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The Sixth International Symposium on Neural Regeneration was held December 6 to 10, 1995, at the Asilomar Conference Center in Pacific Grove, California. The meeting was cosponsored by the Department of Veterans Affairs, the Paralyzed... more
The Sixth International Symposium on Neural Regeneration was held December 6 to 10, 1995, at the Asilomar Conference Center in Pacific Grove, California. The meeting was cosponsored by the Department of Veterans Affairs, the Paralyzed Veterans of America, the National Institutes of Health (National Institute of Neurological Disorders and Stroke, and the National Institute of Child Health and Hu-
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Circuit reorganization after injury was studied in a cerebellar culture model. When cerebellar cultures derived from newborn mice were exposed at explantation to a preparation of cytosine arabinoside that destroyed granule cells and... more
Circuit reorganization after injury was studied in a cerebellar culture model. When cerebellar cultures derived from newborn mice were exposed at explantation to a preparation of cytosine arabinoside that destroyed granule cells and oligodendrocytes and compromised astrocytes, Purkinje cells surviving in greater than usual numbers were unensheathed by astrocytic processes and received twice the control number of inhibitory axosomatic synapses. Purkinje cell axon collaterals sprouted and many of their terminals formed heterotypical synapses with other Purkinje cell dendritic spines. The resulting circuit reorganization preserved inhibition in the cerebellar cortex. Following this reorganization, replacement of the missing granule cells and glia was followed by a restitution of the normal circuitry. Most of these developmental and reconstructive changes were not dependent on neuronal activity, the major exception being inhibitory synaptogenesis. The full complement of inhibitory synap...
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Research Interests: Medicine and Antibodies
When mouse organotypic cerebellar cultures were exposed to anti-GABA agents that increased neuronal activity early in development, there was a doubling of the ratio of inhibitory axosomatic synapse profiles to Purkinje cell somatic... more
When mouse organotypic cerebellar cultures were exposed to anti-GABA agents that increased neuronal activity early in development, there was a doubling of the ratio of inhibitory axosomatic synapse profiles to Purkinje cell somatic profiles after two weeks in vitro, which correlated with a decrease in spontaneous cortical discharges. When similar cultures were maintained in medium with activity blocking agents, Purkinje cell axosomatic synapses were reduced to approximately half of control values and, after recovery from activity blockade, the cultures discharged hyperactively. By contrast, the full complement of excitatory cortical synapses developed in the absence of neuronal activity. These results support the concept that neuronal activity is necessary for the complete development of inhibitory circuitry. When cerebellar cultures were simultaneously exposed to activity blocking agents and to neurotrophins BDNF or NT-4, both of which bound to the TrkB receptor, the numbers of inh...
Research Interests: Biology, Medicine, Signal Transduction, Humans, Cerebellum, and 4 moreAnimals, Neurons, Clinical Sciences, and SYNAPSES
Some neurons, including cerebellar Purkinje cells, are completely ensheathed by astrocytes. When granule cell neurons and functional glia were eliminated from newborn mouse cerebellar cultures by initial exposure to a DNA synthesis... more
Some neurons, including cerebellar Purkinje cells, are completely ensheathed by astrocytes. When granule cell neurons and functional glia were eliminated from newborn mouse cerebellar cultures by initial exposure to a DNA synthesis inhibitor, Purkinje cells lacked glial sheaths and there was a tremendous sprouting of Purkinje cell recurrent axon collaterals, terminals of which hyperinnervated Purkinje cell somata, including persistent somatic spines, and formed heterotypical synapses with Purkinje cell dendritic spines, sites usually occupied by parallel fiber (granule cell axon) terminals. Purkinje cells in such preparations failed to develop complex spikes when recorded from intracellularly, and their membrane input resistances were low, making them less sensitive to inhibitory input. If granule cells and oligodendrocytes were eliminated, but astrocytes were not compromised, sprouting of recurrent axon collaterals occurred and their terminals projected to Purkinje cell dendritic s...
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ABSTRACT Methods are described for preparation of organotypic cultures of cerebellum, cerebral neocortex and spinal cord-dorsal root ganglia. Such cultures are capable of yielding a great amount of information, especially about such... more
ABSTRACT Methods are described for preparation of organotypic cultures of cerebellum, cerebral neocortex and spinal cord-dorsal root ganglia. Such cultures are capable of yielding a great amount of information, especially about such issues as direct effects of substances on a part of the nervous system isolated from systemic, circulatory or hormonal influences, or from other levels of the nervous system. The technique may be difficult and labor intensive, but the effort required may be more than compensated by the knowledge gained from working with a system that has many of the characteristics of the in vivo system from which it was derived. The extrapolatory leap from this culture system to in vivo conditions is far less than with culture systems in which cells are maintained in relative isolation.
