral and small dorsomedial neurons was observed in vivo and in vitro. Neurites developed after 3 days in the presence or absence of nerve growth factor in the medium. In contrast, autoradiographic analysis indicates that [³H]thymidine incorporation in neuronal cultures differed significantly from intact embryos. In vivo, the number of neuronal progenitor cells labeled with [³H]thymidine decreased in older embryos; in vitro, uptake of [³H]thymidine label was not observed in ganglionic progenitor cells regardless of the age of the donor embryo or the type of culture system. Lack of proliferation in ganglionic progenitor cells was not due to degeneration because vital staining and uptake of [³H]deoxyglucose indicated that neurons were metabolically active. Furthermore, the block in mitotic activity in vitro was limited to presumptive ganglionic neuronal cells. In the ependyma of the spinal cord segment connected to the dorsal root ganglia, neuronal progenitor cells were heavily labeled as were non-neuronal cells within both spinal cord and ganglia. Our results suggest that in vitro conditions can promote the differentiation of sensory neurons from early embryos (E3.5–4.5) without proliferation of progenitor cells.     

Electrophysiological Properties of Motor Neurons in a Mouse Model of Severe Spinal Muscular Atrophy: In Vitro versus In Vivo Development

We examined the electrophysiological activity of motor neurons from the mouse model of severe spinal muscular atrophy (SMA) using two different methods: whole cell patch clamp of neurons cultured from day 13 embryos; and multi-electrode recording of ventral horns in spinal cord slices from pups on post-natal days 5 and 6. We used the MED64 multi-electrode array to record electrophysiological activity from motor neurons in slices from the lumbar spinal cord of SMA pups and their unaffected littermates. Recording simultaneously from up to 32 sites across the ventral horn, we observed a significant decrease in the number of active neurons in 5–6 day-old SMA pups compared to littermates. Ventral horn activity in control pups is significantly activated by serotonin and depressed by GABA, while these agents had much less effect on SMA slices. In contrast to the large differences observed in spinal cord, neurons cultured from SMA embryos for up to 21 days showed no significant differences in electrophysiological activity compared to littermates. No differences were observed in membrane potential, frequency of spiking and synaptic activity in cells from SMA embryos compared to controls. In addition, we observed no difference in cell survival between cells from SMA embryos and their unaffected littermates. Our results represent the first report on the electrophysiology of SMN-deficient motor neurons, and suggest that motor neuron development in vitro follows a different path than in vivo development, a path in which loss of SMN expression has little effect on motor neuron function and survival.     

The Inactivation of γ-Aminobutyric Acid Transaminase in Dissociated Neuronal Cultures from Spinal Cord

Abstract: It had previously been shown that dissociated cell cultures from chick embryo spinal cord have a high affinity uptake system for the neurotransmitter γ-aminobutyric acid (GABA) and make functional inhibitory synaptic contacts as determined by electrophysiology (Farb et al., 1979). It is shown here that these cultures can synthesize GABA from added glutamate in a glutamate decarboxylase-dependent reaction. Furthermore, these cultures have a functional GABA transaminase that degrades the neurotransmitter. This enzyme can be specifically and irreversibly blocked with gabaculine. A 15 min incubation with 10⁻⁶ M-gabaculine completely inactivates the enzyme. The inactivation of the enzyme leads to an increase in GABA levels. Long-term incubation (16 days) of gabaculine in the medium does not appear to alter high affinity GABA transport, suggesting that the drug is not toxic to cells capable of accumulating GABA.     

谷氨酸转运抑制剂对器官型培养脊髓片的影响

观察谷氨酸转运体抑制剂苏一羟天冬氨酸(Threo-hydroxyaspartate,THA)对器官型培养的脊髓片的影响,探讨谷氨酸在运动神经元损伤中的作用。取出生后8天乳鼠的腰段脊髓组织切片做脊髓器官型培养,在培养液中加入不同浓度THA(50μmol／L、100μmol／L、5001μmol／L),用神经元的特异性免疫组化染色剂SMI-32,非磷酸化神经丝标记物,对脊髓腹角α运动神经元进行鉴定,用单克隆抗钙网膜蛋白(calretinin)抗体对背角中间神经元进行记数,测定培养液中乳酸脱氢酶(LDH)的含量,并与对照组比较。结果显示对照组α运动神经元数目恒定,THA可以引起剂量依赖性的培养液中LDH含量增高和α运动神经元数目减少,而脊髓背角的中间神经元损伤相对较轻,其中THA100μmol／L组在体外培养4周后出现类似于肌萎缩侧索硬化(ALS)的病理改变：α运动神经元数目较对照组明显减少,而脊髓背角的中间神经元数目无显著变化。细胞外谷氨酸增高主要对运动神经元造成损伤,脊髓运动神经元较感觉神经元对谷氨酸的兴奋毒作用更加敏感。     

