The development of chick spinal cord in tissue culture. II. Cultures of whole chick embryos

By using whole-chick-embryo cultures followed by fragment cultures of spinal-cord primordia, it was possible to reproduce in vitro the whole process of neuronal development beginning with its initiation and continuing up to and including the maturation of neurons. Normal whole embryos were developed to Hamilton-Hamburger stages 17 and 18 by growing embryos from the primitive streak stage on large (28-mm) glass rings. The advantage of whole-embryo cultures is that development can be staged accurately, which is especially important during the early stages when morphogenesis progresses very rapidly. By using such accurately staged embryos and tritiated thymidine, we have determined that some postmitotic neuronal precursor cells appear in chick embryos as early as Hamburger-Hamilton stages 4 and 5, i.e. the definitive streak stages before the neural tube has formed.     

The development of mouse spinal cord in tissue culture

Summary Whole mouse embryos were grown in vitro from Theiler stage 12 (1 to 7 somites) to Theiler stages 15 and 16 (25 to 35 somites). This procedure gives experimental access to precisely staged embryos during the early period of neurogenesis. To follow the further development of neurons in vitro, fragments of spinal primordia were set up from these cultured embryos. In such cultures, the proliferation of precursor cells, the formation of postmitotic cells and, finally, the cytodifferentiation of neurons were observed. A preliminary account of this work was given at the Tissue Culture Association Meeting in 1977, and the Canadian Federation of Biological Societies Meeting in 1977 (1,2). This work was supported by Grant MT 4235 from the Medical Research Council of Canada.     

The development of mouse spinal cord in tissue culture

Summary Neural tubes of mouse embryos at Theiler Stages 14, 15, and 16 were grown in cultures for 21 d with 0.5 μCi/ml tritiated thymidine or cold growth medium. It was found that 50 to 60% of the neurons formed in the outgrowth zone were labeled, indicating that they formed from precursor cells that proliferated in the cultures. The unlabeled neurons must have formed from cells that were already postmitotic when the cultures were started. By comparing the total number of neurons per neuromere formed in vivo and in vitro, it seems that the postmitotic precursor cells survive better in cultures and only a small percentage of proliferative precursor cells in cultures enter the postmitotic stage and form neurons. This work was supported by Grant MT4235 from the Medical Research Council of Canada.     

The development of chick spinal cord in tissue culture. I. Fragment cultures from embryos of various developmental stages

Explants from neural tube and spinal cord of chick embryos at developmental stages 8 through 36 were cultured on collagen-coated cover glasses for 21 days. The cultures of neural tube at stages 10 to 14 contained many neuronal precursor cells which gave rise to mature neurons. This was verified by cumulative labeling of cultures with tritiated thymidine. Explants from spinal cords of stages 26 and 27 contained fewer precursor cells, and at stage 36, only 7% of mature neurons were labeled. Regardless of the stage of development at which explants were made (stages 8 through 36), all cultures had a similar appearance after 21 days, indicating that cells from explants taken from earlier developmental stages (before neurons were formed) "caught up" with the explants from later developmental stages, which already had formed neurons at the time of explantation.     

Axonal growth characteristics of nerve cells of the chick embryo spinal cord in tissue culture

Numerous axons of associated cells were found in the cultures of 13-day old chick embryo spinal cord. These axons formed loops, while leaving the explants, and returned into the cultivated piece. This phenomenon can be due to pronounced specific influence of the explant on the growth of axons of the associated cells via the motoneurons which play the role of target cells.     

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.     

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.     

The development of synapses in vitro between previously dissociated chick spinal cord neurons

Summary The formation and development of synaptic contacts between dissociated chick spinal cord neurons has been investigated. By the 6th day in vitro immature profiles with few vesicles were observed. By 14–18 days mature types with numerous vesicles were found, indistinguishable from those of newly hatched chick spinal cord. After this period degeneration occurred, and was especially marked in the post-synaptic element. Such degeneration could be postponed by the addition of small numbers of somatic muscle cells. The Kanaseki and Kadota (1969) technique was applied to the study of coated vesicles at various stages of synaptic development.     

