Gerhard Hager

Astrocytes in the hippocampus of patients with temporal lobe epilepsy display changes in potassium conductances

Functional properties of astrocytes were investigated with the patch‐clamp technique in acute hippocampal brain slices obtained from surgical specimens of patients suffering from pharmaco‐resistant temporal lobe epilepsy (TLE). In patients with significant neuronal cell loss, i.e. Ammons horn sclerosis, the glial current patterns resembled properties characteristic of immature astrocytes in the murine or rat hippocampus. Depolarizing voltage steps activated delayed rectifier and transient K+ currents as well as tetrodotoxin‐sensitive Na+ currents in all astrocytes analysed in the sclerotic human tissue. Hyperpolarizing voltages elicited inward rectifier currents that inactivated at membrane potentials negative to ‐130 mV. Comparative recordings were performed in astrocytes from patients with lesion‐associated TLE that lacked significant histopathological hippocampal alterations. These cells displayed stronger inward rectification. To obtain a quantitative measure, current densities were calculated and the ratio of inward to outward K+ conductances was determined. Both values were significantly smaller in astrocytes from the sclerotic group compared with lesion‐associated TLE.

Neuronal MCP-1 Expression in Response to Remote Nerve Injury

Direct injury of the brain is followed by inflammatory responses regulated by cytokines and chemoattractants secreted from resident glia and invading cells of the peripheral immune system. In contrast, after remote lesion of the central nervous system, exemplified here by peripheral transection or crush of the facial and hypoglossal nerve, the locally observed inflammatory activation is most likely triggered by the damaged cells themselves, that is, the injured neurons. The authors investigated the expression of the chemoattractants monocyte chemoattractant protein MCP-1, regulation on activation normal T-cell expressed and secreted (RANTES), and interferon-gamma inducible protein IP10 after peripheral nerve lesion of the facial and hypoglossal nuclei. In situ hybridization and immunohistochemistry revealed an induction of neuronal MCP-1 expression within 6 hours postoperation, reaching a peak at 3 days and remaining up-regulated for up to 6 weeks. MCP-1 expression was almost exclusively confined to neurons but was also present on a few scattered glial cells. The authors found no alterations in the level of expression and cellular distribution of RANTES or IP10, which were both confined to neurons. Protein expression of the MCP-1 receptor CCR2 did not change. MCP-1, expressed by astrocytes and activated microglia, has been shown to be crucial for monocytic, or T-cell chemoattraction, or both. Accordingly, expression of MCP-1 by neurons and its corresponding receptor in microglia suggests that this chemokine is involved in neuron and microglia interaction.

Lesion-induced changes of electrophysiological properties in astrocytes of the rat dentate gyrus

Reorganization of the adult dentate gyrus following unilateral entorhinal cortex lesion (ECL) is a well‐established model for studying mechanisms of trauma‐induced neuronal plasticity. The lesion induces deafferentiation of the outer molecular layer, which is accompanied by a strong astroglial reaction. This glial response is thought to contribute to subsequent repair processes, but the underlying mechanisms are poorly understood. In this study we addressed the question whether denervation leads to modifications in the electrophysiological properties of astrocytes, assuming that such changes might be involved in the remodeling of neural circuitry. Patch‐clamp recordings were obtained from astrocytes in the dentate gyrus of adult rats that underwent ECL and compared to corresponding data from control animals. We observed a significant reduction of inward rectifier K+ current densities, a positive shift of resting potentials, and an increase in input resistance in astrocytes of the denervated molecular layer. Current densities were reduced between 6 and 19 days postlesion (dpl), reaching a minimum at 10 dpl. Voltage‐gated outward K+ currents were not affected by the lesion. Inward rectifier K+ currents increase with maturation in astrocytes. Thus, our results provide evidence that, following ECL, mature astrocytes dedifferentiated and readapted an immature current pattern. Presumably, these changes lead to stronger and prolonged depolarization of glial cells and neurons in response to activity‐dependent K+ release, which in turn might enhance the synthesis of neurotrophic factors and contribute to a permissive environment for neuronal reorganization. GLIA 28:166–174, 1999.

Neuregulin-1 isoforms are differentially expressed in the intact and regenerating adult rat nervous system.