ABSTRACT Cerebellar tissue cultures retain or develop gross structural characteristics that identify the tissue as cerebellar in origin and allow definition of specific cortical and subcortical regions. The cortex is laminated, the... more
ABSTRACT Cerebellar tissue cultures retain or develop gross structural characteristics that identify the tissue as cerebellar in origin and allow definition of specific cortical and subcortical regions. The cortex is laminated, the formation of laminae occurring after explantation. A zone of subcortically converging Purkinje cell axons that myelinate in vitro is equivalent to a white matter area in situ. Intracerebellar nuclei are the targets of converging Purkinje cell axons, and gross anatomical features of such nuclei make their presumptive identification possible. Characteristic neuronal types are present in cortical and subcortical portions of cerebellar explants. Ultrastructural studies of cortical neurons have defined interneuronal relationships as being similar to those in situ and have demonstrated the orderly development of synapses in vitro. Chemical and metabolic studies have further validated the similarity of the in vitro cerebellum to its in situ counterpart. Extracellular electrophysiological recordings have defined spontaneous and stimulus-elicited cortical activity. Spontaneous discharges are phasic and regular in character, predominantly the former, and are like spontaneous discharges recorded from cerebellar cortex in vivo. Stimulus-elicited cortical activity is most commonly characterized as sequences of excitation-inhibition-excitation, resembling similar sequences recorded in situ.
... CPT Fredrick J. Seil, MC, USAR; CPT Sydney S. Schochet, Jr., MC, USAR; and Kenneth M. Earle, MD, Washington, DC ... in young children.1-8 The clinical picture most often associated with this disease has been one of developmental... more
... CPT Fredrick J. Seil, MC, USAR; CPT Sydney S. Schochet, Jr., MC, USAR; and Kenneth M. Earle, MD, Washington, DC ... in young children.1-8 The clinical picture most often associated with this disease has been one of developmental retardation and en-largement of the head. ...
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ABSTRACT Demyelinating antibodies are generally defined by their destructive effects on myelin in vitro. Such antibodies are of interest because of a possible role in demyelinating diseases in vivo, including multiple sclerosis, and... more
ABSTRACT Demyelinating antibodies are generally defined by their destructive effects on myelin in vitro. Such antibodies are of interest because of a possible role in demyelinating diseases in vivo, including multiple sclerosis, and because of their potential use as probes for the study of myelination.
Demyelination can be defined as a process in which the pathological events that lead to destruction of myelin are directed at myelin sheaths or at cells that form and maintain myelin, whereas axons are relatively spared. Animal models of... more
Demyelination can be defined as a process in which the pathological events that lead to destruction of myelin are directed at myelin sheaths or at cells that form and maintain myelin, whereas axons are relatively spared. Animal models of immunologically mediated demyelination are described and tissue culture studies of serum antimyelin factors are reviewed.
A possible mechanism for recovery of function after central nervous system (CNS) injury is collateral sprouting. Uninjured axons innervating the same target territory as axons that have sustained damage may sprout collateral axons that... more
A possible mechanism for recovery of function after central nervous system (CNS) injury is collateral sprouting. Uninjured axons innervating the same target territory as axons that have sustained damage may sprout collateral axons that innervate the target sites vacated by the damaged fibers. Collateral sprouting represents a regenerative response in which there is a territorial expansion of innervation by surviving intact axons. Studies beginning in the late 1950s have shown that collateral sprouting is a mechanism that is available for circuit reorganization in the injured CNS. Several examples of CNS collateral sprouting after injury are reviewed and possible consequences of collateral sprouting and circuit reorganization for the injured subject are discussed.
Studies of myelination after transplantation of mature oligodendrocytes to cerebellar cultures in which oligodendrocyte maturation and myelination had been irreversibly inhibited by exposure to cytosine arabinoside were reviewed.... more
Studies of myelination after transplantation of mature oligodendrocytes to cerebellar cultures in which oligodendrocyte maturation and myelination had been irreversibly inhibited by exposure to cytosine arabinoside were reviewed. Transplanted oligodendrocytes were derived from three sources, including cerebellar explants treated with kainic acid, dissociated oligodendrocyte cultures, and optic nerve fragments. Oligodendrocytes from all sources migrated into the host explants and myelinated appropriate axons. The time of appearance of myelin and the percentage of host cultures myelinated differed for the three sources of oligodendrocytes, however. Myelin was visible earliest and in the highest percentage of host explants transplanted with cultured dissociated oligodendrocytes, which were presumably the most free to migrate into the host tissue, and latest and in the lowest percentage of host cultures transplanted with optic nerve, from which oligodendrocytes were presumably least fre...
