Acetylcholine synthesis by chick spinal cord neurons in dissociated cell culture

Acetylcholine (ACh) synthesis was examined in cultures of chick spinal cord cells to follow the development of the cholinergic neurons. The cells, prepared from 4-day-old embryonic chick spinal cords, were grown either alone in dissociated cell cultures (SC cultures) or with chick myotubes (SC-M cultures). ACh synthesis was measured by incubating the cultures in [³Hcholine and using high-voltage paper electrophoresis to quantitate the amount of [³H]ACh present in cell extracts prepared from the labeled cultures. The amount of [³H]ACh synthesized in SC-M cultures was strictly proportional to the number of spinal cord cells used to prepare the cultures, and was linear with the time of incubation in [³H]choline for periods up to 1 hr. Maximal rates of synthesis were observed with [³H]choline concentrations in excess of 100 μM. Such rates for 1-week-old SC-M cultures were approximately 10–20 pmoles of [³H]ACh/hr/10⁵ spinal cord cells. Studies on the stability of the intracellular [³H]ACh revealed the presence of a major pool with a half-time of 20–30 min. A second, small pool decayed more rapidly. No detectable [³H]ACh was spontaneously released from the cells, suggesting that most of the decay represented intracellular degradation. Development of cholinergic neurons as monitored by [³H]ACh synthesis continued over a 2-week period in SC-M cultures and paralleled general cell growth. When examined at 1 week, SC-M cultures had about a 50% greater capacity for [³H]ACh synthesis and 60% more choline acetyltransferase activity than did SC cultures. No difference was observed in the stability of the [³H]ACh formed for the two types of cultures at 1 week, and no further difference was observed in the rates of [³H]ACh synthesis at 2 weeks. Growth of SC cultures in medium containing different amounts of chick embryo extract (2–10%) or in medium with fetal calf serum (10%) instead of extract produced only small differences in the measured rates of [³H]ACh synthesis. Thus chick spinal cord cells can undergo some of the early stages of cholinergic development in cell culture without sustained contact with skeletal myotubes, one of the normal postsynaptic target cells for the cholinergic neuron population. No absolute requirement for muscle factors was revealed under these conditions, although such factors may have been provided by other cell types in the spinal cord population or may have been present in other additions to the culture medium.     

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.     

Acetylcholinesterase distribution in chick spinal cord cultures

Summary Explants of 10–12 day chick embryo spinal cord were cultured by coverslip-roller tube method for 3–80 days. The cellular and subcellular localization of acetylcholinesterase activity in cultured neurons was studied by the thiocholine techniques of Karnovsky and Roots and Lewis and Shute.At the light microscopic level, acetylcholinesterase was demonstrated in the neurons of both ventral and dorsal horn regions. Occasionally neurons migrated in the outgrowth zone exhibited strong intracellular activity.At the electron microscopic level, acetylcholinesterase activity was found in the nuclear envelope, granular endoplasmic reticulum and the Golgi apparatus of the neurons. No enzyme reaction was detected in the glial cell cytoplasm.     

Selective adherence of neurons and glial cells from dissociated cerebral and spinal cord microcarrier cultures.

In stationary cultures of dissociated brain and spinal cord grown on microcarriers (MCs), the neuronal and ependymal cells attached to the MCs forming floating aggregates in which they grow in a three-dimensional pattern. The glial and meningeal elements on the contrary, tend to dissociate from the aggregates and adhere to the plastic dish where they divide to form a monolayer. This different behavior of CNS components is not observed in rotating cultures in which all CNS cells remain attached to the MCs and develop into mature floating structures. This cell separation in stationary MC-cultures which is documented here by SEM and immunocytochemistry, may be useful for analysis and evaluation of the metabolic biochemical events of each of the cellular components derived from the same culture.     

