Regenerative failure in the mammalian CNS.
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Animals↗
Biomedical subjects
C C Stichel
Publications and source records attributed to C C Stichel.
Transplants of immature astrocytes promote axonal regeneration in the adult rat brain.
To study beneficial effects of immature astrocytes on axonal regeneration in the injured adult mammalian brain, we have stereotactically implanted cultured astrocytes from embryonic (E 14-16) rat cerebral cortex into the lesion site following transection of the postcommissural fornix. The spatio-temporal pattern of axonal degeneration and regrowth in the proximal fornix stump was investigated using wheat germ agglutinin-horseradish peroxidase tracing techniques and quantitative analysis of myelinated axon profiles. Transection of the postcommissural fornix tract caused disintegration of the axons in the distal stump as well as rapid and pronounced retrograde axonal degeneration up to 800-1,200 microns proximal to the lesion site. While a small bundle of subicular fibers spontaneously extended to the lesion site within 4 weeks after injury, axonal regeneration was markedly stimulated in those animals that had received an astroglial implant. Following the former pathway, regenerating axons sprouted towards the implant but did not penetrate the graft. Instead, the axons elongated over the surface of the transplant, avoiding growth into the surrounding neuropil or into the distal fornix segment. In grafted animals we further observed a substantial increase in the number of myelinated axons of approximately 31.5% (at the level of 800 microns) and approximately 40% (at the 400 microns level) compared with the injured tract lacking a transplant. Our results indicate the capacity of juvenile astrocytes to stimulate axonal regeneration after injury of the post-commissural fornix tract in the adult rat brain. We further demonstrate myelination of the regenerated axons.
Animals↗
Extensive and long-lasting changes of glial cells following transection of the postcommissural fornix in the adult rat.
Injury to the adult brain results in the formation of a glial scar that involves both activated astroglia and microglia/macrophages. Although reactive gliosis has been linked to failure of axonal regeneration in the adult mammalian CNS, the spatio-temporal pattern of the postlesion responses in morphology and distribution of the major cellular constituents of the gliosis has attracted little attention. In an attempt to define these relationships, we have undertaken a systematic study of astrocytic and microglial/macrophagic responses after stereotactic transection of the postcommissural fornix in rat. To visualize astrocytes, microglia, and macrophages, antibodies against glial fibrillary acidic protein (GFAP), vimentin (VIM), complement receptor type 3 (OX42), and ED1 were used. The cellular responses occurring between 2 h and 1 year postlesion (PL) at various distances distal and proximal to the lesion site were studied. Transection of the postcommissural fornix leads to: 1) a very early microglial reaction both distal and proximal to the lesion, as indicated by OX42-immunoreactivity, followed by a massive astrocytic response; 2) a transient GFAP(-) but VIM(+) region around the lesion center; 3) the appearance of numerous ED1(+) macrophages at PL3d; and 4) long-lasting (at least 1 year) persistence of both astrocytes and microglia/macrophages. The timing and extension of the sequential glial reactions after postcommissural fornix transection are discussed in relation to the myelin degradation and spontaneous sprouting of injured axons that have previously been observed in this lesion model (Wunderlich et al: Glia 10:49-58, 1994).
Animals↗
Relationship between injury-induced astrogliosis, laminin expression and axonal sprouting in the adult rat brain.
Lesion-induced regenerative sprouting of CNS axons is accompanied by structural and metabolic changes of astrocytes. In order to evaluate the effects of these astrocytic changes on axonal regeneration, we investigated the spatio-temporal relationship of gliosis, laminin expression and axonal sprouting in the postcommissural fornix of the adult rat. Using immunocytochemical methods we observed (1) a perilesional area with a transient lack of astrocytes and axons, (2) the reappearance of reactive astrocytes followed by the ingrowth of sprouting fibres and finally an increase in laminin-immunoreactivity, (3) the absence of lesion-induced laminin-expression in reactive astrocytes and (4) the formation and long-lasting (at least 28 months) persistence of a dense plexus of laminin-immunopositive blood vessels at the site of transection and in the proximal and distal stumps. These data indicate that astrogliosis is permeable for regrowing axons and that injury-induced axonal sprouting in the transected postcommissural fornix may be mediated by laminin-independent mechanisms.
