Edith R. Peterson

The onset of synapse formation in spinal cord cultures as studied by electron microscopy

The development of synapses in cultured nervous tissue was studied by explanting cross sections of fetal rat spinal cord prior to first synapse formation. Periodic electron microscopic examination confirmed that the initial explants were free of synapses and indicated that synapses first appeared regularly after 70 h of culture of 14 day fetal cord. Initial synaptic profiles cover a small area, contain few vesicles, lack mitochondria, and are exclusively axodendritic. These early synapses coexist with a number of forms of plasma membrane junctions; some but not all of the junctions may represent precursors of synapses. The time at which the first synapses are seen in the electron microscope correlates well with the appearance of bioelectric activity indicative of the presence of functioning synaptic networks7. The detection of this close correlation and the finding that synaptic networks form in tissue completely isolated from normal afferent and efferent connections are considered the most significant contributions of this communication.

Differentiation and prolonged maintenance of bioelectrically active spinal cord cultures (rat, chick and human)

SummaryExplants of embryonic and fetal spinal cords (rat, chick, and human) develop and maintain in vitro many cytologic and bioelectric properties characteristic of central nervous tissues in situ. Despite the thickness of the cord explants, a condition which appears to be necessary for differentiation, considerable neuronal development becomes visible to light microscopic examination.The mature expiant consists of large concentrations of small neurons interspersed with large neurons and neuroglia, bounded by a broad tract of nerve fibers and capped by a neuropil. Myelination in rat cultures usually begins 2–3 weeks after explantation in both the explant and outgrowth zone. Myelin is of the central glial type unless the cord explants are grown with their meningeal covering. In the latter case the myelination pattern abruptly changes to the peripheral Schwannian type as the axons penetrate the meninges.Bouton-like endings are observed in the neuropil of rat cord explants (whole-mounts) impregnated with silver; but most neurons are only partially blackened. In 20 μ sections, neuronal somas and dendrites are identified in negative image with blackened bouton-like endings suggesting synapses.Chick spinal cord, when grown in Rose chambers, becomes more thinly spread so that more detailed interrelationships can be visualized in the living neuronal somas, neuritic processes and termini. Bouton-like endings on neuronal somas have been selectively stained, vitally, with methylene blue.Complex bioelectric activity can be evoked in these long-term spinal cord explants by electric stimuli localized to various regions of the cord tissues as well as to attached dorsal-root ganglia. The long-lasting “after-discharge” patterns and the neuropharmacologic sensitivity of the responses show remarkable similarity to the activity of synaptic networks of the central nervous system in situ. These functions develop gradually during the first week after explantation of fetal rat cord tissues — more slowly in cultures explanted before the establishment of reflex arcs in utero, and more rapidly in cord explants from older fetuses. Reference is made to the companion electron microscopy study of older fetuses, which shows that characteristic synaptic structures, although extremely rare at the time of explantation, are abundant in later stages of the cultures development. This confirms the functional evidence that synapses are able to develop in organized culture conditions.

NOTES ON SYNAPTIC VESICLES AND RELATED STRUCTURES, ENDOPLASMIC RETICULUM, LYSOSOMES AND PEROXISOMES IN NERVOUS TISSUE AND THE ADRENAL MEDULLA

This paper reviews aspects of the origin and fate of synaptic vesicles and of the related catecholamine-containing secretion granules of the adrenal medulla. Most attention is given to evidence concerning the proposal that the membrane surrounding synaptic vesicles can originate from axonal agranular reticulum, participate in exocytosis and endocytosis and eventually undergo degradation in lysosomes of axons and perikarya. A number of relevant details of the roles of several organelles in neurons and other cells of the nervous system are discussed. The paper centers around microscopic and cytochemical work on a few experimental systems.