The development of a population of spinal cord neurons and their axonal projections revealed by GABA immunocytochemistry in frog embryos

The development of a population of cerebrospinal-fluid-contacting neurons in the spinal cord of the Xenopus embryo ('Kolmer-Agduhr' cells) has been followed by using an immunocytochemical procedure that identifies GABA in fixed nervous tissue. Stained Kolmer-Agduhr cells containing GABA first appeared at stage 25 and their numbers increased steadily with the developmental age of the embryo. The Kolmer-Agduhr neurons had ascending ipsilateral axons that often terminated in growth cones. These axons and growth cones could be stained by the GABA antiserum from the earliest stages of outgrowth from the Kolmer-Agduhr cell body. We measured the angle of the earliest axons' outgrowth relative to the rostrocaudal axis of the spinal cord. The initial outgrowth of axons was always rostral over a narrow range of angles. This observation is inconsistent with the hypothesis of random initial outgrowth followed by later selection of the correct orientation, which would predict that axons would initially grow out over a wide range of angles. Instead, it suggests that, even from the earliest moments, axon outgrowth from the Kolmer-Agduhr cells is directed rostrally in a specific stereotyped manner.     

Autonomous early differentiation of neurons and muscle cells in single cell cultures

The extent to which early differentiation of neurons and muscle cells is autonomous or governed by soluble factors released from other cells has been examined by following development of single cells plated alone in a simple, defined culture medium. The differentiation of electrical excitability and sensitivity to neurotransmitters of amphibian spinal neurons and trunk muscle in Xenopus embryos has already been described. For both cell types, differentiation in cultures containing relatively large numbers of dispersed cells parallels development in vivo, with respect to qualitative changes in membrane properties and the time course of development. Cell contacts are not required for this process. Here we show that the differentiation of membrane properties of single, isolated cells exhibits a similar set of changes, although muscle cells develop more slowly in some respects and all cells survive for a shorter period of time. The results suggest that the continued presence of specific extracellular differentiation-promoting factors is not required for these early steps of neuronal development, although a role for such factors in development of myocytes cannot be excluded. In contrast, survival factors secreted by other cells may be necessary to prolong the lifetimes of dissociated cells.     

Regulation of cholinergic expression in cultured spinal cord neurons

Factors regulating development of cholinergic spinal neurons were examined in cultures of dissociated embryonic rat spinal cord. Levels of choline acetyltransferase (CAT) activity in freshly dissociated cells decreased rapidly, remained low for the first week in culture, and then increased. The decrease in enzyme activity was partially prevented by increased cell density or by treatment with spinal cord membranes. CAT activity was also stimulated by treatment with MANS, a molecule solubilized from spinal cord membranes. The effects of MANS were greatest in low-density cultures and in freshly plated cells, suggesting that the molecule may substitute for the effects of elevated density and cell-cell contact. CAT activity in ventral (motor neuron-enriched) spinal cord cultures was similarly regulated by elevated density or treatment with MANS, whereas enzyme activity was largely unchanged in mediodorsal (autonomic neuron-enriched) cultures under these conditions. These observations suggest that development of cholinergic motor neurons and autonomic neurons are not regulated by the same factors. Treatment of ventral spinal cord cultures with MANS did not increase the number of cholinergic neurons detected by immunocytochemistry with a monoclonal CAT antibody, suggesting that MANS did not increase motor neuron survival but rather stimulated levels of CAT activity per neuron. These observations indicate that development of motor neurons can be regulated by cell-cell contact and that the MANS factor may mediate the stimulatory effects of cell-cell contact on cholinergic expression.     

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.     

Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets

Extracellular matrix molecules--including chondroitin sulfate proteoglycans, hyaluronan, and tenascin-R--are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type-dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice-like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin-expressing interneurons, which developed after 2-3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN-expressing neurons revealed that these are large fast-spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity-dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca2+ permeable AMPA subtype of glutamate receptors or L-type Ca2+ voltage-gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca2+ influx via AMPA receptors and L-type channels is necessary for activity-dependent formation of PNs. To study functions of chondroitin sulfate-rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity.     

