Search PubMed⌕ Search

Biomedical subjects

S M Crain

Publications and source records attributed to S M Crain.

At least 91 records · Page 5Linked to original sources
PubMed1981

Neurites from mouse retina and dorsal root ganglion explants show specific behavior within co-cultured tectum or spinal cord.

We have utilized extracellular microiontophoretic injections of horseradish peroxidase into fetal mouse retinal explants to label retinal ganglion cell axons innervating co-cultured tectal explants in a solid Golgi-like manner. Using dorsal root ganglia-tectum and retina-spinal cord co-cultures as controls, our results indicate that retinal neurites show selective growth and arborizations within their appropriate tectal, target tissue. Retinotectal explant co-cultures may be a useful model system for studying aspects of neuronal specificity.

Animals↗
PubMed1981

Development of ganglion cells and their axons in organized cultures of fetal mouse retinal explants.

Retinas from 13-15 day fetal mice were explanted alone, with adjacent eyeball tissue, or with nearby superior colliculus explants. The organotypic structure of the retina developed in situ, including photoreceptors, interneurons, plexiform layers, ganglion cells, and an optic fibre layer. Electrophysiologic recordings demonstrated that functional synaptic networks developed resembling bioelectric response patterns seen in situ. Within half-retinas, arrays of optic fibers converged to the optic nerve head; in co-cultures with tectum they could become myelinated. Large bundles of long, naked neurites--1 degree primary retinal fibers--emerged from the explant in the first few days in vitro; these could often be traced back to the optic nerve head and a detailed survey of their properties using horseradish peroxidase (HRP) tracing methods identified tham as ganglion cell axons. When growing upon collagen substrata, 1 degree fibers began to disintegrate during the second week in vitro; however, many 1 degree fibers that grew into superior colliculus explants were maintained for at least 5 weeks in vitro, where they formed elaborate, functional terminal arborizations. In a few cases, 1 degree fibers grew across skeletal muscle fibers and appeared to induce them to contract. A second type of neuritic outgrowth pattern appeared after the first week in vitro: 2 degrees retinal fibers. This was composed of a mixed population of interneuronal neurites; a small percentage was catecholaminergic. Our characterization of the morphologic properties of retinal ganglion cells and their axons in organotypic cultures provides the necessary background to interpret electrophysiologic mapping and neural-specificity analyses of retino-CNS co-cultures. This in vitro model system may have biological relevance to understanding the cues that control the development of the retinotectal projection in situ.

Animals↗
PubMed1981

Selective innervation of target regions within fetal mouse spinal cord and medulla explants by isolated dorsal root ganglia in organotypic co-cultures.

Correlative electrophysiologic and cytologic analyses demonstrate that a significant group of neurons in isolated (NGF-enhanced) fetal mouse dorsal root ganglia (DRGs) can grow across a collagen substrate and selectively innervate specific dorsal horn and dorsal column nuclei regions in co-cultured explants of deafferented spinal cord and medulla. Neurites from this group of DRG cells from connections in CNS target zones that generate characteristic primary afferent network responses to sensory stimuli, as observed in cultures of spinal cord with attached DRGs. Systematic microelectrode stimulus-mapping tests revealed that many DRG neurites were preferentially distributed in sensory target zones of co-cultured cord and medulla explants and that few collaterals of these DRG neurons were present in neighboring inappropriate regions, especially in the ventral cord. Another group of DRG neurons appears to be responsible for the less prominent, but clear-cut, innervation that developed in some of the co-cultured ventral cord explants. Fetal DRGs were also able to establish characteristic primary afferent dorsal horn or dorsal column nuclei networks when introduced into cultures of deafferented spinal cord and medulla that had been explanted alone for 1-3 weeks prior to introduction of the DRGs. These experiments demonstrate that CNS target neurons remain receptive to DRG innervation even after 1-3 weeks of maturation in vitro. Our electrophysiologic and cytologic analyses of DRG and CNS explants in organotypic co-cultures provide the first systematic attempt to establish conditions under which preferential neuritic growth to and functional innervation of specific CNS target tissues can occur in vitro. This model system should facilitate analyses of mechanisms underlying development, as well as regeneration, of specific synaptic connections in the CNS.

Afferent Pathways↗
PubMed1981

Preferential growth of neurites from isolated fetal mouse dorsal root ganglia in relation to specific regions of co-cultured spinal cord explants.