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Granuloprival cerebellar cultures derived from neonatal mice were transplanted at nine days in vitro with granule cells and glia, and the changes induced in the host explants were examined daily with the electron microscope from one to... more
Granuloprival cerebellar cultures derived from neonatal mice were transplanted at nine days in vitro with granule cells and glia, and the changes induced in the host explants were examined daily with the electron microscope from one to nine days post-transplantation. Granule cells and astrocytes had migrated into the host cultures within 24 h, and astrocytic processes began to ensheath Purkinje cells and to interpose themselves between axon terminals and Purkinje cell somata, reducing the number of axosomatic synapses. Occasional degenerating Purkinje cells were present. At two days post-transplantation, synapse formation between parallel fibre terminals and Purkinje cell dendritic spines was initially evident, and Purkinje cells began to proliferate dendritic spines near astrocytic processes. Degenerating Purkinje cells were more frequently encountered. Myelin was first observed in host cultures at three days after transplantation, and astrocytes continued to ensheath Purkinje cell...
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Cerebellar granule cells and oligodendrocytes are destroyed and astrocytes are functionally compromised by exposure of organotypic cerebellar cultures derived from newborn mice to cytosine arabinoside for the first 5 days in vitro.... more
Cerebellar granule cells and oligodendrocytes are destroyed and astrocytes are functionally compromised by exposure of organotypic cerebellar cultures derived from newborn mice to cytosine arabinoside for the first 5 days in vitro. Consequently, myelin does not form and Purkinje cells survive in increased numbers, but without astrocytic ensheathment. In the absence of glial sheaths, Purkinje cells have altered membrane properties and reduced input resistance. Their inhibitory recurrent axon collaterals sprout enormously and hyperinnervate the unensheathed somata of other Purkinje cells and form heterotypical synapses with Purkinje cell dendritic spines normally occupied by homotypical excitatory parallel fiber (granule cell axon) terminals. This reorganization of the cortical circuitry, in which recurrent axon collaterals are the dominant inhibitory elements, allows retention of some inhibition in the absence of parallel fiber excitation of the inhibitory interneurons. In the absenc...
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Studies of Purkinje cell dendritic spine proliferation after transplantation of cytosine arabinoside (Ara C) treated organotypic cerebellar cultures with glia and granule cells, either separately and in combination, were reviewed.... more
Studies of Purkinje cell dendritic spine proliferation after transplantation of cytosine arabinoside (Ara C) treated organotypic cerebellar cultures with glia and granule cells, either separately and in combination, were reviewed. Exposure of cerebellar explants to Ara C for the first 5 days in vitro results in the destruction of granule cells, the only excitatory cortical neurons, and oligodendroglia, and functionally compromises surviving astrocytes so that they do not appose neuronal membranes. In the absence of granule cells, there is a sprouting of Purkinje cell recurrent axon collaterals, the terminals of which project to and form heterotypical synapses with Purkinje cell dendritic spines, which are usually occupied by terminals of granule cell axons (parallel fibers). After this reorganization has been achieved, the explants can be transplanted with the missing elements to induce a second round of reorganization, with approximate restoration of the usual interneuronal relatio...
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Neonatal mouse cerebellar cultures depleted of granule cells and functional glia by exposure to cytosine arabinoside were transplanted with either granule cells and glia or with granule cells in the absence of functional glia. Myelination... more
Neonatal mouse cerebellar cultures depleted of granule cells and functional glia by exposure to cytosine arabinoside were transplanted with either granule cells and glia or with granule cells in the absence of functional glia. Myelination was evident in cultures transplanted with granule cells and glia, excess sprouted cortical neurites were reduced, Purkinje cells acquired astrocytic sheaths and had a near normal complement of axosomatic synapses, and homotypical parallel fiber-Purkinje cell dendritic spine synapses were present in a 2.4:1 ratio to heterotypical recurrent axon collateral-Purkinje cell dendritic spine synapses. Cultures transplanted with granule cells were not myelinated, sprouted cortical neurites were not reduced. Purkinje cells lacked astrocytic sheaths and their somata remained hyperinnervated, and the ratio of homotypical to heterotypical dendritic spine synapses was 1.4:1. In the absence of functional glia there was a greater persistence of heterotypical recur...
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Guinea pigs received a suboptimal transfer of lymphocytes sensitized to myelin basic protein (BP) and were then immunized with guinea pig BP, BP plus chicken brain or chicken myelin, or chicken brain alone. Sera from these animals were... more
Guinea pigs received a suboptimal transfer of lymphocytes sensitized to myelin basic protein (BP) and were then immunized with guinea pig BP, BP plus chicken brain or chicken myelin, or chicken brain alone. Sera from these animals were tested for the presence of myelinotoxic antibodies, as detected by the myelination inhibition assay. Myelination inhibition activity correlated with the histologic severity of demyelination.