Myelin formation in cultures of previously dissociated mouse spinal cord

Myelin formation in cultures of previously dissociated spinal cord from foetal mice is described. In addition to the expected pattern of myelination, in which axons are closely wrapped by myelin lamellae, redundant folds of myelin have been found, as have double sheaths surrounding a single axon. Hypotheses concerning the generation of these appearances are discussed. It is suggested that certain intracytoplasmic laminar bodies found in oligodendrocytes in vitro may be of mitochondrial origin.     

Urotensin-II regulates intracellular calcium in dissociated rat spinal cord neurons

Urotensin-II (U-II), a peptide with multiple vascular effects, is detected in cholinergic neurons of the rat brainstem and spinal cord. Here, the effects of U-II on [Ca2+]i was examined in dissociated rat spinal cord neurons by fura 2 microfluorimetry. The neurons investigated were choline acetyltransferase-positive and had morphological features of motoneurons. U-II induced [Ca2+]i increases in these neurons with a threshold of 10-9 m, and a maximal effect at 10-6 m with an estimated EC50 of 6.2 x 10-9 m. The [Ca2+]i increase induced by U-II was mainly caused by Ca2+ influx from extracellular space, as the response was markedly attenuated in a Ca2+-free medium. Omega-conotoxin GVIA (10-7 m), a N-type Ca2+ channel blocker, largely inhibited these increases, whereas the P/Q Ca2+ channel blocker, omega-conotoxin GVIIC (10-7 m) and the l-type Ca2+ channel blocker, verapamil (10-5 m) had minimal effects. Down-regulation of protein kinase C by 4-alpha-phorbol 12-myristate 13-acetate (10-6 m) or enzyme inhibition using the specific inhibitor bisindolylmaleimide I (10-6 m) did not inhibit the observed effects. Similarly, inhibition of protein kinase G with KT5823 (10-6 m) or Rp-8-pCPT-cGMPS (3 x 10-5 m) did not modify U-II-induced [Ca2+]i increases. In contrast, protein kinase A inhibitors KT5720 (10-6 m) and Rp-cAMPS (3 x 10-5 m) reduced the response to 25 +/- 3% and 42 +/- 8%, respectively. Present results demonstrate that U-II modulates [Ca2+]i in rat spinal cord neurons via protein kinase A cascade.     

Pharmacology of GABA-mediated inhibition of spinal cord neurons in vivo and in primary dissociated cell culture

Summary In this paper it is shown that the postsynaptic GABA-receptor chloride ion channel complex is composed of several functional subunits. There are probably at least two stereospecific locations on the receptor for GABA-binding and both must be occupied to obtain an increase in chloride conductance. The interaction between these sites is uncertain but there could be either positive cooperativity between the sites or only a requirement that both sites are occupied without occupation of either site affecting the affinity for GABA of the other site. There is a chloride conductance channel coupled to the GABA receptor which opens for an average of 20 msec and has an average conductance of 18 pS. The GABA-coupled chloride channel may or may not have the same composition as the glycine coupled chloride channel.In addition to the GABA-recognition site and the chloride ion channel, GABA-receptors must have additional binding sites or modulator sites where drugs can bind to modify GABA activation of the GABA-receptor. The convulsant PICRO binds to a site which is independent of the GABA-recognition site and PICRO reduces GABA responses. Barbiturates and benzodiazepines augment GABA-responses without reducing GABA-binding and thus they must bind to a modulator site independent of the GABA recognition site. Whether or not this is the same site as the PICRO binding site is uncertain. Thus, the GABA-receptorchloride ion channel complex is composed of at least: 1) two GABA-binding sites; 2) a chloride ion channel; 3) a convulsant binding site (PICRO-binding site) and 4) an anticonvulsant binding site. This organization serves several obvious purposes. First, since two GABA-molecules are required to activate GABA-coupled chloride ion channels, the dose-response relationship for GABA is sigmoidal and steep. Thus minor shifts in GABA affinity will produce large alterations in GABA-responses and the GABA receptor can be easily modulated. Second, since the receptor has binding sites for convulsant and anticonvulsant compounds which decrease and increase GABA-responses, GABAergic inhibition can easily be modulated.     

