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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.

The discovery that missense mutations in the alpha-synuclein gene represent a rare genetic cause of Parkinson's disease (PD) has had significant impact on the development of research into neurodegenerative disorders. It is becoming increasingly clear that alpha-synuclein plays a central role in the pathological process, which causes Lewy body formation and neurodegeneration in PD. Importantly, there is evidence to suggest that mutated alpha-synuclein is toxic to both nerve cells and glia. However, the regulation and function of wild-type alpha-synuclein are as yet ill defined. Using the facial nerve axotomy model, we have addressed the question whether the expression of alpha-synuclein in nerve cells may change in response to injury. We were particularly interested in testing the hypothesis that the severity of neuronal injury had an effect on alpha-synuclein metabolism. Facial nerve cut and crush, respectively, were performed in adult rats where normal facial motoneurones do not express alpha-synuclein. Following axotomy, a subset of facial motoneurones newly expressed high levels of alpha-synuclein immunoreactivity in their cell body and, occasionally, their nucleus. Significantly more nerve cells were labelled following facial nerve transection than following facial nerve crush. Confocal microscopy revealed a granular pattern of alpha-synuclein aggregation in degenerating nerve cells. Interestingly, the observed cell death phenotype was clearly non-apoptotic and developed over days or weeks rather than hours. Thus, axotomy of adult rat facial motoneurones triggers de novo expression of alpha-synuclein and this expression is associated with a non-apoptotic, slow form a neurodegeneration. In addition, the extent of alpha-synuclein expression is related to the severity of neuronal injury.

Microglial motility was studied in living mammalian brain tissue using infrared gradient contrast microscopy in combination with video contrast enhancement and time lapse video recording. The infrared gradient contrast allows the visualization of living cells up to a depth of 60 microm in brain slices, in regions where cell bodies remain largely uninjured by the tissue preparation and are visible in their natural environment. In contrast to other techniques, including confocal microscopy, this procedure does not require any staining or labeling of cell membranes and thus guarantees the investigation of tissue which has not been altered, apart from during preparation. Microglial cells are activated and increase in number in the facial nucleus following peripheral axotomy. Thus we established the preparation of longitudinal rat brainstem slices containing the axotomized facial nucleus as a source of activated microglial cells. During prolonged video time lapse recordings, two different types of microglial cell motility could be observed. Microglial cells which had accumulated at the surface of the slice remained stationary but showed activity of the cell soma, developing pseudopods of different shape and size which undulated and which were used for phagocytosis of cell debris. Microglial phagocytosis of bacteria could be documented for the first time in situ. In contrast, ameboid microglia which did not display pseudopods but showed migratory capacity, could be observed exclusively in the depth of the tissue. Some of these cells maintained a close contact to neurons and appeared to move along their dendrites, a finding that may be relevant to the role of microglia in "synaptic stripping", the displacement of synapses following axotomy. This approach provides a valuable opportunity to investigate the interactions between activated microglial cells and the surrounding cellular and extracellular structures in the absence of staining or labeling, thus opening a wide field for the analysis of the cellular mechanisms involved in numerous pathologies of the CNS.

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a widely expressed glycoprotein, which plays an important role in leukocyte extravasation and in the interaction of lymphocytes with antigen-presenting cells. In the current study we examined the regulation of ICAM-1 in the mouse facial motor nucleus after facial nerve transection, using immunohistochemistry, confocal laser microscopy and electron microscopy. In the normal facial nucleus ICAM-1 immunoreactivity was restricted to vascular endothelium. Transection of the facial nerve led to a strong and selective upregulation of ICAM-1 on activated microglia. Quantitation of microglial ICAM-1 immunoreactivity revealed a biphasic increase. The first peak 1-2 days post operation paralleling the early stage of microglial activation was followed by a decline at 4-7 days. The second induction of ICAM-1 occurred at day 14 accompanying the period of neuronal cell death and microglial phagocytosis of neuronal debris. Immunoelectron microscopy showed strong ICAM-1 reactivity on the cell membrane of activated microglia at day 2. During the second peak (day 14), ICAM-1 was also observed on lymphocytes adhering to phagocytotic microglia forming aggregates around neuronal debris. No immunolabelling was observed on neurons, astrocytes or oligodendroglia. These data suggest the involvement of ICAM-1 in the adhesion of activated microglia, in their phagocytosis of neuronal debris, and also in the interaction with infiltrating lymphocytes following this injury.

