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. Ammon's 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. During normal development of rodent brain, astroglial inward rectification gradually increases. It thus appears reasonable to suggest that astrocytes in human sclerotic tissue return to an immature current pattern. Reduced astroglial inward rectification in conjunction with seizure-induced shrinkage of the extracellular space may lead to impaired spatial K+ buffering. This will result in stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent K+ release, and may thus contribute to seizure generation in this particular condition of human TLE.

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.

Although the spatial and temporal patterns of neuronal migration have been analyzed in great detail, little direct evidence is available as to what extracellular matrix molecules are involved. Because there is indirect evidence implicating the extracellular matrix protein laminin in neuronal migration, we investigated the effects of antibodies against a synthetic peptide derived from a neurite outgrowth domain of the B2 chain of laminin on neuronal migration in living cerebellar slices. We show by using infrared video microscopy that divalent Fab2 fragments of these antibodies inhibit granule neuronal movement in living slices of (P8) rat cerebellum. This inhibition of neuronal movement manifests itself by cessation of both radial and horizontal translocations of nuclei inside the granule neuronal processes. Fab2 fragments of antibodies against the intact (native) laminin molecule or Fab2 fragments from the preimmune serum do not affect nuclear translocation. Immunocytochemistry shows binding of the divalent Fab2 fragments of the B2 chain-specific antibodies to the Purkinje and Bergmann glial cell areas, and as punctate deposits in between the cells of the external granule cell layer. Native laminin antibodies bind to the basement membranes, and binding of the Fab2 fragments from the preimmune sera cannot be demonstrated. These results indicate that neuronal migration in the postnatal rat cerebellum in vivo involves nuclear translocation that can be inhibited by antibodies against a neurite outgrowth domain of the B2 chain of laminin. Thus, migration of cerebellar granule neurons may depend on the interaction between a neurite outgrowth domain of the B2 chain of laminin and neuronal cytoskeleton involved in nuclear movement.

Infrared video microscopy of neonatal rat cerebellum (P0-P14) was used to directly visualize migrating granule neurons in relation to other cerebellar cells in a brain slice for up to 24 hr. Initially (P0-P5), granule neurons move along radial migration pathways of other neuronal fibers. These pathways are probably established by the bipolar granule neurons that attach to the external basement membrane via one process and extend another process toward the Purkinje cell layer. At P5-P8, a substantial number of granule neurons move horizontally and extend long parallel fibers. Both radially and horizontally migrating granule neurons move by nuclear translocation inside their preformed processes with a speed that varies between 6 and 120 microns/hr. In P10-P12 animals, the horizontally oriented granule neurons start to migrate radially. They move into the internal granule cell layer either along the radial pathways of other neuronal fibers or in contact with the matured glial processes. The radial neuronal migration pathways disappear by P14 whereas the glial cell processes are maintained and reach the basal lamina. These results describe novel radial and horizontal modes of neuronal migration that proceed independently of the physical glial guidance.

In the CNS, microglia become activated, i.e. change their functional state and phenotype, in response to a wide variety of pathological stimuli. Since this activation is triggered at a very low threshold and at the same time remains territorially restricted, the spatial distribution of activated microglia can be used as a sensitive, generic measure of the anatomical localisation of ongoing disease processes. One protein complex, undetectable in resting microglia but highly up-regulated upon activation in vivo and in vitro, is the 'peripheral benzodiazepine binding site', as measured by binding of the isoquinoline derivate PK11195. Particularly numerous in the outer membrane of mitochondria, this binding site has also been referred to as the 'mitochondrial benzodiazepine receptor'. The de novo expression of this receptor by activated microglia suggests that the process of activation may be associated with important qualitative changes in the state of mitochondria. Here, we provide confocal light- and electron microscopic evidence that the activation of microglia indeed entails conspicuous mitochondrial alterations. In cultured rat microglia stained with the fluorescent probe, JC-1, a sensitive indicator of mitochondrial membrane potential, we demonstrate that stimulation by bacterial lipopolysaccharide and interferon-gamma increases the number of microglial mitochondrial profiles and leads to marked changes in their morphology. Prominent elongated, "needle-like" mitochondria are a characteristic feature of activated microglia in vitro. Electron microscopically, an abundance of abnormal profiles, including circular cristae or ring- and U-shaped membranes, are found. Our observations support the notion that the previously reported increase in microglial binding of PK11195, that labelled with carbon-11 ([11C] (R)-PK11195) has clinical use for the visualisation of activated microglia in vivo by positron emission tomography, may at least in part relate to an increased number and altered functional state of microglial mitochondria.