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ABSTRACT Methods are described for preparation of organotypic cultures of cerebellum, cerebral neocortex and spinal cord-dorsal root ganglia. Such cultures are capable of yielding a great amount of information, especially about such... more
ABSTRACT Methods are described for preparation of organotypic cultures of cerebellum, cerebral neocortex and spinal cord-dorsal root ganglia. Such cultures are capable of yielding a great amount of information, especially about such issues as direct effects of substances on a part of the nervous system isolated from systemic, circulatory or hormonal influences, or from other levels of the nervous system. The technique may be difficult and labor intensive, but the effort required may be more than compensated by the knowledge gained from working with a system that has many of the characteristics of the in vivo system from which it was derived. The extrapolatory leap from this culture system to in vivo conditions is far less than with culture systems in which cells are maintained in relative isolation.
Sensitization of guinea pigs with purified myelin basic protein induces experimental allergic encephalomyelitis (EAE) but does not induce a serum factor which inhibits myelin formation in vitro. This factor, induced by some unidentified... more
Sensitization of guinea pigs with purified myelin basic protein induces experimental allergic encephalomyelitis (EAE) but does not induce a serum factor which inhibits myelin formation in vitro. This factor, induced by some unidentified constituent of whole central nervous system tissue, should not be characterized as a component of "EAE serum."
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Synthetic galactodihydrocerebrosides with widely different fatty acid components can evoke myelination-inhibiting antibodies in rabbits. Whether these are the only such haptens involved in experimental immunizations of other species or in... more
Synthetic galactodihydrocerebrosides with widely different fatty acid components can evoke myelination-inhibiting antibodies in rabbits. Whether these are the only such haptens involved in experimental immunizations of other species or in spontaneous human diseases is not yet known.
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Unmyelinated mouse cerebellar cerebellar cultures in which oligodendrocyte differentiation had been suppressed by exposure to cytosine arabinoside developed axonal myelin after superimposition of kainic acid-treated cerebellar explants... more
Unmyelinated mouse cerebellar cerebellar cultures in which oligodendrocyte differentiation had been suppressed by exposure to cytosine arabinoside developed axonal myelin after superimposition of kainic acid-treated cerebellar explants devoid of myelin-receptive axons. The latter explants contained differentiated oligodendrocytes. The operation of a diffusible myelin-stimulating factor was ruled out by the failure of myelination in cytosine arabinoside-exposed explants not in direct contact with oligodendrocyte-containing transplants.
Research Interests: Science, Multidisciplinary, Cerebellum, Tissue culture, Collagen, and 5 moreMice, Animals, Axons, Myelin Sheath, and Kainic acid
Neurites of mesencephalic V nucleus neurons, which in vivo have a peripheral process, were observed to grow away from cerebellar explants. This pattern of neurite growth was contrasted with a previously observed pattern of looping and... more
Neurites of mesencephalic V nucleus neurons, which in vivo have a peripheral process, were observed to grow away from cerebellar explants. This pattern of neurite growth was contrasted with a previously observed pattern of looping and returning to the explant characteristic of central processes of other neurons in cerebellar cultures. The described tissue culture system could serve as a model for elucidating mechanisms which determine the directional growth of neurites.
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During the Persian Gulf War, pyridostigmine bromide (PB), a reversible inhibitor of acetylcholinesterase, was used as prophylaxis against exposure to nerve gas. Exposure to PB has been suggested as a potential cause of the persistent... more
During the Persian Gulf War, pyridostigmine bromide (PB), a reversible inhibitor of acetylcholinesterase, was used as prophylaxis against exposure to nerve gas. Exposure to PB has been suggested as a potential cause of the persistent fatigue reported among Gulf War veterans. The aim of this study was to evaluate the effects of acute and continuous exposure to low doses of PB on the neuromuscular junction. Organotypic spinal cord-muscle cocultures were used to examine in vitro the effects of PB under controlled conditions. Acute exposure to PB potentiated neuromuscular activity. Continuous exposure to PB produced a progressive decrease in the contractile activity of muscle fibers. Ultrastructural examination by electron microscopy revealed no abnormalities in the neuromuscular junctions after 1 week of exposure. Nerve terminal degeneration and atrophy of the postjunctional folds were evident after 2-week exposure to low-dose PB. The effects of PB were reversible following withdrawal. The reversibility of the PB-induced changes in vitro suggests that such changes are causally unrelated to the fatigue reported by Persian Gulf War veterans years after exposure to PB.