Brain lesions, even of the most subtle type, are accompanied by the activation of microglia, the main immune cells of the brain. Microglial cells dramatically increase in number through proliferation and adhere to the injured neurons, where they displace the synaptic input. After proliferation, microglia gradually migrate into the nearby parenchyma and appear to decrease in number. Here we examined the possible involvement of apoptosis in the regulation of the microglial cell number using Terminal transferase mediated d-UTP Nick End-Labelling (TUNEL). In vitro, cell death is a common phenomenon in microglial cell cultures, and is enhanced by the withdrawal of the mitogen, granulocyte-macrophage colony stimulating factor. In vivo, application of the TUNEL-reaction revealed TUNEL-positive microglia beginning at day 4, with a peak 7 days after transection of the facial nerve. Surprisingly, TUNEL-labelling in vivo was localized on the outer side of the nuclear membrane and in the microglial cytoplasm, with very little staining within the nucleus itself. These TUNEL-labelled cells also lacked other classic morphological signs of apoptosis, like membrane blebbing, chromatin condensation and apoptotic bodies. These data suggest that the regulation of post-mitotic microglia is not mediated by the classic pathway of apoptosis.

The isoquinoline PK11195 has been suggested as a marker of glial pathology in the lesioned brain. The aim of the present study is to clarify the precise cellular location of its binding site in the central nervous system. Here, we report that in the facial nucleus after facial nerve axotomy-a lesion causing a retrograde neuronal reaction without nerve cell death while keeping the blood-brain barrier intact--activated microglia are the predominant source of lesion-induced increases of PK11195 binding. Likewise, increased PK11195 binding is seen in the gracile nucleus after anterograde neuronal injury following sciatic nerve transection. The peak of PK11195 binding, using the single isomer R-PK11195, was observed 4 days after the peripheral nerve lesion, consistent with the well-known time course of microglial activation. Photoemulsion microautoradiography confirmed the restriction of PK11195 binding to activated microglia. The increase of PK11195 binding in the facial nucleus seen after selective cell death of facial motoneurons by retrograde suicide transport of toxic ricin, a lesion that is accompanied by the rapid transformation of microglia into phagocytes, was no higher than that seen following axotomy. This suggests that the full transformation of microglia into parenchymal phagocytes is not necessary to reach maximal levels of PK11195 binding. PK11195, therefore, is a well-suited marker to detect microglial activation in areas of subtle brain pathology, where neither a disturbance of the blood-brain barrier function nor the presence of macrophages and inflammatory cells indicate an on-going disease process.

In the molecular layer of the mouse cerebellum, the histochemical activity of the adenosine-producing ectoenzyme 5'-nucleotidase discloses a parasagittal pattern of alternating enzyme-rich and enzyme-poor bands. In the rat, 5'-nucleotidase activity transiently labels cerebellar synapses during postnatal development and shifts later on towards an exclusive glial location in the molecular layer. We therefore asked whether different ultrastructural expression of 5'-nucleotidase would account for the light microscopic pattern seen in the adult mouse cerebellum. Using an enzyme cytochemical method, we localized 5'-nucleotidase activity on the glial cells and at the main types of asymmetrical synapses in the developing and mature cerebellum of the mouse. The percentage of labelled synapses increased until adulthood within the 5'-nucleotidase-positive bands. Here, the vast majority (86%) of the synapses were labelled against only 27% within the negative bands in the adult. Thus, 5'-nucleotidase appears as a marker of glia and of Purkinje cell synapses across cerebellar compartments. Changes in purinergic neuromodulation and/or cell adhesion mediated by 5'-nucleotidase across bands might participate in the functional differentiation of the cerebellar parasagittal subsets.