Animals↗
Spontaneous activity and recurrent inhibition in cultured hippocampal networks.
As a model for an integrated neuronal network based on the concept of modular units, we have investigated the occurrence of spontaneous activity and the formation of synaptic circuits in primary cultures of dissociated hippocampal neurons from the embryonic rat. Sodium-dependent action potentials (APs) could be elicited after 1 day in vitro (DIV), whereas spontaneous postsynaptic potentials (PSPs), "miniature" PSPs and APs appeared after 3-6 DIV. The number of cells with spontaneous APs and the rate of APs increased during development of the neuritic network. In addition to a stochastic spike interval distribution, pyramid-shaped neurons could be identified after 10-12 DIV, which fired preferentially at interspike intervals between 20-120 ms and 190-400 ms. This distinctive bimodal interspike interval pattern was sensitive to GABA-A antagonists. Simultaneous recordings of pairs of neurons demonstrated recurrent inhibitory, GABA-ergic synaptic circuits. In addition, a subpopulation of GABAergic neurons could be visualized by immunocytochemistry. These results are discussed in relation to the hypothesis that spontaneous firing of connected neurons is network-driven, based on synaptic "noise" and patterned by recurrent inhibition.
Action Potentials↗
Expression of inherent neuronal shape characteristics after transient sensitivity to epigenetic factors.
We investigated effects of different substrates and culture media on the early morphological differentiation of rat neocortical neurons in culture. In particular, we examined the effects of homotypic astrocytes, the adhesive glycoprotein laminin and the polycationic substrate poly-L-lysine, as well as diffusible astrocyte-derived conditioned medium factors and serum on (1) soma area, (2) total neuron area and (3) primary neurite number. To assess variations in morphological reactions of neurons with a defined neurotransmitter phenotype, we analyzed the differentiation of GABAergic neurons. The morphology of young neocortical neurons was dramatically affected by both substrate and culture medium. Replacement of the astrocytic monolayer or the astrocyte-conditioned medium by other substrates or non-conditioned medium, respectively, was accompanied by (1) spreading and flattening of neuronal somata, (2) a marked decrease in total neuron area and (3) an increase in the number of primary neurites. The various morphological parameters studied exhibited different sensitivities to changes of these external factors. Moreover, the influences of epigenetic factors on the generation of primary neurites depended on the transmitter phenotype of the neuron. The induced morphological alterations were transient. At the end of the first week in culture, the surviving neurons underwent substantial remodeling of their morphology leading to an expression of in vivo shape characteristics. These observations suggest that despite an early, transient sensitivity to environmental influences, the neuronal differentiation with respect to the morphological parameters studied in culture is to a large degree determined by intrinsic factors.
Animals↗
Dissociated cell culture of rat cerebral cortical neurons in serum-free, conditioned media: GABA-immunopositive neurons.
The gamma-aminobutyric acid (GABA)ergic properties of embryonic (E15d) rat cortical neurons were studied in dissociated serum-free culture by immunohistochemical methods. GABA-like immunoreactivity was found in a subpopulation of neurons from the first day onwards. The number of GABA-positive neurons reached mature values (10.5-12.6%) within the first week, while their morphological differentiation was not found to be fully completed until the 11th day of culture and was characterized by several discrete developmental stages. First, GABA-positive neurons gained their mature complement of neurites at 3 days in vitro (DIV). Three days later somal maturation became evident, followed at least by the maturation of the neuritic arbor. Double-labelling studies revealed the coexpression of GABA and tyrosine hydroxylase within the same cells. The similarities of relative number, morphology, time course of development and biochemistry of cultured GABAergic neurons compared with those in situ suggest that the applied culture system is a useful model to investigate several aspects of GABAergic neurotransmission at the cellular level.
Animals↗
Distribution of glial fibrillary acidic protein and vimentin immunoreactivity during rat visual cortex development.
