Stanley M. Crain

Development of "organotypic" bioelectric activities in central nervous tissues during maturation in culture.

Publisher Summary This chapter discusses the bioelectric activities that develop in explants of vertebrate central nervous tissues during differentiation and maturation in vitro . The chapter also focuses on the remarkable degree of intrinsic self-differentiation, which has been demonstrated in long-term cultures of explants from various parts of the vertebrate neuraxis—spinal cord to cerebral cortex. Correlative electrophysiological and electron-microscopic analyses demonstrate that neurons in embryonic spinal-cord explants can form organized synaptic networks in vitro . Dorsal-root ganglia attached to these cord cross sections can develop functional connections with cord neurons. Axons from the latter may, in turn, grow into attached skeletal muscle tissue, leading to characteristic neuromuscular transmission. The entire spinal reflex arc is thus available for direct study during its formation in vitro . Bioelectric and ultrastructural data obtained during the growth of neonatal mouse cerebral cortex in culture reveal that a similar process of self-differentiation occurs. Primary evoked potentials and secondary oscillatory afterdischarges develop in cerebral explants and display a remarkable similarity to recordings from cerebral cortex in situ . Characteristic sensitivity of these complex bioelectric activities to various neuropharmacological agents provides further evidence of their organotypic nature.

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.

Functional Studies of Cultured Brain Tissues as Related to "Demyelinative Disorders"

The serums from animals with experimental allergic encephalomyelitis and humans with multiple sclerosis produce, in addition to demyelination, rapid, reversible alterations in complex, evoked bioelectric (synaptic) responses of cultured cerebral cortex and spinal cord tissues of the mouse. The active factors are dependent on complement and are not present in serums from normal animals and humans.

Bioelectric activity of neonatal mouse cerebral cortex during growth and differentiation in tissue culture

Electrophysiologic studies with extracellular microelectrodes demonstrate the development of characteristic bioelectric activity in cultured fragments of mouse cerebral neocortex during their growth and differentiation in vitro. Within the first 2–3 days after explantation, only simple spike potentials can be evoked by electric stimuli. By 4 days, responses of much greater complexity and duration begin to appear in the cultures. Facilitation effects at long test intervals can now be demonstrated and can also be augmented by strychnine. During the following week in culture, bioelectric responses increase in amplitude, complexity and regularity. Barrages of spikes and slow waves occur sporadically as well as in response to electric stimuli. d-Tubocurarine and strychnine may greatly enhance these activities, whereas procaine blocks them at levels which still permit simple spike responses. Simultaneous records of evoked-potential patterns from various regions of the explants suggest maintenance of laminar organization of neural elements parallel to the original surface of the cerebral cortex. This degree of functional integrity may be retained for more than 2 months in vitro. In many respects, the bioelectric properties of cultured cerebral explants are remarkably similar to those of chronic, neuronally isolated slabs of neonatal cerebral cortex in situ. Application of this method to problems of cerebral function at the cellular level are discussed.

Innervation of hippocampal explants by central catecholaminergic neurons in co-cultured fetal mouse brain stem explants

The ability of central catecholaminergic neurons to grow into and establish functional connections with the hippocampus in vitro was studied using organotypic tissue culture. Brain stem explanted from the region of the locus coeruleus and hippocampal explants, from 18-day fetal mice, were maintained as co-cultures and were also grown separately. After 1-4 weeks these tissues were analyzed by glyoxylic acid-induced histofluorescence, by light and electron microscopic radioautography after incubation with [3H]norepinephrine, and by electrophysiology. Brain stem explants exhibited specifically fluorescent catecholaminergic cell bodies and varicose fibers after 2-4 weeks in culture. In contrast, no fluorescent cells or neurites could be seen in isolated hippocampal cultures grown for 2-3 weeks in vitro. When hippocampal explants were grown near brain stem explants, catecholaminergic fibers grew out of the brain stem and entered the hippocampus. In additional experiments, co-cultures of brain stem and hippocampus were incubated with [3H]norepinephrine (0.5 micron) and the monoamine oxidase inhibitor nialamide (100 micron). Radioautographic analyses revealed that brain stem neurites which entered the hippocampus took up norepinephrine, whereas neurites in the isolated hippocampal explants did not. Electron microscopic studies of the hippocampus showed varicose axon terminals within the hippocampus to be preferentially labeled. Although close relationships could be seen between labeled axons and dendrites, junctions exhibiting the membranous modifications associated with synapses were never seen. Electrophysiological studies suggested that the catecholaminergic neurites within the hippocampus were functional. Complex synaptically mediated slow wave discharges could be evoked by electrical stimuli in isolated hippocampal explants. Introduction of the beta adrenergic antagonist propranolol (0.4-4.3 micron) did not alter, or slightly depressed, these hippocampal discharges. On the other hand, in hippocampus-brain stem co-cultures, these concentrations of propranolol enhanced the complex hippocampal responses to brain stem or hippocampal stimuli. Similar enhancement of hippocampal responses by propranolol also occurred in these cocultures after acute surgical extirpation of the brain stem explant. The data suggest, therefore, that the action of propranol was probably to block adrenergic inhibitory connections with hippocampal synaptic networks. These experiments provide morphological and electrophysiological evidence that catecholaminergic neurons from fetal mouse brain stem maintained in organotypic tissue culture can grow into and functionally innervate the hippocampus.

SOME CYTOLOGIC EFFECTS OF SALIVARY NERVE-GROWTH FACTOR ON TISSUE CULTURES OF PERIPHERAL GANGLIA.

T h e growth-stimulating e f f ec t s of mouse sal ivary nerve-growth factor (NGF) on peripheral ganglia both in t i s s u e cul ture and in situ h a v e been w e l l demonstrated (Levi-Mcntalcini, 1958; Cohen, 1958; Levi-Montalcini & Cohen, 1960; Levi-Montalcini (k Booker, 1960; Levi-Montalcini et d., 1963). We h a v e attempted to extend t h i s work in two directions: f i rs t , by exploring short-term, N G F treated sp ina l ganglion cul tures under the electron microscope and secondly, by studying t h e long-term deve lopgen t of sympathet ic ganglion cul tures in t h e l iving s t a t e , under t h e l ight microscope, during repeated N G F administration.

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.