Our knowledge on Neuregulin-1 (Nrg-1) during development of the nervous system is increasing rapidly, but little is known about Nrg-1-ErbB signaling in the adult brain. Nrg-1 is involved in determination, proliferation, differentiation, and migration of neurons and glial cells in the developing brain. In the peripheral nervous system, Nrg-1 signaling is required for Schwann cell differentiation and myelination, and establishment of neuromuscular junctions (NMJs). Multiple alternative splicing of Nrg-1 was shown, but correlation of its structural and functional diversity was rarely addressed. Therefore, we investigated the expression of Nrg-1 isoforms in the rat brain and brain-derived cell types, and their involvement in regeneration of the adult brain, using immunohistochemistry, in situ hybridization, and semiquantitative RT-PCR. We found expression of at least 12 distinct Nrg-1 isoforms in the brain and altered expression of several isoforms in the facial motor nucleus after peripheral transection of the seventh cranial nerve. An upregulation of Nrg-1 type-I mRNA, probably type-I-α, was observed in reactive astrocytes of the facial nucleus 1 d postaxotomy. Nrg-1 type-III and the splice variants β1 and β5 are dramatically downregulated in axotomized motoneurons, which lack contact to their target tissue. Baseline expression levels were reestablished when the first axons reached the facial muscles and reformed NMJs. Nrg-1-β1 and -β5 might act in maintenance of NMJs. The splice variants β2 and β4 display an initial downregulation of mRNA levels, followed by an increase during the period of axon remyelination. Thus, Nrg-1-β2 and -β4 might be involved in myelination.

Cellular activation in neuroregeneration.

Publisher Summary A peripheral nerve lesion leads to a characteristic retrograde reaction with substantial changes in morphology, metabolism, and gene expression of the injured neurons and the surrounding glial cells. The process causing the induction of axonal regrowth and re-establishment of the contact to the target is gradually becoming understood. Stepwise changes seem to lead to a transition from the “normal” physiological status to the induction of sets of genes necessary for successful re-innervation in the involved cell types: neurons, astrocytes and microglial cells. This molecular activation is cell-specific and selective. In addition, reaction to injury involves cellular interaction resulting in the modification of intracellular signaling cascades. During the regeneration process the neuron recapitulates parts of the embryonic program associated with axonal outgrowth. Hence, re-activation of regulatory mechanisms controlling neuronal differentiation and axonal outgrowth during development has been observed. The astrocytic metabolism is shifted to support survival and regeneration of the neuron. This includes the biosynthesis of growth factors and cytokines, as well as the remodeling of the extracellular matrix. The microglial cell, as the intrinsic immune cell of the brain, exhibits its activation program as an unspecific repertoire to defend brain injury.

Direct observation of neurotoxicity in brain slices with infrared videomicroscopy

We employed the novel technique of infrared videomicroscopy to study the morphological changes induced by the neurotoxicity of high concentrations of L-glutamate and by anoxia. The infrared videomicroscopy system described uses an inverted microscope and employs a combination of infrared illumination, differential interference contrast (DIC) and contrast enhancement by video. With this system, we were able to observe swelling of neurons 50 microns deep in rat neocortical slices after bath application of glutamatergic agonists or during anoxia. By recording in time lapse mode it was possible to visualize the dynamics of cell swelling and to demonstrate neuroprotection by glutamatergic antagonists. The method may be of use in screening of potential neuroprotective drugs for stroke therapy.

c-Jun regulation in rat neonatal motoneurons postaxotomy.

Motoneurons respond to peripheral nerve transection by either regenerative or degenerative events depending on their state of maturation. Since the expression of c‐Jun has been involved in the early signalling of the regenerative process that follows nerve transection in adults, we have investigated c‐Jun on rat neonatal axotomized motoneurons during the period in which neuronal death is induced. Changes in levels of c‐Jun protein and its mRNA were determined by means of quantitative immunocytochemistry and in situ hybridization. Three hours after nerve transection performed on postnatal day (P)3, c‐Jun protein and mRNA is induced in axotomized spinal cord motoneurons, and high levels were reached between 1 and 10 days after. This response is associated with a detectable c‐Jun activation by phosphorylation on serine 63. No changes were found in the levels of activating transcription factor ‐2. Most of dying motoneurons were not labelled by either a specific c‐Jun antibody or a c‐jun mRNA probe. However, dying motoneurons were specifically stained by a polyclonal anti c‐Jun antibody, indicating that some c‐Jun antibodies react with unknown epitopes, probably distinct from c‐Jun p39, that are specifically associated with apoptosis. J. Neurosci. Res. 63:469–479, 2001.