Morphological studies of synapses and varicosities in dissociated cell cultures of sympathetic neurons

Brain Cell Biology - Neurons dissociated from the superior cervical ganglia of newborn rats can be grown under conditions which support either adrenergic or cholinergic differentiation. In both...     

3H]-Nitrendipine binding in developing dissociated fetal mouse spinal cord neurons

The development of [3H]-nitrendipine binding was investigated in spinal cord neurons. Kinetic studies indicated two classes of binding sites which were present throughout development and the dissociation constants (Kd) and Bmax increased during development. [3H]-nitrendipine binding during development was characterized by a plateau on days 3-5 with maximal binding observed on day 19 after plating.     

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.     

Synaptic connections in a dissociated mixed culture of rat dorsal root ganglion and spinal cord neurons

Postsynaptic currents and action potentials recorded from neurons in a mixed culture of rat dorsal root ganglion and spinal cord cells are described. The existence of mutual synaptic connections between the above two types of neurons is demonstrated. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 358–360, July–August, 2006.     

L-glutamate activated membrane conductivity in spinal cord neurons during early chick embryogenesis

Transient and steady-state components of L-glutamate-activated membrane currents were investigated using intracellular perfusion, voltage clamp, and concentration clamp techniques in spinal cord neurons of 6–11 day chick embryos. Hill's coefficient was found to equal 1 for transient and 2 for steady-state components. It was shown that the L-glutamate-activated receptors are present, which appear in the membrane of spinal neurons at the early stages of development.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 251–258, March–April, 1987.     

Differentiated properties of identified serotonin neurons in dissociated cultures of embryonic rat brain stem

Serotonin neurons in 14-d embryonic rat brain stem were identified by peroxidase-antiperoxidase immunocytochemistry with an affinity-purified antiserotonin antibody. Brain-stem tissue was dissected from 14- or 15- d embryonic rats, dissociated and grown in cell culture for up to 5 wk, and serotonin neurons were identified by immunocytochemistry. Within 24 h of plating, serotonin immunoreactivity was present in 3.3% of neurons. Immunoreactivity in neuronal cell bodies decreased with time, whereas staining of processes increased. The number of serotonin- immunoreactive neurons remained constant at 3-5% over the first 14 d in culture. From 14 to 28 d, the total number of neurons decreased with little change in the number of serotonin neurons, such that, by day 28 in culture, up to 36% of surviving neurons exhibited serotonin immunoreactivity. Similar percentages of cultured brain stem neurons accumulating 3H-serotonin were identified by autoradiography. Uptake was abolished by the serotonin-uptake inhibitor, clomipramine, but was unaffected by excess norepinephrine, or by the norepinephrine-uptake inhibitor, maprotiline. Synthesis of 3H-serotonin was detected after incubation of cultures with 3H-tryptophan, and newly synthesized serotonin was released by potassium depolarization in a calcium- dependent manner. More than 95% of serotonin neurons were destroyed after incubation of cultures with 5,6-dihydroxytryptamine. Brain-stem cultures contained virtually no neurons with the ability to accumulate 3H-norepinephrine or 3H-dopamine. Approximately 40% of brain-stem neurons were labeled with gamma-aminobutyric acid (3H-GABA). However, there was almost no overlap in the surface area of neurons accumulating 3H-serotonin or 3H-GABA.     

Developmental decrease in neurite extension in cultured chick embryo retina and spinal cord neurons

Retina and spinal cord neurons from chick embryos attach to culture substrates and extend neurites. There is a statistically significant age-related decrease in the percentage and average length of neurites formed in 24-hr cultures of chick retina and spinal cord neurons between 6 and 16 days of embryonic age. The developmental decrease of neurite extension may be important for synaptogenesis in the developing nervous system.     