The distribution of the neural-specific growth associated protein B-50 (GAP-43), which persists in the mature spinal cord and dorsal root ganglia, has been studied by light and electron microscopic immunohistochemistry in the cat. Throughout the spinal cord, B-50 immunoreactivity was seen confined to the neuropil, whereas neuronal cell bodies were unreactive. The most conspicuous immunostaining was observed in the dorsal horn, where it gradually decreased from superficial laminae (I-II) toward more ventral laminae (III-V), and in the central portion of the intermediate gray (mainly lamina X). In these regions, the labelling was localized within unmyelinated, small diameter nerve fibres and axon terminals. In the rest of the intermediate zone (laminae VI-VIII), B-50 immunoreactivity was virtually absent. The intermediolateral nucleus in the thoracic and cranial lumbar cord showed a circumscribed intense B-50 immunoreactivity brought about by the labelling of many axon terminals on preganglionic sympathetic neurons. In motor nuclei of the ventral horn (lamina IX), low levels of B-50 immunoreactivity were present in a few axon terminals on dendritic and somal profiles of motoneurons. In dorsal root ganglia, B-50 immunoreactivity was mainly localized in the cell bodies of small and medium-sized sensory neurons. The selective distribution of persisting B-50 immunoreactivity in the mature cat throughout sensory, motor, and autonomic areas of the spinal cord and in dorsal root ganglia suggests that B-50-positive systems retain in adult life the capacity for structural and functional plasticity.

Resident endoneurial macrophages form a prominent, but little recognized component of the PNS. We have studied immunocytochemically the distribution, morphology and immunophenotype of endoneurial macrophages in several normal peripheral nerves of the rat. In addition, we investigated the macrophage response following crush injury of the sciatic nerve. Resident endoneurial macrophages had a ramified morphology with processes oriented parallel to the long axis of nerve fibres. They were positive for several monocyte/macrophage markers such as ED1, ED2 and the recently-described MUC 101 and MUC 102 antibodies. They furthermore expressed the complement type three receptor, the CD4 antigen and MHC class I and II molecules. These results were consistent in all the peripheral nerves studied. In addition, 1000 rad of gamma-irradiation led to a strong reduction in the number of MHC class II-positive ramified cells in the peripheral nerves similar to that observed in other peripheral organs such as the heart. A considerable percentage of resident macrophages in the PNS and/or their precursor cells are therefore radiosensitive and could be related to the lineage of dendritic cells. Following crush injury, ED1-3-, OX-42-, MUC 101- and MUC 102-positive round macrophages were observed from 24 h postlesion onward at the site of trauma. In the distal part, they were observed to form strings of round, foamy macrophages probably involved in myelin phagocytosis. In contrast, the number of MHC class II-positive resident macrophages was only slightly increased at the site of trauma and in the distal part. These cells transformed from a ramified to a round morphology, but did not appear as typical strings of foamy macrophages. These results demonstrate that the PNS is provided with a resident macrophage population analogous in many respects to microglial cells in the CNS. These constitutively MHC class II-positive PNS microglial-like cells could act as the major antigen-presenting cells in the peripheral nerve. They may thus constitute a local immune defense system of the PNS with a function similar to that of microglial cells in the CNS.

Laminin, the major non-collagenous protein of basement membranes, has been shown to be a potent stimulator of neurite outgrowth, to potentiate neuronal survival and to stimulate Schwann cell division in vitro. The aim of the present study was to determine the ultrastructural localization of laminin in the mouse sciatic nerve during development and regeneration in order to elucidate whether laminin might also evoke similar effects in vivo. For this purpose polyclonal antibodies against laminin were used for pre-embedding electron microscopic immunocytochemistry of tissue sections from mouse sciatic nerves. In the adult, although laminin immunoreactivity was found to be predominantly associated with basement membranes as expected, the surface membranes of Schwann cells also displayed weak labelling. This distribution pattern was similar in developing sciatic nerves with the exception that laminin immunoreactivity was generally higher and also found to be present on interstitial collagen fibres. One week following sciatic nerve transection, strong laminin immunoreactivity was seen on regenerating axons growing along laminin-positive basement membrane tubes in the distal stump of the transected nerve. Our results demonstrate that laminin immunoreactivity is not restricted to basement membranes of the mouse sciatic nerve, but is also found in direct contact with adult, developing and regenerating axons as well as on the surface of Schwann cells. The finding of laminin immunoreactivity on extracellular matrix components, axons and Schwann cell membranes under conditions of growth and regeneration makes it more likely that axons are able to interact with laminin not only in vitro but also in vivo.

Concomitant with the proliferation of satellite microglial cells occurring in the process of motor neuron regeneration, an astrocytic hypertrophy is also seen. A remarkable increase of glial fibrillary acidic protein (GFAP) immunoreactivity is demonstrated in astrocytes of the facial nucleus within a few days following nerve transection. The increase of GFAP antigenicity is associated with an increased appearance of glial filaments and astrocytic processes. We suggest that resident protoplasmic astrocytes become involved in retrograde changes in facial motor neurons and transform into reactive astrocytes. They are of the fibrous type and highly positive for GFAP.

