Differential expression of creatine kinase and phosphoglycerate mutase isozymes during development in aneural and innervated human muscle culture.

Several enzymes that occur in multimolecular forms undergo transitions during myogenesis. Studies of such developmentally regulated isozymes (e.g. creatine kinase) indicate that muscle cells, cultured in the absence of neural tissue never develop fully mature isozyme patterns, but continue to express large amounts of 'housekeeping' isozymes that are characteristically present in fetal muscle. We studied two developmentally controlled isozymes, creatine kinase (CK) and phosphoglycerate mutase (PGAM) in normal human muscle, both aneurally cultured and co-cultured with fetal mouse spinal cord complex. Innervated cultures attain a greater degree of maturity than non-innervated cultures, as revealed by light and electron microscopy, showing well-developed sarcomeres and motor endplates after several weeks in vitro. During early stages of muscle regeneration in co-culture, characteristic fetal isozyme patterns of CK-BB and PGAM-BB activity predominate, as in aneural cultures. The muscle-specific isozymes (CK-MM; PGAM-MM) begin to appear as the muscle differentiates, and after 2-3 months in co-culture only, virtually all enzyme activity is due to the muscle-specific forms of CK and PGAM, as is normally observed in mature skeletal muscle in vivo.

Duchenne dystrophic muscle develops lesions in long-term coculture with mouse spinal cord.

When strips of human skeletal muscle from biopsies of normal children and donors with Duchenne muscular dystrophy (DMD) are explanted in organotypic coculture with fetal mouse spinal cord, many regenerating muscle fibers develop, become innervated, and maintain a remarkable degree of mature structure and function for more than 3-6 months in vitro. Sequential light microscopy in correlation with electron-microscopic and electrophysiologic analyses showed that despite cross-species innervation, these human muscle fibers develop stable cross-striations, peripherally positioned myonuclei, and mature, functional motor endplates. Of special interest is the onset of significant progressive abnormalities, e.g., unusual focal myofibrillar lesions, in substantial numbers of innervated mature DMD muscle fibers after 2-4 months in culture. The focal myofibrillar lesions were not detected in normal muscle fibers maintained as long as 6 months in coculture, nor are they comparable to the generalized loss of cross-striations observed in muscle atrophy following in vitro denervation of mature DMD fibers.

Taxol effects on glia in organotypic mouse spinal cord-DRG cultures.

Exposure of organotypic mouse spinal cord-dorsal root ganglion cultures to 1-2 microM taxol for up to 6 days results in a remarkable increase in cytoplasmic microtubules in differentiated oligodendroglia. In contrast, there is a notably smaller change in the microtubule complement of astroglia. This suggests that the regulatory and/or synthetic systems for tubulin in these glial types may differ, and that oligodendroglia appear more responsive to modulation in the levels of tubulin subunits as a result of the action of taxol.

Monoclonal antibodies binding to nuclear structures and axons in adult avian dorsal root ganglia.

Four monoclonal antibodies raised against embryonic chick dorsal root ganglia (5) recognize epitopes in neuronal and supporting cell nuclei, and in axons and contiguous cytoplasmic elements in some neurons of the adult chicken. Binding, analyzed at the ultrastructural level, is to reaction sites on the nuclear matrix, nucleolar complex, nuclear bodies, and filamentous elements in axons and some perikaryal regions.

Morphological alterations in dorsal root ganglion neurons and supporting cells of organotypic mouse spinal cord-ganglion cultures exposed to taxol.

Explants of 14-day fetal mouse spinal cord with attached dorsal root ganglia, which had become differentiated over 2-3 weeks in culture, were exposed to 1-2 microM taxol for up to 6 days. The culture medium was supplemented with nerve growth factor (300 units/ml) during exposure to the drug. By 3-6 days in taxol, unusually numerous microtubules were seen in peripheral perikaryal and proximal neuritic regions of ganglion neurons. Microtubules also engirdled massive aggregations of pleomorphic vesicular/cisternal elements in many neurons. These aggregates were visible as unusual 'clear' spheroidal regions in the living cells, and were often as large as the nuclei. Some of the elements comprising these striking vesicular/cisternal accumulations appeared to be portions of disrupted Golgi complexes normally polarized around the cytocentrum, as well as hypertrophied smooth endoplasmic reticulum formations. In other neuronal areas, Golgi complexes and other organelles were altered or disrupted to lesser degrees. Ordered microtubular arrays occurred along endoplasmic reticulum cisternae both in neuron somata and neurites. Over time, a plethora of microtubules assembled throughout the perikarya in various orientations apparently unrelated to microtubule organizing centers. Unlike the effects of other plant alkaloids that interact with tubulin, there was no discernible increase in filaments, although their distribution appeared altered. Concentric ordered microtubular-macromolecular lamellated complexes were seen only in neurites. Neuronal nuclei were misshapen, often displaced, and displayed fine structure reminiscent of chromatolysis. Satellite and Schwann cells contained atypically abundant microtubules, abnormal cisternae, disrupted Golgi complexes, and increased lysosomes. Some nuclei displayed abnormal chromatin, and in rare cases even microtubules. We suggest that taxol alters the distribution, integrity, and/or organization of organelle systems in dorsal root ganglion cells by engendering unusually abundant microtubules in abnormal groupings and aberrant locations in these cells.

