T. Deller

LESION-INDUCED PLASTICITY OF CENTRAL NEURONS: SPROUTING OF SINGLE FIBRES IN THE RAT HIPPOCAMPUS AFTER UNILATERAL ENTORHINAL CORTEX LESION

In response to a central nervous system trauma surviving neurons reorganize their connections and form new synapses that replace those lost by the lesion. A well established in vivo system for the analysis of this lesion-induced plasticity is the reorganization of the fascia dentata following unilateral entorhinal cortex lesions in rats. After general considerations of neuronal reorganization following a central nervous system trauma, this review focuses on the sprouting of single fibres in the rat hippocampus after entorhinal lesion and the molecular factors which may regulate this process. First, the connectivity of the fascia dentata in control animals is reviewed and previously unknown commissural fibers to the outer molecular layer and entorhinal fibres to the inner molecular layer are characterized. Second, sprouting of commissural and crossed entorhinal fibres after entorhinal cortex lesion is described. Single fibres sprout by forming additional collaterals, axonal extensions, boutons, and tangle-like axon formations. It is pointed out that the sprouting after entorhinal lesion mainly involves unlesioned fibre systems terminating within the layer of fibre degeneration and is therefore layer-specific. Third, molecular changes associated with axonal growth and synapse formation are considered. In this context, the role of adhesion molecules, glial cells, and neurotrophic factors for the sprouting process are discussed. Finally, an involvement of sprouting processes in the formation of neuritic plaques in Alzheimers disease is reviewed and discussed with regard to the axonal tangle-like formations observed after entorhinal cortex lesion.

Differential Regulation of Ciliary Neurotrophic Factor (CNTF) and CNTF Receptor α Expression in Astrocytes and Neurons of the Fascia Dentata after Entorhinal Cortex Lesion

Neurotrophic factors have been implicated in reactive processes occurring in response to CNS lesions. Ciliary neurotrophic factor (CNTF), in particular, has been shown to ameliorate axotomy-induced degeneration of CNS neurons and to be upregulated at wound sites in the brain. To investigate a potential role of CNTF in lesion-induced degeneration and reorganization, we have analyzed the expression of CNTF protein and CNTF receptor α (CNTFRα) mRNA in the rat dentate gyrus after unilateral entorhinal cortex lesions (ECLs), using immunocytochemistry and nonradioactive in situhybridization, respectively. In sham-operated as in normal animals, CNTF protein was not detectable by immunocytochemistry. Starting at 3 d after ECL, upregulation of CNTF expression was observed in the ipsilateral outer molecular layer (OML). Expression was maximal at around day 7, and at this stage immunoreactivity could be specifically localized to astrocytes in the ipsilateral OML. By day 14 postlesion, CNTF immunoreactivity had returned to control levels. CNTFRα mRNA was restricted to neurons of the granule cell layer in controls. Three days postlesion, prominent CNTFRα expression was observed in the deafferented OML. A similar but less prominent response was noticed in the contralateral OML. After 10 d, CNTFRα expression had returned to control levels. Double labeling for CNTFRα mRNA and glial fibrillary acidic protein (GFAP) showed that upregulation of CNTFRα occurred in reactive, GFAP-immunopositive astrocytes of the OML. A substantial reduction of CNTFRα expression in the deafferented granule cells was transiently observed at 7 and 10 d postlesion. Our results suggest a paracrine or autocrine function of CNTF in the regulation of astrocytic and neuronal responses after brain injury.

Sprouting in the hippocampus is layer-specific.

Partial removal of layer-specific afferents of the hippocampus is said to induce sprouting of intact fibers from neighboring layers that invade the zone of the degenerating axons. However, recent in vivo and in vitro studies using sensitive anterograde tracers have failed to demonstrate sprouting across laminar boundaries. Sprouting does occur; but, it mainly involves unlesioned fiber systems terminating within the layer of fiber degeneration in addition to the degenerating afferents. These findings point to rigid laminar cues attracting certain fiber systems while repelling others in normal development and after partial deafferentation.