The postnatal maturation of astrocytes in the rat visual cortex was analysed by immunostaining the astroglial proteins vimentin and glial fibrillary acidic protein with poly- and monoclonal antibodies. Vimentin immunoreactivity was present in the visual cortex up to the third postnatal week, whereas immunolabelling first disappeared in the cortical layers and then in the white matter. In the early postnatal period, vimentin antibodies labelled radial glial fibres. After the first postnatal week staining of radial glial fibres gradually disappeared and vimentin immunoreactivity was localized in a few protoplasmic astrocytes in the grey matter and fibrous astrocytes in the white matter. The development of glial fibrillary acidic protein-positive astrocytes was not fully complete until postnatal day 50. Glial fibrillary acidic protein-positive radial glial fibres were present after birth and disappeared towards the end of the third postnatal week. Staining of astrocytes in the white matter and in cortical layers I and VI reached an adult density at postnatal days 8 and 20, respectively. A progressively later development of glial fibrillary acidic protein-positive astrocytes was observed in cortical layers II-V which was completed between postnatal days 47 and 50. In the adult rat visual cortex glial fibrillary acidic protein-positive astrocytes were especially dense in layers I and VI, moderate in layers II/III and V and nearly absent in layer IV and lower layer III. The time course of the loss of vimentin and the gradual appearance of glial fibrillary acidic protein immunoreactivity in the visual cortex is considered as an index of astrocytic maturation and the spatiotemporal sequence of this maturation pattern is discussed in terms of reciprocal neuron-astrocyte interactions during brain development.
Animals↗
Ultrastructure of PkC(II/III)-immunopositive structures in rat primary visual cortex.
In the primary visual cortex of adult rats the cellular and subcellular distribution of protein kinase C isozymes II and III (PkCII/III) was examined by immunohistochemical methods with a monoclonal antibody against PkCII/III. Strong PkC(II/III)-immunoreactivity was found in neurons and astrocytes. Immunopositive neurons exhibited morphological features characteristic for both pyramidal and non-pyramidal cells. They were distributed in layers II through VI but were concentrated in layers II/III. At the electron microscopic level immunoprecipitate was found predominantly in distinct regions of the somata, except the nuclei, and only a few labeled dendrites and axons were seen. Two different patterns of cytoplasmic immunoreactivity could be distinguished. In most neurons, PkC(II/III)-staining was confined to cytoplasmic spots associated with the Golgi complex, while a few neurons exhibited additional labeling in the vicinity of the cell membrane. Moreover, PkC(II/III)-immunoreactivity was present in numerous astroglial processes and in the perikaryal cytoplasm of a subpopulation of astrocytes. The present data provide morphological indications for specific functions of PkC isozymes II and III in neurons as well as in astrocytes.
Animals↗
Light and electron microscopic immunocytochemical localization of parvalbumin in the dorsal lateral geniculate nucleus of the cat: evidence for coexistence with GABA.
The coexistence in individual neurons of parvalbumin and gamma-aminobutyric acid (GABA) was studied in the dorsal lateral geniculate nucleus (dLGN) of the cat using pre- and postembedding immunocytochemical methods. PV(+) cell bodies and processes were found in the perigeniculate nucleus (PGN) and throughout all laminae of the dLGN. PV(+) neurons were relatively small and had circular to fusiform shapes. Electron microscopy revealed PV(+) reaction product within the perikarya, axons, and dendrites of labeled cells. It was associated preferentially with microtubules, postsynaptic densities, and intracellular membranes. PV(+) presynaptic boutons were identified on the basis of their synaptic relations and ultrastructure as retinal terminals (RLP) and as profiles originating from inhibitory interneurons (F1 and F2). Immunopositive somata and dendrites received asymmetric synaptic contacts from labeled RLP and non-identified, non-immunoreactive synaptic boutons. Moreover, PV(+) dendrites were postsynaptic to labeled F profiles. In the PGN all neurons were both PV(+) and GABA-immunoreactive and in the dLGN the vast majority of PV(+) neurons showed GABA-immunoreactivity. It is suggested that the high incidence of PV in GABAergic neurons is related to the particular activation patterns of these neurons and the resulting demand for calcium buffer systems.
Animals↗
Localization of isoenzymes II/III of protein kinase C in the rat visual cortex (area 17), hippocampus and dentate gyrus.