Kinetic characteristics of inhibitory postsynaptic currents in cultured chick embryo spinal cord neurons

Decay of inhibitory postsynaptic currents (IPSC) was analyzed in dissociated culture of chick embryo spinal cord. Differences in the kinetic characteristics of low-amplitude and giant IPSC were revealed. Decay of currents in the first group was single-exponential, while decay in the second group was double-exponential. The time constant of single-exponential current decay increased during membrane depolarization and decreased during rise in temperature of the solution. Decay of the double-exponential currents depended little on potential, while temperature changes acted only on its slow component. Strychnine in submaximum concentrations produced not only a decrease in amplitude of giant IPSC, but also a deceleration of decay due to the slow component. The regularity of these phenomena suggests that decay of giant IPSC, as distinguished from that of low-amplitude currents, is determined by removal of transmitter from the synaptic cleft.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 427–435, July–August, 1991.     

The differentiation of glial cells and glia limitans in organ cultures of chick spinal cord

Summary Differentiation of glial cells and the glia limitans in organ cultures of chick spinal cord explanted at early neural tube stages, alone or with adjacent tissues, was studied by electron microscopy. Oligodendrocytes and astrocytes comparable to those seen in the chicken in vivo were observed, mainly in areas of good neuronal differentiation. A glia limitans with basal lamina, comparable to that in vivo, was found when spinal cord was bordered by normally adjacent tissues. When it was surrounded by vitelline membrane only, a characteristic limiting layer of glial processes, but no basal lamina, was seen. Contact with a filter membrane (Millipore) elicited excessive differentiation of glial filaments and modified cell fine structure; no glia limitans was formed. Supported by Grant 5 RO 1 NB 0637 from the United States Public Health Service.     

Spontaneous synaptic activity in cell culture of chick embryo spinal cord

The spontaneous development of synaptic activity (SSA) was studied in cell cultures of chick embryo spinal cord. The complicated time structure of the SSA, an important early-stage characteristic of which was giant inhibitory postsynaptic currents (IPSC), was demonstrated. The ionic nature and pharmacological sensitivity of these IPSC suggest that glycine is their transmitter. Emergence of excitatory postsynaptic currents (EPSC) and complex antagonistic relationships between excitatory and inhibitory SSA was detected later. Possible mechanisms for maintenance of synaptic activity during the inhibitory function are discussed. Correlations between the regularities of synaptic transmission development that we have disclosed and neuronal circuit electrical activity are examined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 280–290, May–June, 1991.     

Conditions increasing the adrenergic properties of dissociated chick superior cervical ganglion neurons grown in long-term culture

Neurons dissociated from the embryonic chick superior cervical ganglion (SCG) were separated from ganglionic nonneuronal cells using a density gradient formed with Percoll. The sympathetic neurons were then grown for 3-4 weeks in serum containing medium on a polyornithine substrate precoated with heart-conditioned medium. Both catecholamine (CA) and acetylcholine (ACh) are synthesized and accumulated by these neurons, but the amount of CA is higher and increases much more over time in culture than the amount of ACh. The cultures become therefore more adrenergic with time. We report here that the adrenergic properties of these cells can be enhanced. A 3-fold increase in CA synthesis, as expressed on a per neuron basis, is obtained by increasing neuron cell density 3- to 4-fold. ACh synthesis, however, is decreased at high neuronal density. Optimal CA production is obtained at densities of 120-150,000 neurons/cm2. This effect is due to direct cell contact since it cannot be transferred to low density cultures by medium conditioned by high density cultures. Nerve growth factor concentrations 5-10-fold higher than the amount necessary for optimal neuronal survival (1 microgram/ml 7S NGF) increases CA production but do not affect ACh synthesis. This effect is highest at low plating densities (20-30,000 neurons/cm2, 10-fold increase) and progressively decreases with increasing neuronal density. No increase is obtained in high density cultures where CA production is maximal. In addition, we made the novel observation that medium conditioned by chick liver cells in culture (LCM) increases CA production approximately 4-fold, whereas it does not increase ACh production by the SCG neurons. Work is in progress to biochemically characterize the active component(s) present in the LCM and to determine whether they favor the survival of a subpopulation of adrenergic neurons possible present in these ganglia. Alternatively, the adrenergic differentiation of neurons initially capable of synthesizing both CA and ACh could be selectively increased by LCM.