The chondroitin sulphate proteoglycan brevican is upregulated by astrocytes after entorhinal cortex lesions in adult rats

The chondroitin sulphate proteoglycan brevican is one of the most abundant extracellular matrix molecules in the adult rat brain. It is primarily synthesized by astrocytes and is believed to influence astroglial motility during development and under certain pathological conditions. In order to study a potential role of brevican in the glial reaction after brain injury, its expression was analysed following entorhinal cortex lesion in rats (12u2003h, 1, 2, 4, 10, 14 and 28u2003days and 6u2003months post lesion). In situ hybridization and immunohistochemistry were employed to study brevican mRNA and protein, respectively, in the denervated outer molecular layer of the fascia dentata and at the lesion site. In both regions brevican mRNA was upregulated between 1 and 4u2003days post lesion. The combination of in situ hybridization with immunohistochemistry for glial fibrillary acidic protein demonstrated that many brevican mRNA‐expressing cells are astrocytes. In the denervated zone of the fascia dentata, immunostaining for brevican was increased by 4u2003days, reached a maximum by 4u2003weeks and remained detectable up to 6u2003months post lesion. Electron microscopic immunocytochemistry showed that brevican is a component of the extracellular matrix compartment. At the lesion site a similar time course of brevican upregulation was observed. These data demonstrate that brevican is upregulated in areas of brain damage as well as in areas denervated by a lesion. They suggest a role of brevican in reactive gliosis and are compatible with the hypothesis that brevican is involved in the synaptic reorganization of denervated brain areas.

Actin-associated protein synaptopodin in the rat hippocampal formation: localization in the spine neck and close association with the spine apparatus of principal neurons.

Dendritic spines are sites of synaptic plasticity in the brain and are capable of remodeling their shape and size. However, little is known about the cellular mechanisms that regulate spine morphology and motility. Synaptopodin is a recently described actin‐associated protein found in renal podocytes and dendritic spines (Mundel et al. J Cell Biol. [1997] 139:193–204), which is believed to play a role in spine plasticity. The presentstudy analyzed the distribution of synaptopodin in the hippocampal formation. In situ hybridization histochemistry revealed a high constitutive expression of synaptopodin mRNA in the principal cell layers. Light microscopic immunohistochemistry showed that the protein is distributed throughout the hippocampal formation in a region‐ and lamina‐specific manner. Postembedding immunogold histochemistry demonstrated that synaptopodin is exclusively present in dendrites and spines, specifically in the spine neck in close association with the spine apparatus. Spines lacking a spine apparatus are not immunoreactive for synaptopodin. These data suggest that synaptopodin links the spine apparatus to actin and may thus be involved in the actin‐based plasticity of spines. J. Comp. Neurol. 418:164–181, 2000.

Up-regulation of astrocyte-derived tenascin-C correlates with neurite outgrowth in the rat dentate gyrus after unilateral entorhinal cortex lesion

The extracellular matrix protein tenascin-C has been implicated in the regulation of axonal growth. Using unilateral entorhinal cortex lesions, which induce a massive sprouting response in the denervated outer molecular layer of the rat fascia dentata, the role of tenascin-C for axonal growth was investigated in vivo. Monoclonal antibodies against the neurite outgrowth and anti-adhesive domains of the molecule were employed. Immunostaining was increased throughout the denervated outer molecular layer by day 2, reached a maximum around day 10, and was back to control levels by four weeks post lesion. Growth cone deflecting as well as neurite outgrowth promoting isoforms of tenascin-C were up-regulated after the lesion. Using electron microscopy, single intensely tenascin-C immunoreactive cells were identified as reactive astrocytes that phagocytose degenerated terminals. In situ hybridization histochemistry for tenascin-C messenger RNA revealed numerous cellular profiles in the denervated outer molecular layer of the ipsilateral and contralateral dentate gyrus two days post lesion. Tenascin-C messenger RNA-positive cells in the outer molecular layer were identified as astrocytes using double-labelling for tenascin-C messenger RNA and glial fibrillary acidic protein immunohistochemistry. Thus, a tenascin-C-rich substrate is present in the outer molecular layer during the time of sprouting and a sharp boundary is formed against the inner molecular layer. This pattern may contribute to the layer-specific sprouting response of surviving afferents after entorhinal lesion. Neurite outgrowth may be promoted within the denervated zone, whereas axons trying to grow into the denervated outer molecular layer, for example from the inner molecular layer, would be deflected by a tenascin-C-rich barrier.

Dentate granule cells in reeler mutants and VLDLR and ApoER2 knockout mice.