A peptide derived from a neurite outgrowth-promoting domain on the γ1 chain of laminin modulates the electrical properties of neocortical neurons

Laminins form a family of large multidomain glycoproteins of the extracellular matrix. The cellular distribution of laminin immunoreactivity in the adult mammalian central nervous system suggests an important role for laminins in mature brain function in addition to their role during brain development. To characterize the effects of this group of extracellular matrix molecules on mature brain function, intracellular recording techniques were applied to in vitro slice preparations of the rat neocortex. The experiments show that a peptide homologous to the C-terminal part of the gamma 1 chain of laminin modulates the electrical activity of pyramidal neurons in the adult neocortex of the rat. The peptide is part of the neurite outgrowth-promoting domain of the gamma 1 chain on the E8 fragment of laminin and it displays the neurite outgrowth-promoting activity of the native laminin molecule. Perfusion of in vitro brain slices with the peptide increased the input resistance of the neuronal membrane. In addition, a rise in inward rectification could be observed. These events were accompanied by a strong increase in direct excitability of the treated neurons. Immunohistochemistry techniques were applied to sections of the adult rat neocortex and hippocampus to demonstrate the presence of both the neurite outgrowth-promoting domain and the native laminin in the adult brain. An antiserum raised against the neurite outgrowth-promoting domain on the gamma 1 chain of laminin, which also recognized the free synthetic peptide, showed immunoreactivity on neurons. In addition, a population of glial fibrillary acidic protein-positive astrocytes in the hippocampus displayed immunoreactivity for this antibody. These results were confirmed by using several antibodies directed against the whole laminin-1 molecule. Neurons in the neocortex and hippocampus, as well as astrocytes in the hippocampus, demonstrated immunoreactivity for antibodies directed against the whole laminin-1 molecule. The results suggest that laminins containing the gamma 1 chain have the potential to modulate neuronal activity. This effect may be mediated either by direct cell-cell contact from surrounding cells, or through the neuronal expression of laminin or laminin-like molecules which are inserted into the neuronal cell membrane.

Suppression of nidogen-1 translation by antisense targeting affects the adhesive properties of cultured astrocytes

The multidomain glycoprotein nidogen‐1 is a common component of basal membranes. Nidogen‐1 is produced by the endothelial cells and the mesenchymal cells of the developing central nervous system. Recent results give evidence that nidogen‐1 may also be secreted by cultured Schwann cells to basement membranes of peripheral nerves. We were interested in ascertaining whether astrocytes, which have the capacity to produce laminin and fibronectin and are an important source of extracellular matrix (ECM) molecule secretion in the brain, might also produce nidogen‐1. Immunocytochemistry, in combination with polymerase chain reaction and in situ hybridization techniques, revealed that astrocytes in culture synthesize nidogen‐1. To show the functional significance of the nidogen‐1 secretion by astrocytes, antisense targeting techniques were applied. These experiments showed that nidogen‐1 may be an essential modulator of astrocytic adhesion to the substrate. The suppression of nidogen‐1 synthesis by the application of antisense oligonucleotides induced a morphological transition from a flat, polygonal to a round cell and was accompanied by the detachment of the astrocytes from the substrate. Hence, nidogen‐1 might be an important component of the ECM secreted by astrocytes. The suppression of nidogen‐1 synthesis may disturb the aggregation of ECM molecules to a functional basement membrane and thus reduce the astrocytic adhesion to the substrate. Nidogen‐1 secretion to basement membranes by astrocytes may have important functional implications during blood–brain barrier and scar formation. GLIA 28:138–149, 1999.

Semiquantitative analysis of low levels of mRNA expression from small amounts of brain tissue by nonradioactive reverse transcriptase-polymerase chain reaction

We have developed an easy and fast method to semiquantify low levels of mRNA from small amounts of brain tissues based on nonradioactive reverse transcription-polymerase chain reaction (RT-PCR). The regulation of mRNA for the growth associated protein GAP-43/B-50 and the homeodomain protein islet-1 was examined in the facial nucleus of the rat after a unilateral transection of the nerve. In both cases a similar sensitivity for radioactive and nonradioactive RT-PCR methods was found. The expression of the housekeeping gene, cyclophilin A was used to normalize total mRNA amounts and PCR conditions. After amplification the PCR products were separated electrophoretically on polyacrylamide gels. For nonradioactive semiquantification gels were stained with ethidium bromide and recorded using a CCD camera and transillumination. The recordings were evaluated with specialized software. Using nonradioactive RT-PCR, the increase in GAP-43/B-50 mRNA in response to axotomy was easily detectable in the small volume of tissue obtained from the facial nucleus. In contrast, the low expression of islet-1 mRNA made it necessary to develop a two-step amplification procedure in order to provide a reliable semiquantitative analysis. The procedure included preamplification of the cDNA and subsequent purification of the cDNA. Using this method, the down-regulation of islet-1 could be demonstrated with a similar sensitivity to that previously shown with radioactive RT-PCR.