We have studied beta-nerve growth factor (beta-NGF) receptor expression in the injured and regenerating chick PNS using [125I]-iodinated beta-NGF as a radioactive probe to map and quantitate autoradiographically the in situ distribution of specific [125I] beta-NGF binding. Two different mechanisms are involved in the reappearance of specific [125I] beta-NGF binding on the normally unlabelled adult peripheral nerves. The anterograde and retrograde axonal transport of beta-NGF binding sites leads to a rapid but transient accumulation of [125I] beta-NGF binding on both sides of crushed or transected sciatic and brachial nerves. There is a dramatic decrease in the axonal transport of beta-NGF binding sites, starting 1 day after nerve injury (1 DPO) and reaching basal levels of 10-20% of the control values at 3 to 10 DPO. Gradual but complete recovery of this axonal transport was noted in the sciatic neurites allowed to regain contact with their peripheral targets. A very different regulation pattern was observed for the local reappearance of specific [125I] beta-NGF binding on the endoneurial Schwann cells throughout the distal part of the axotomized nerve. It was first observed at 4 DPO, becoming maximal at 6 DPO. Reinnervation of the nerve after crush led to a rapid decrease of this specific [125I] beta-NGF binding, which followed a proximo-distal temporal gradient. These results show that axotomy leads to a drastic decrease in the axonal expression of [125I] beta-NGF binding, while causing its appearance on the Schwann cells of the denervated endoneurium. They suggest that these endoneurial cells may become the primary target for beta-NGF following axotomy and during regeneration.

The possible cellular mechanism of action of systemically administered monosodium-L-glutamate and the projections of glutamate-sensitive area postrema neurons have been studied in rats. Parenteral administration of monosodium-L-glutamate induced a selective degeneration of a particular population of AChE-containing area postrema neurons. Electron microscopic cytochemistry and X-ray microanalysis revealed the presence of calcium-containing electron-dense deposits in the mitochondria of degenerating area postrema neurons indicating the possible pathogenetic role of an enhanced intracellular calcium level in the mechanism of monosodium-L-glutamate-induced nerve cell degeneration. Degeneration of area postrema neurons was followed by the appearance of degenerating axon terminals in a well-defined region of the nucleus of the solitary tract, the area subpostrema. Degenerating area postrema neurons and axon terminals were rapidly engulfed by phagocytes predominantly of microglial character. AChE activity, localized to the basal lamina of the capillaries of the area subpostrema under normal conditions, could no longer be detected in rats treated with monosodium-L-glutamate 3-4 weeks previously. These findings provide evidence for the existence of a particular population of glutamate-sensitive, AChE-containing area postrema neurons which project and transport AChE to the nucleus of the solitary tract. This specific neuronal pathway connecting the area postrema with the nucleus of the solitary tract may play an important role in some of the functions attributed to the area postrema. The results also strengthen the hypothesis that brain capillary AChE activity may be of neuronal origin.

In response to concanavalin A (Con A), neuroblastoma cells undergo marked morphological changes which involve the retraction of neurites and the induction of broad and extensive lamellar regions around the cell periphery. From the use of FITC-Con A it was shown that the membrane formed on the induced lamellar regions lacked receptors to Con A from the onset of lamella formation. These receptors were confined to the cell body; they initially showed a uniform distribution and were subsequently collected into patches and finally into aggregates or caps. When the aggregates occurred on the cell periphery their position coincided with areas free of lamellae. Investigations of the lamellar regions in Triton-extracted cell monolayers showed them to consist of a meshwork of actin filaments containing radiating thin filament bundles or microspikes. With increasing time in the presence of Con A there was a progressive increase in the number of radiating microspikes. Previous studies have shown the actin in these lamellar regions to be singly polarized with respect to the cell body. From the segregation of Con A receptors away from areas of actin polymerization in the lamellae it is concluded that actin is involved in some indirect way in surface receptor movement.