Modification of development in isolated dorsal root ganglia by nutritional and physical factors

Abstract Dorsal root ganglia isolated from embryonic chicks of 8–10 days normally maintain tissue interrelationships in culture that lead to the progressive differentiation of the cellular community. By modifying the nutritional or physical conditions of cultivation in this system it is possible to dissociate certain elements of the developmental complex, suppressing one cell function while advancing another. By this means it has been shown that the production and storage of Nissl substance in the neuron is independent of the development of supporting cells and the differentiation of other structures. The formation of reticulin capsule and sheath is dependent upon a specific relationship of satellite and Schwann cells to the neuron, but quite independent of the maturation of the soma. The formation of myelin appears to depend upon both the chromidial maturation of the neuron soma and an appropriate relationship between Schwann cell and axis cylinder. The altered shape which some cultural conditions impose on neurons may be a limiting factor to their later development and survival. Several chemically defined media that are adequate for long-term propagation of strains of mouse fibroblasts are not adequate for differentiation or maintenance of this heterogeneous developing chick system.

Bioelectric Activity in Long-Term Cultures of Spinal Cord Tissues

Fragments of embryonic spinal cord (human, rat, and chick) can regenerate and differentiate in tissue culture. Complex bioelectric activity evoked by electric stimuli indicates that nerve cells in cultures may maintain, for months in vitro, not only the capacity to propagate impulses along their neurites but also a remarkable degree of functional organization resembling the activity of synaptic networks of the central nervous system.

PATTERNS OF PERIPHERAL DEMYELINATION IN VITRO

Isolated dorsal root ganglia of fetal rat will regenerate, mature and differentiate in vitro. After two to three weeks of cultivation, myelin begins to form in widely scattered segments. An active period of myelination continues for several weeks until large numbers of long nerve fibers growing singly or in well-organized fiber tracts, become ensheathed continuously, in thick well-differentiated myelin. These extensively myelinated cultures (FIGURE 1) can be maintained for several months, a period more than adequate for experimental demyelination procedures.

Ultrastructural studies of the dying-back process: VI. Examination of nerve fibers undergoing giant axonal degeneration in organotypic culture

Organotypic tissue cultures, composed of structurally and functionally coupled explants of mouse spinal cord, dorsal root ganglia, and striated muscle, have been used to create a model of the distal (dying-back) axonopathy found in animals and humans with aliphatic hexacarbon neuropathy. Mature explants were treated with 50–650 μg/ml of the following hexacarbons dissolved in nutrient fluid: H-hexane, 2-hcxanol, 2,5-hcxanediol, methyl n-butyl ketone, 5-hydroxy-2-hcxanone, 2,5-hexanedione (all neurotoxic), or 2,4-hexanedione (a non-neurotoxic diketone). High concentrations (400–650 μg/ml) induced pancytotoxic damage and necrosis of tissue within days, while the lower doses (50–100 μg/ml) induced no pathological changes over a period of several weeks. Continuous exposure of explants to 245–325 μg/ml (2.8 mM) of the neurotoxic hexacarbons caused specific pathological changes to develop in distal nerve fibers after three to six weeks. Initial changes seen in distal, nonterminal regions of myelinated fibers included: nodal elongation, axonal swellings on proximal-side paranodes, and paranodal myelin retraction. Prolonged treatment was associated with Wallerian-like degeneration of distal nerve fibers. Denuded paranodal swellings in more proximal regions of affected myelinated fibers adopted a more-normal size and underwent remyelination; this occurred during and after the course of treatment. Remyelination by lateral extension from adjacent Schwann cells was documented in living and fixed tissue. The observations confirm the spatial-temporal evolution of hexacarbon distal axonopathy previously suggested from comparable studies in vivo.

THE EFFECTS OF 6-MERCAPTOPURINE, 8-AZAGUANINE, AND 1,4-DIMETHANESULFONYLOXYBUTANE ON AN EXPERIMENTAL BRAIN TUMOR: PRELIMINARY OBSERVATIONS

In the study to be reported, the effects of 6-mercaptopurine (Purinethol, mercaptopurine), 8-azaguanine (guanazolo) and 1,4-dimethanesulfonyloxybutane (Myleran, GT41) on a transplantable brain tumor in mice were determined both in zrioo and in nlilro. The results have demonstrated that two of the drugs inhibit the growth of this malignant tumor. Evidence will be presented which suggests that a metabolic product of mercaptopurine may be the active antitumor agent, whereas the parent compound is inactive against this particular experimental neoplasm.