Disappearance of Rohon-Beard neurons from the spinal cord of larval Xenopus laevis.

Rohon-Beard neurons are primary sensory cells located in the spinal cord of embryonic lower vertebrates. The kinetics of their normal, gradual, but complete disappearance in Xenopus tadpoles has been followed. Levels of acid phosphatase activity, a common histochemical correlate of cell death, were assayed and found to increase at the time of onset of disappearance of Rohon-Beard cells. Ultrastructural examination revealed the presence of numerous secondary lysosomes, swelling of endoplasmic reticulum and mitochondria, and a decrease in nuclear density. The disappearance of Rohon-Beard neurons may be attributed to autophagic cell death involving lysosomal acid hydrolases. This process begins only a few days after the maturation of voltage- and neurotransmitter-dependent membrane conductances and the electrical uncoupling of these neurons. The loss of Rohon-Beard neurons in embryos whose development was arrested by crowding was appropriate for the developmental stage of the animals rather than their chronological age.

Development of a high-affinity GABA uptake system in embryonic amphibian spinal neurons.

High-affinity uptake systems for amino acid neurotransmitter precursors have been highly correlated with the use of the particular amino acid or its derivative as a transmitter. We have found interneurons in the Xenopus embryo spinal cord which accumulate GABA by a high-affinity uptake system. They originate near the end of gastrulation and their ability to accumulate GABA first appears at the early tail bud stage. By position and appearance they are comparable to some of the embryonic interneurons described by A. Roberts and J. D. W. Clarke (1982, Phil. Trans. R. Soc. London Ser. B 296, 195-212). GABA-accumulating neurons also develop in dissociated cell cultures made from the presumptive spinal cord of neural plate stage Xenopus embryos. GABA accumulation in cultured neurons, as in cells in vivo, occurs via a high-affinity uptake system; GABA-accumulating cells have the same time of origin as the cells in vivo, and the ability to accumulate GABA in the population of cultured neurons appears at a time equivalent to that observed in intact sibling embryos. Thus it seems likely that the population of GABA-accumulating neurons developing in cell culture corresponds to the GABA-accumulating interneurons in vivo. The development of these neurons in dissociated cell cultures permits perturbation experiments that would be difficult to perform in vivo. We have examined the development of high-affinity GABA uptake in conditions that permit no electrical impulse activity in the cultures. The onset and extent of development of GABA accumulation in the neuronal population are normal under these conditions.

Rohon-beard cells and other large neurons in Xenopus embryos originate during gastrulation.

The time of origin (birthday) of Rohon-Beard cells in Xenopus laevis was studied by 3H-thymidine autoradiography. Rohon-Beard cells were selected because they are a morphologically identifiable population of neurons in which the development of chemical and electrical excitability has been studied. A single injection of a radioactive DNA precursor was given to animals in successive stages of development from blastula to late tail bud (Nieuwkoop and Faber stages 8--33/34). The label was available throughout the stage of injection and longer. The labeling pattern was examined when animals had reached stage 42, when Rohon-Beard cells are easily recognized. All neurons including Rohon-Beard cells were labeled in animals injected with 3H-thymidine before stage 10 1/2 (early gastrula). Unlabeled Rohon-Beard cells were observed in animals injected with 3H-thymidine in and after stage 10 1/2. The percentage of unlabeled Rohon-Beard cells increased as development progressed. About 80% were born by the completion of gastrulation (stage 13). The other approximately 20% were born during neurulation and early tail bud stages. By stage 27, no Rohon-Beard neuron incorporated 3H-thymidine. In addition to Rohon-Beard neurons, five other neuronal populations begin generation during gastrulation: Mauthner neurons (Vargas-Lizardi and Lyser, '74), trigeminal ganglion cells, large basal plate cells of the medulla, extramedullary neurons, and primary motor neurons. The first birthdays in any of the six populations are temporally close to but appear to be independent of the others.

Ultrastructural development of Rohon-Beard neurons: loss of intramitochondrial granules parallels loss of calcium action potentials.

We have examined the ultrastructure of the cell body of a vertebrate spinal neuron, the Rohon-Beard cell of Xenopus laevis, at four stages during its development (Nieuwkoop and Faber stages: 22, 29/30, 37/38 and 42). At this time it has attained its electrical excitability and the action potential mechanism in the cell body is maturing through a sequence of stages in which the inward current is carried by Ca++ (stages 20-25), later by Ca++ and Na+ (stages 25-40), and finally by Na+ (stages 40-51) (Spitzer and Baccaglini, '76; Baccaglini and Spitzer, '77). There is a change in the abundance and distribution of the organelles in the perikaryon during this period, characteristic of other developing neurons. Mitochondria and Golgi apparatus become localized progressively more in the interior of the cells, and rough endoplasmic reticulum progressively more to the periphery where it often appears in orderly tiers parallel to the plasma membrane. The mitochondria contain dense intramitochondrial granules which are known in other cells to contain concentrations of divalent cations. The number of granules declines over the course of the developmental period studied. The presence of the intramitochondrial granules was examined quantitatively because electrophysiological data indicate that the amount of Ca++ entering the cells in early stages should raise the internal Ca++ concentration by several orders of magnitude, and that Ca++ is rapidly sequestered (Baccaglini and Spitzer, '77). A minimum of 200 mitochondrial profiles from at least four Rohon-Beard cells were scored for the presence of dense intramitochondrial granules at each stage studied. In stage 22 Rohon-Beard cells 75 +/- 5% (mean +/- SD, n = 4) of the mitochondrial profiles scored contained granules; in stage 29/30, 56 +/- 10% (n = 7); in stage 37/38, 3 +/- 3% (n = 5); and in stage 42, 0.5 +/- 0.25% (n = 4). Therefore, dense intramitochondrial granules, an indication of calcium accumulation in mitochondria, decrease in parallel with the loss of the Ca++ component of the inward current of the action potential in Rohon-Beard neurons.

The development of the action potential mechanism of amphibian neurons isolated in culture.

Nerve and muscle cells differentiated morphologically, in cultures of dissociated cells prepared from amphibian neural plate and underlying mesoderm (Xenopus laevis, Nieuwkoop and Faber stage 15). Cultures were grown in a defined medium containing sterile Steinberg's salt solution and 0.1% bovine serum albumin, and maintained for periods up to 5 days.