Conjugates of the B4 isolectin from Griffonia simplicifolia seeds and horseradish peroxidase were used as a histochemical reagent for the specific visualization of microglial cells in the rat CNS. Resident microglia bearing galactose-containing glycoconjugates were stained throughout the brainstem and cerebellum. In the first week following axotomy of the facial nerve, a profound and rapid accumulation of reactive microglia, as evidenced by increasing lectin reactivity, was seen to take place in the facial nucleus. Light microscopy of paraffin sections demonstrated binding of lectin-horseradish peroxidase conjugates to microglial cytoplasmic processes. When ultrastructural cytochemistry was performed, reaction product was found localized on microglial plasma membranes, as well as on intracytoplasmic membranes. The glial reaction to axotomy was studied further with double labelling of microglia and astrocytes by lectin histochemistry and immunostaining for glial fibrillary acidic protein, respectively. Our results demonstrated the presence of membrane-associated glycoconjugates containing terminal alpha-D-galactose residues on microglia, but not on other glial cell types. The possible nature and function of these glycoconjugates are discussed.

We have studied the expression of the platelet-derived growth factor (PDGF) receptors in the injured chick PNS using [125I]-iodinated PDGF as a radioactive probe to map autoradiographically the in situ distribution of specific [125I]PDGF binding. Crush or transection of the sciatic nerve led to a rapid and massive induction of specific [125I]PDGF binding on fibroblast-like cells of the injured endoneurium, already observed 2 h postoperatively. It is initially characterized by a symmetrical appearance both below and above the site of injury, spreading throughout the distal part of the lesioned nerve 1 to 2 days postoperatively. Comparison with distribution of specific [125I]beta-nerve growth factor (beta-NGF) binding (see preceding paper) revealed a number of important differences: unlike the specific [125I]beta-NGF binding, which rapidly disappears after reinnervation of the distal nerve, this was not observed in the case of [125I]PDGF binding. [125I]PDGF binding also correlated poorly with the extent of axonal injury. The segmental removal of the perineurium, resulting in heavy interstitial oedema without widespread axonal injury, led to a strong, local induction of [125I]PDGF binding, while causing moderate beta-NGF binding to only the few degenerating nerve fibre tubes. These results suggest the existence of different pathophysiological mechanisms that regulate the expression of PDGF and beta-NGF receptors in the lesioned and regenerating PNS.

Transection of the facial nerve causes proliferation of microglial cells in the facial nucleus. The miroglial cells can be seen in perineuronal and perivascular positions. A high activity of 5'-nucleotidase is demonstrated cytochemically in the plasma membranes of these cells. The increase of enzymatic sites for the production of adenosine during axonal reaction might be of functional significance for the regenerating motor neuron.

Localization of 5'-nucleotidase in the retina of four rodent species was investigated using histochemical and cytochemical techniques. It was revealed by light microscopy that the enzyme is present in the receptor layer, the external nuclear layer and the external plexiform layer. It was found by electro microscopy that the glial cells of the Müller type have a strong activity on their plasmalemma in the enzyme-positive layers. With the exception of weak and scattered activity on some Müller cell processes forming horizontal fibres and the honeycomb meshwork in the ganglion cell layer, none of the internal layers contain 5'-nucleotidase. The asymmetric distribution of this ectoenzyme on the surface of the glial cells is a novel finding and demonstrates a surprising heterogeneity of glial plasmalemma.

Transection of the facial nerve causes a rapid increase of glial fibrillary acidic protein in reactive astrocytes and a proliferation of local microglial cells. The latter is associated with a detachment of synaptic terminals from the regenerating motor neurons. About 3 weeks following axotomy the reactive astrocytes begin to form thin, sheet-like lamellar processes which cover virtually all neuronal surfaces. A high 5'-nucleotidase enzymic activity can be demonstrated in the plasma membrane of these thin cell processes. Subsequently, the lamellar processes become arranged in stacks which persist for several months and thus isolate the regenerating motor neurons from their afferent synaptic input. It is speculated that the process may protect the motor neurons during regeneration.

Unlike astrocytes and oligodendrocytes, microglia are extremely plastic making them the chameleon among the glial cells in the CNS. This great mutability of the microglial cell shape suggests the presence of an elaborate cytoskeleton which is demonstrated here by applying a new ultrastructural method. Electron microscopic immunocytochemistry shows the presence of vimentin at intermediate filament sites in reactive microglia stimulated by rat facial nerve axotomy. It is suggested that vimentin-expression may serve as a marker for activated states of microglia, including brain macrophages.