Expression analysis of chick Frizzled receptors during spinal cord development

Frizzleds (Fzds) are transmembrane receptors that can transduce signals dependent upon binding of Wnts, a large family of secreted glycoproteins homologous to the Drosophila wingless gene. FZDs are critical for a wide variety of normal and pathological developmental processes. In the nervous system, Wnts and Frizzleds play an important role in anterior-posterior patterning, cell fate decisions, proliferation, and synaptogenesis. Here, we preformed a comprehensive expression profile of Wnt receptors (FZD) by using situ hybridization to identify FZDs that are expressed in dorsal-ventral regions of the neural tube development. Our data show specific expression for FZD1,2,3,7,9 and 10 in the chick developing spinal cord. This expression profile of cFZD receptors offers the basis for functional studies in the future to determine roles for the different FZD receptors and their interactions with Wnts during dorsal-ventral neural tube development in vivo. Furthermore, we also show that co-overexpression of Wnt1/3a by in vivo electroporation affects FZD7/10 expression in the neural tube. This illustrates an example of Wnts-FZDs interactions during spinal cord neurogenesis.     

The development of voltage-dependent ionic conductances in murine spinal cord neurones in culture

The development of voltage-dependent ionic conductances of foetal mouse spinal cord neurones was examined using the whole-cell patch-clamp technique on neurones cultured from embryos aged 10-12 days (E10-E12) which were studied between the first day in vitro (V1) to V10. A delayed rectifier potassium conductance (Ik) and a leak conductance were observed in neurones of E10, V1, E11, V1, and E12, V1 as well as in neurones cultured for longer periods. A rapidly activating and inactivating potassium conductance (IA) was seen in neurones from E11. V2 and E12, V1 and at longer times in vitro. A tetrodotoxin (TTX) sensitive sodium-dependent inward current was observed in neurones of E11 and E12 from V1 onwards. Calcium-dependent conductances were not detectable in these neurones unless the external calcium concentration was raised 10-to 20-fold and potassium conductances were blocked. Under these conditions calcium currents could be observed as early as E11. V3 and E12, V2 and at subsequent times in vitro. The pattern of development of voltage-dependent ionic conductances in murine spinal neurones is such that initially leak and potassium currents are present followed by sodium current and subsequently calcium current.     

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.     

The development of mouse spinal cord in tissue culture. I. Cultures of whole mouse embryos and spinal-cord primordia.

Whole mouse embryos were grown in vitro from Theiler stage 12 (1 to 7 somites) to Theiler stages 15 and 16 (25 to 35 somites). This procedure gives experimental access to precisely staged embryos during the early period of neurogenesis. To follow the further development of neurons in vitro, fragments of spinal primordia were set up from these cultured embryos. In such cultures, the proliferation of precursor cells, the formation of postmitotic cells, and, finally, the cytodifferentiation of neurons were observed.     

Variation of glycine-activated conductivity of the membranes of spinal cord neurons in the chick embryo in culture

The method of leading off whole-cell currents is used to study the neuron responses elicited by applications of glycine at different stages of culturing of the chick embryo dissociated spinal cord. For two types of cells described earlier [4] differently directed shifts of the concentration dependence curves of the transmitter effect are shown to be characteristic. "Mature" cells, surviving several days in culture and represented by motoneurons, typically show a lowered sensitivity to glycine. The sensitivity of little-differentiated neurons increases more than tenfold by the end of the second week of culturing. The desensitization kinetics of the glycine-activated current also slows down with age. Blocking of the spontaneous electrical activity during development prevented these changes, and the presence of agonist in the culture medium did not affect them. Analogous patterns of formation of transmitter sensitivity are assumed to be exhibited by differentiating neurons of the intact nervous system.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 580–587, September–October, 1991.     

Growth pattern of pioneering chick spinal cord axons

The early growth pattern of axons in the embryonic chick spinal cord was studied by electron microscopy. Serial perisagittal thin sections were obtained from the lateral margins of spinal cords of stage 17 (S17) and S19 embryos. A simple stereotypic pattern of axonal growth was found. Axons originated from a dispersed population of presumptive interneurons located along the lateral spinal cord margin. They first grew ventrally in a nonfasciculative pattern and later turned at right angles and grew in a fasciculative manner longitudinally in the ventrolateral fasciculus. Growth along the circumferential pathway was analyzed in detail by reconstructing individual axons and growth cones from the S17 specimen. Most circumferential axons, regardless of their site of origin, grew in a parallel orientation, and each of their growth cones projected ventrally. This pattern suggested that circumferential growth cones were guided at many, if not all, points along their path. Study of the region in front of these seven growth cones, however, revealed no apparent structural basis for their guidance. Alternative guidance mechanisms are discussed. In conjunction with previous studies (e.g., Windle and Baxter, 1936; Lyser, 1966), these findings suggest that the circumferential-nonfasciculative and the longitudinal-fasciculative patterns of axonal growth are the two fundamental patterns followed by most early forming axons in the brain stem and spinal cord of all higher vertebrates.