Microtubule arrays in taxol-treated mouse dorsal root ganglion-spinal cord cultures.

Exposure of organotypic dorsal root ganglion-spinal cord cultures to taxol, a potent microtubule promoting and stabilizing agent, results in an unusual abundance of microtubules in neurons, and the presence of microtubule-endoplasmic reticulum arrays in their perikarya and processes. Ordered concentric arrays of microtubules alternating with marcromolecular material are observed in dorsal root ganglion neurites. Short linear structures are discernible between some microtubules and the macromolecular material, as well as between microtubules and endoplasmic reticulum cisternae. Analyses of such unusual microtubule arrays in taxol-treated cultures may provide valuable insights into tubulin-related systems in neurons, as well as in other cells.

Origin, development, and nature of intranuclear rodlets and associated bodies in chicken sympathetic neurons.

Correlative data are presented here on the developmental history, dynamics, histochemistry, and fine structure of intranuclear rodlets in chicken sympathetic neurons from in vivo material and long-term organized tissue cultures. The rodlets consist of bundles of approximately 70 +/- 10 A proteinaceous filaments closely associated with approximately 0.4-0.8 micro spheroidal, granulofibrillar (gf) bodies of a related nature. These bodies are already present in the developing embryo a week or more in advance of the rodlets. In early formative stages rodlets consist of small clusters of aligned filaments contiguous with the gf-bodies. As neuronal differentiation progresses these filaments increase in number and become organized into well-ordered polyhedral arrays. Time-lapse cinemicrography reveals transient changes in rodlet contour associated with intrinsic factors, changes in form and position of the nucleolus with respect to the rodlet, and activity of the gf-bodies. With the electron microscope filaments may be seen extending between the nucleolus, gf-bodies, and rodlets; nucleoli display circumscribed regions with fine structural features and staining reactions reminiscent of those of gf-bodies, We suggest that the latter may be derivatives of the nucleolus and that the two may act together in the assemblage and functional dynamics of the rodlet. The egress of rodlet filaments into the cytoplasm raises the possibility that these might represent a source of the cell's filamentous constituents.

Cytological studies of organotypic cultures of rat dorsal root ganglia following X-irradiation in vitro. I. Changes in neurons and satellite cells.

Long-term organotypic cultures of rat dorsal root ganglia were exposed to a single 40 kR dose of 184 kvp X-rays and studied in the living and fixed states by light or electron microscopy at 1-14 day intervals thereafter. Within the first 4 days following irradiation, over 30% of the neurons display chromatolytic reactions (eccentric nuclei, peripheral dispersal of Nissl substance, central granular zone) as well as abnormal nucleolar changes and dissociation of ribosomes from endoplasmic reticulum cisternae. Some satellite cells undergo retraction or acute degeneration, leaving only basement membrane to cover the neuron in these areas. 8 days after irradiation, neurons also exhibit (a) areas in which ribosomes are substantially reduced, (b) regions of cytoplasmic sequestration, (c) extensive vacuolization of granular endoplasmic reticulum and Golgi complex, and (d) diversely altered mitochondria (including the presence of ribosome-like particles or association with abnormal glycogen and lipid deposits). Nucleolar components become altered or reoriented and may form abnormal projections and ringlike configurations. Sizeable areas of the neuronal soma are now denuded of satellite cells; underlying these areas, nerve processes are found abnormally invaginated into the neuronal cytoplasm. By the 14th day following irradiation, most neurons display marked degenerative changes including extensive regions of ribosome depletion, sequestration, vacuolization, autolysis, and, in some areas, swirls of filaments, myelin figures, and heterogeneous dense bodies. These observations demonstrate that X-irradiation produces profound cytopathological changes in nervous tissue isolated from the host and that many of these changes resemble the effects of radiation on nervous tissue in vivo.