Monoclonal antibodies against type II and type III subspecies of protein kinase C PkC(II/III) were used to map the distribution of these isoenzymes in the visual cortex (area 17), hippocampus and dentate gyrus of the rat. PkC(II/III)-immunocytochemistry resulted in a specific staining of neuropil and of neuronal somata with their proximal dendrites. The majority of immunopositive cells exhibited a punctate distribution of reaction product, while only a few neurons were homogeneously labeled. In the visual cortex stained neurons were distributed throughout all laminae and reached a particularly high density in layers II/III. Moreover, PkC(II/III)-positive neurons were found within the strata pyramidale and radiatum of the hippocampus proper and in the stratum granulosum, the subgranular zone and the hilar region of the dentate gyrus. The present results suggest that PkC(II/III)-positive neurons constitute a distinct population of both projection and local circuit neurons that are not exclusively associated with any one neurotransmitter system.
Animals↗
Ontogenetic changes in the level and subcellular distribution of protein kinase C in cat visual cortex.
Ontogenetic changes of the total activity and the subcellular distribution of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) were investigated in the cat visual cortex. Following homogenization of the tissue in Ca2+-free buffer and separation of membrane-bound and soluble protein kinase C, the enzyme was partially purified by diethylaminoethyl-cellulose chromatography. Total, membrane-bound and soluble enzyme activity were determined by measuring the phosphate incorporation into lysine-rich histone, a substrate for protein kinase C. It was found that the total activity level, while being low in the first 4 weeks, increased rapidly to peak values at 5 weeks of age and declined slightly thereafter. Although at all developmental stages most of the enzyme was recovered in the soluble fraction, the membrane-bound activity exhibited a considerable increase at 5 weeks of age, reflecting a relative shift of protein kinase C from the cytosol to the membranes. The increase and redistribution of protein kinase C activity coincide in time with the developmental phase during which the visual cortex is particularly susceptible to undergo use-dependent modifications. This finding is compatible with the hypothesis that protein kinase C-mediated phosphorylation processes are involved in activity-dependent modifications of neuronal transmission.
Aging↗
Immunohistochemical localization of calcium-binding proteins, parvalbumin and calbindin-D 28k, in the adult and developing visual cortex of cats: a light and electron microscopic study.
In the cat primary visual cortex, we investigated with immunohistochemical techniques the developmental changes in the cellular and subcellular localization of the Ca2+-binding proteins parvalbumin (PV) and calbindin-D 28K (CBP), in order to determine whether there is a correlation between the expression of Ca2+-dependent processes and the time course of the critical period for use-dependent plasticity. On the 54th day of gestation and at 1 week postnatally, both calcium-binding proteins were present only in a subpopulation of neurons in layers V and VI. During subsequent maturation, the number of PV(+) and CBP(+) neurons increased significantly and labeled cells were detected in more superficial layers. Moreover, the homogeneous labeling of some CBP(+) neurons in layers IV to VI decreased and changed to a punctate pattern. In adult cats PV(+) neurons were evenly distributed throughout layers II to VI, whereas CBP(+) neurons were concentrated in layers II/III. Only a few immunoreactive cells had morphological features characteristic of pyramidal cells; the large majority were nonpyramidal. Electron microscopy confirmed the presence of PV- and CBP-reaction product within the perikarya, axons, and dendrites of labeled cells. It was associated preferentially with microtubules, postsynaptic densities, and intracellular membranes. Immunoreactive neurons received immunonegative asymmetric synapses on their dendritic shafts and made symmetric synaptic contacts with labeled and unlabeled somata and with unlabeled dendritic shafts. The large number and widespread distribution of immunoreactive neurons implies that PV and CBP play an important role in the regulation of calcium-dependent processes in the visual cortex. Furthermore, the developmental redistribution of PV and CBP points to changes in the organization of Ca2+-dependent processes during maturation.
Aging↗
Quantitative analysis of the choline acetyltransferase-immunoreactive axonal network in the cat primary visual cortex: I. Adult cats.