Clusters of dorsal root ganglia (DRGs) from 13- to 14-day fetal mice were co-cultured with specific fragments of deafferented spinal cord (0.5-1 mm apart) on collagen-coated coverslips in Maximow slide chambers. Nerve growth factor (NGF) was added to the culture medium (1000 biological units/ml, at explantation) to ensure optimal survival and growth of a large fraction of the fetal DRG neurons. Sequential microscopic observations of the living cultures and cytologic studies after silver impregnation demonstrate that many neurites from isolated DRGs can invade dorsal (DC) regions of co-cultured spinal cord explants, whereas they are deflected from ventral cord (VC) tissue and its neuritic-glial outgrowth. Furthermore, some DRG neurites may become redirected towards distant DC target explants even after long circuitous detours around proximally arrayed VC explants. DRG neurites also show remarkably sharp projections to DC tissue and more complete avoidance of adjacent VC tissue when the DRG neurites approach a suitably arranged DC-VC-DC interface, e.g. forming de novo 'dorsal roots' at the end of a longitudinal strip of whole spinal cord. These experiments suggest that DC-VC boundaries may be particularly effective in guiding DRG neurites to specific regions of the CNS. The present studies of co-cultured fetal mouse DRG and spinal cord explants provide the first demonstration of preferential neuritic growth in vitro in relation to specific CNS target tissues.

Animals↗
PubMed1981

Specific neuritic pathways and arborizations formed by fetal mouse dorsal root ganglion cells within organized spinal cord explants in culture: a peroxidase-labeling study.

Extracellular microiontophoretic injections of horseradish peroxidase (HRP) into NGF-enhanced fetal mouse dorsal root ganglia (DRGs) produced an anterograde solid Golgi-like labeling of DRG neurites and their terminal arborizations within co-cultured spinal cord explants. In cultures of spinal cord transverse cross-sections with attached DRGs, the large NGF-enhanced DRGs remained in close proximity to the cord, often adjacent to both dorsal and ventral cord regions. Despite this, nearly all DRG neurites that entered the cord did so via dorsal root fascicles. They branched and ramified extensively within the dorsal region, taking on a wavy or kinky course and showed various types of arborizations. The density of cord innervation was much lower when isolated DRGs and cord explants were co-cultured 0.5-1 mm apart. Although fewer entering DRG fibers were labeled by our HRP injections the same qualitative growth and arborization patterns were seen within dorsal and ventral cord regions as in explants of cord with attached DRGs. When the facing edge contained both dorsal and ventral tissues, HRP-labeled DRG fibers entered dorsal regions selectively. DRG fibers readily entered, ramified and arborized within isolated strips of dorsal cord, whereas they sharply avoided isolated ventral cord explants. The avoidance of ventral cord cannot simply be due to the paucity of specific synaptic targets within the tissue, for larger numbers of DRG fibers entered completely inappropriate CNS target tissues, e.g. superior colliculus explants--though they did not ramify or arborize to any degree comparable to that seen within dorsal cord regions.

Animals↗
PubMed1981

Presence of leucine-enkephalin in organotypic explants of fetal mouse spinal cord.

Spinal cord explants with attached dorsal root ganglia (DRGs), from 14-day fetal mice were fixed at 1-3 weeks in vitro and incubated for leucine-enkephalin (LE) immunoreactivity by the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Results show long processes with labeled varicosities seen more often in dorsal regions of the cord explants. Stained punctate bodies and varicosities were often seen close to large cells in these cultures, whereas no label was detected in neuronal perikarya. A prominent laminar array of stained punctate bodies was noted in one cord explant, concentric with the perimeter of the explant. No LE label was detected in the neuritic outgrowths from the cord-DRG explants, whereas high levels of opiate receptors develop in these outgrowths, primarily on the DRG neurites, by 1-2 weeks in culture. The results indicate the presence of LE in explants of fetal mouse spinal cord with attached DRGs and offer an in vitro model system in which the onset and development of peptidergic neurons can be studied as they form functional cellular interrelationships with neurons bearing opioid and monoaminergic receptors in these organotypic cultures.

Animals↗
PubMed1979

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.

Animals↗
PubMed1978

Regional localization of patterned spontaneous discharges during maturation in culture of fetal mouse medulla and spinal cord explants.

Development and regional localization of patterned spontaneous bioelectric activities, recorded extracellularly, were studied in cultures of frontal sections of spinal cord and medulla, explanted from 13- to 15-day-old fetal mice. Spontaneous single- and multi-unit tonic and phasic spike discharges were recorded after 4-21 days in vitro. Stereotyped slow waves with positive or negative polarity, and durations up to several seconds, often occurred concomitant with spike burst discharges. Microelectrode mapping demonstrated regional localization of spontaneous activity patterns in these cross-sectional central nervous system explants, which were oriented so that dorsal and ventral borders could be regularly identified. Phasic discharges were widely distributed throughout the explants, whereas tonic discharges were located primarily in the dorsal region of the medulla; negative slow waves were generated in central and ventral regions of spinal cord and medulla explants, whereas positivities were located in the peripheral regions of the explants. Phasic discharges were often synchronous when recorded at two symmetrical regions of an oriented explant, as early as 4 days in vitro. The major developmental change in spontaneous activities of these explants was a progressive shortening of both burst and cycle durations of the phasic discharges.

Animals↗