We have studied the organization and cellular differentiation of dentate granule cells and their axons, the mossy fibers, in reeler mutant mice lacking reelin and in mutants lacking the reelin receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). We show that granule cells in reeler mice do not form a densely packed granular layer, but are loosely distributed throughout the hilar region. Immunolabeling for calbindin and calretinin revealed that the sharp border between dentate granule cells and hilar mossy cells is completely lost in reeler mice. ApoER2/VLDLR double-knockout mice copy the reeler phenotype. Mice deficient only in VLDLR showed minor alterations of dentate organization; migration defects were more prominent in ApoER2 knockout mice. Tracing of the mossy fibers with Phaseolus vulgaris leukoagglutinin and calbindin immunolabeling revealed an irregular broad projection in reeler mice and ApoER2/VLDLR double knockouts, likely caused by the irregular wide distribution of granule cell somata. Mutants lacking only one of the lipoprotein receptors showed only minor changes in the mossy fiber projection. In all mutants, mossy fibers respected the CA3-CA1 border. Retrograde labeling with DiI showed that malpositioned granule cells also projected as normal to the CA3 region. These results indicate that ( 1 ) reelin signaling via ApoER2 and VLDLR is required for the normal positioning of dentate granule cells and (2) the reelin signaling pathway is not involved in pathfinding and target recognition of granule cell axons.

Developmental distribution of a reeler gene-related antigen in the rat hippocampal formation visualized by CR-50 immunocytochemistry.

During histogenesis of the neocortex, Cajal Retzius cells in the marginal zone express the glycoprotein reelin which is developmentally regulated and involved in the formation of the inside out mode of cortical layering. Cajal Retzius cells are also present in the developing hippocampus. There, inhibition of reelin by blocking with CR-50, an antibody which recognizes the N-terminus of this protein, leads to abnormal development of layer-specific connections. Here we report the developmental distribution pattern of reelin expressing neurons in the rat hippocampal formation using CR-50 immunocytochemistry. Labelled Cajal Retzius cells were located near the hippocampal fissure in neonate rats. Many of these cells were still present in the adult. From postnatal day 4 on, neurons in other layers were stained with the CR-50 antibody. In adult rats immunopositive neurons were found in all hippocampal subfields and in the entorhinal cortex. These observations indicate that in the rat hippocampal formation reelin is expressed in different neuronal types during development and in adulthood. Moreover, Cajal Retzius cells in the marginal zone near the hippocampal fissure are still found in adult animals.

Different primary target cells are important for fiber lamination in the fascia dentata: a lesson from reeler mutant mice.

The factors determining the lamina-specific termination of entorhinal and commissural afferents to the fascia dentata are poorly understood. Recently it was shown that early generated Cajal-Retzius (CR) cells in the outer molecular layer and reelin, synthesized by CR cells, play a role in the lamina-specific termination of entorhinal fibers which form transient synapses with CR cells before establishing their definite contacts with granule cell dendrites (J. A. del Rio et al., 1997, Nature 385, 70-74). By using anterograde tracing with Phaseolus vulgaris leukoagglutinin we show that the normal, sharply delineated entorhinal projection to the outer molecular layer is retained in reeler mutant mice lacking reelin. This coincides with the regular presence of CR cells, the primary, transient target cells of entorhinal fibers. In contrast, the commissural fibers were found to terminate in an abnormal broad, not clearly defined area. This widespread projection coincides with the distribution of granule cells which in the mutant do not form a dense cell layer but are scattered all over the hilus due to a migration defect. Unlike the entorhinal fibers, the commissural fibers arrive in their target layer late in development, when granule cell dendrites are already there. We hypothesize from these results that the presence of the adequate postsynaptic element at the time of fiber ingrowth, CR cells for the early ingrowing entorhinal fibers and granule cells for the late-arriving commissural fibers, is crucial for the normal formation of these layer-specific projections.

Sprouting of crossed entorhinodentate fibers after a unilateral entorhinal lesion: Anterograde tracing of fiber reorganization with Phaseolus vulgaris-leucoagglutinin (PHAL)

Fibers from the contralateral entorhinal cortex (EC) to the dentate gyrus partially replace the input lost after an ipsilateral EC lesion. To study the morphology and course of single sprouted crossed entorhinodentate fibers, the anterograde tracer Phaseolus vulgaris‐leucoagglutinin (PHAL) was used. Rats that survived for 4 to 8 weeks after a unilateral entorhinal lesion received PHAL deposits into the entorhinal cortex contralateral to the lesion. Control animals received a similar PHAL deposit. Single PHAL‐labeled fibers in the molecular layer of the contralateral (EC lesion) fascia dentata were drawn with a camera lucida, and an axon‐branching index (branch points/100 μm axon length) was calculated for these crossed entorhinodentate fibers in controls and operated animals.