The laminar distribution of cholinergic axons was analyzed quantitatively in the visual cortex of adult cats by using immunocytochemistry with a monoclonal antibody against choline acetyltransferase (ChAT). ChAT(+) fibers and varicosities were counted at different locations within area 17 and the distribution patterns in various animals were compared. Choline acetyltransferase-immunoreactivity was localized in fine, varicose fibers, which were present in all layers of the visual cortex. The density of labeled fibers was highest in layer I, which contained 14% of all fibers and 19.5% of all varicosities, and decreased toward deeper layers. The number of varicosities decreased more markedly toward deeper layers than the frequency of fibers. These distribution patterns were very consistent, showing only slight intra- and interindividual variability.
Animals↗
Quantitative analysis of the choline acetyltransferase-immunoreactive axonal network in the cat primary visual cortex: II. Pre- and postnatal development.
The pre- and postnatal development of cholinergic projections was investigated in the cat striate cortex by applying immunohistochemical methods based on a monoclonal antibody against choline acetyltransferase (ChAT). The earliest age investigated was gestational day 54. At this stage a sparse network of ChAT(+) fibers was distributed throughout the striate cortex. Subsequent postnatal maturation of ChAT(+) fibers was characterized by an increase in fiber density that started in layer VI and gradually progressed toward more superficial layers. By 4 weeks of age the density of ChAT(+) fibers and varicosities had reached adult levels in layers V and VI but was still subnormal in layers I-IV. The mature pattern of cholinergic innervation was established by 13 weeks of age. There was no evidence for developmental gradients in the anteroposterior and mediolateral directions within area 17. These results indicate that the cholinergic projection to striate cortex develops continuously in an inside-out sequence as is characteristic for most cortical maturation processes. There was no indication that striate cortex receives an especially dense cholinergic input during the critical period.
Animals↗
A search for choline acetyltransferase-like immunoreactivity in neurons of cat striate cortex.
The goal of this study was to determine whether cat striate cortex contains neurons with choline acetyltransferase-like immunoreactivity. Two different monoclonal antibodies were applied with various fixation protocols. As controls served selected regions of the cat brain and the rat neocortex where cholinergic neurons had been demonstrated previously with independent methods. All experimental protocols labelled the presumptive cholinergic neurons in the central regions but revealed only a few weakly stained neurons in cat striate cortex. These had a non-pyramidal morphology and were scattered throughout all layers.
Animals↗
Parvalbumin in cat brain: isolation, characterization, and localization.
Because of the increasing evidence that Ca2+-binding proteins have important regulating functions in nerve cells and because of the indications that there are species differences in the structures of these proteins, parvalbumin was purified from cat brain and muscle. Brain and muscle parvalbumins were found to be indistinguishable from each other in their biochemical and immunological properties. However, cat parvalbumin differs from all other mammalian parvalbumins by its apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 10-11K (compared to rat parvalbumin, 12K), and a lower pI of 4.6 (rat parvalbumin, 4.9), in the tryptic peptide maps, and in the immunological properties, indicating a distinct primary structure. With the purified parvalbumin as antigen, polyclonal antibodies were raised in rabbits and these were subsequently used for immunohistochemical localizations of parvalbumin in the cat brain. In the visual cortices of adult cats immunoreactive neurons were present throughout layers II and IV. In cerebellar cortex, Purkinje, basket, and stellate cells were immunoreactive. Comparison with staining patterns obtained with antiserum against rat parvalbumin revealed some cross-reactivity but confirmed the existence of species differences in the antigenic structure of rat and cat parvalbumin.
Amino Acids↗
Organization and morphological characteristics of choline acetyltransferase-containing fibers in the visual thalamus and striate cortex of the cat.
The morphological characteristics and the distribution of cholinergic fibers in the cat visual system were studied with immunohistochemical methods employing monoclonal antibodies directed against choline acetyltransferase (ChAT). ChAT-positive fibers form dense meshworks within the main laminae of the dorsal lateral geniculate nucleus (LGNd), the perigeniculate nucleus (PGN) and the visual cortex. The fibers are of fine caliber, branch extensively and possess numerous varicosities in close contact with cellular elements. The density of ChAT + fibers is not uniform and is particularly high in cortical laminae I-III, the PGN complex and laminae A and A1 of the LGNd.
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