Biochemical characterization of [3H]norepinephrine uptake in dissociated brain cell cultures from chick embryos

The uptake of [³H]norepinephrine ([³H]NE) was studied in dissociated brain cell cultures prepared from 8-day-old chick embryos using the whole brain (minus optic lobes). Uptake of [³H]NE, 5×10^–9 M, 10 min incubation, in freshly dissociated noncultured embryonic chick brain cells, was detected in 6-day-old embryos; it was temperature and drug (cocaine, metanephrine) sensitive and increased with brain development. In cultured cells, which were assayed at various days in culture, the increase in [³H]NE accumulation per culture was less than that seen in freshly dissociated noncultured embryonic cells. When [³H]NE uptake was expressed per mg protein, a decrease with days in culture was observed, reflecting perhaps a dilution of growth or proliferation of cells not accumulating NE. Metanephrine, 5×10^–6 M, an inhibitor of extraneuronal uptake, inhibited [³H]NE in 5-day-old cultures whereas desmethylimipramine, an inhibitor of neuronal uptake, inhibited [³H]NE uptake in 15- and 20-day-old cultures. Cocaine, another neuronal inhibitor, inhibited [³H]NE at 10 and 15 days only. We interpret these findings to suggest that during early growth in culture most neuroblasts accumulate NE nonspecifically and, as neuronal maturation proceeds, NE accumulation becomes specific.     

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.     

Recent advances in neural cell culture

Cells isolated from the avian and mammalian central and peripheral nervous system and cultured in vitro provide an opportunity to study in situ properties of neurons and glial cells under relatively simple and carefully controlled conditions. Since Harrison's success in maintaining in vitro embryonic frog spinal cord 80 years ago, neural tissue culture has developed into an important and versatile discipline of neuroscience. The techniques developed in the past fall into four broad classes: Explant cultures, which are explanted from specific neuroanatomic loci to substrates as small tissue fragments. Dissociated cell cultures, which involve the seeding of enzymatically or mechanically dispersed cells on various attachment substrates. Reaggregate cultures, which require re-association of dissociated cells into small aggregates. Purified cell populations, which are prepared by the isolation of different cell types by gradient centrifugation or other separation techniques. These cultures have been utilized in studying various aspects of brain development and function. In this review several areas of significant and stimulating development in neural cell culture have been documented. They include formulation of serum-free medium, effects of growth factors, utilization of cell type-specific markers, and isolation and culture of purified neuronal/glial cells.     

Toxicity of 1-Methyl-4-Phenylpyridinium for Rat Dopaminergic Neurons in Culture: Selectivity and Irreversibility

Cultures of dissociated embryonic rat mesencephalic cells were exposed to 10 microM 1-methyl-4-phenylpyridinium (MPP+), a concentration shown earlier to result in loss of greater than 85% of tyrosine hydroxylase (TH)-positive neurons without affecting the total number of cells observed by phase-contrast microscopy. To characterize better the selectivity of the toxic action of MPP+, other parameters were measured reflecting survival and function of dopaminergic or nondopaminergic neurons. Exposure of cultures to 10 microM MPP+ for 48 h reduced TH activity to 11% of control values without reducing protein levels. [3H]Dopamine uptake was reduced to less than 4% of control values, whereas the uptake of gamma-[3H]aminobutyric acid ([3H]GABA) was not affected in these cultures. This same treatment failed to reduce the number of cholinergic cells visualized in septal cultures and did not affect either choline acetyltransferase activity or high-affinity choline uptake. To assess for possible recovery of dopaminergic neurons, cultures were exposed to 10, 1.0, or 0.1 microM MPP+ for 48 h and then kept for up to 6 days in MPP(+)-free medium. After exposure to 10 microM MPP+, the number of TH-positive neurons, their neurite density, TH activity, and [3H]dopamine uptake remained at constant, reduced levels throughout the period of observation after termination of exposure, whereas GABA uptake remained normal. Treatment with lower concentrations of MPP+, i.e., 1.0 and 0.1 microM, induced less pronounced dopaminergic toxic effects. However, no recovery was seen after posttreatment incubation in toxin-free medium. These findings provide evidence that MPP+ treatment results in highly selective and irreversible toxicity for cultured dopaminergic neurons.