Microglia and synapses

  1. Cunningham C, Deacon R, Wells H, Boche D, Waters S, Diniz CP, Scott H, Rawlins JN, Perry VH (2003) Synaptic changes characterize early behavioural signs in the ME7 model of murine prion disease. Eur.J.Neurosci. 17:2147-2155
    Abstract: Prion diseases are fatal, chronic neurodegenerative diseases of mammals, characterized by amyloid deposition, astrogliosis, Microglial activation, tissue vacuolation and neuronal loss. In the ME7 model of prion disease in the C57BL/6 J mouse, we have shown previously that these animals display behavioural changes that indicate the onset of neuronal dysfunction. The current study examines the neuropathological correlates of these early behavioural changes. After injection of ME7-infected homogenate into the dorsal hippocampus, we found statistically significant impairment of burrowing, nesting and glucose consumption, and increased open field activity at 13 weeks. At this time, Microglia activation and PrPSc deposition was visible selectively throughout the limbic system, including the hippocampus, entorhinal cortex, medial and lateral septum, mamillary bodies, dorsal thalamus and, to a lesser degree, in regions of the brainstem. No increase in apoptosis or neuronal cell loss was detectable at this time, while in animals at 19 weeks postinjection there was 40% neuronal loss from CA1. There was a statistically significant reduction in synaptophysin staining in the stratum radiatum of the CA1 at 13 weeks indicating loss of presynaptic terminals. Damage to the dorsal hippocampus is known to disrupt burrowing and nesting behaviour. We have demonstrated a neuropathological correlate of an early behavioural deficit in prion disease and suggest that this should allow insights into the first steps of the neuropathogenesis of prion diseases

  2. Hansson E, Ronnback L (2003) Glial neuronal signaling in the central nervous system. FASEB J. 17:341-348
    Abstract: Glial cells are known to interact extensively with neuronal elements in the brain, influencing their activity. Astrocytes associated with synapses integrate neuronal inputs and release transmitters that modulate synaptic sensitivity. Glial cells participate in formation and rebuilding of synapses and play a prominent role in protection and repair of nervous tissue after damage. For glial cells to take an active part in plastic alterations under physiological conditions and pathological disturbances, extensive specific signaling, both within single cells and between cells, is required. In recent years, intensive research has led to our first insight into this signaling. We know there are active connections between astrocytes in the form of networks promoting Ca2+ and ATP signaling; we also know there is intense signaling between astrocytes, Microglia, oligodendrocytes, and neurons, with an array of molecules acting as signaling substances. The cells must be functionally integrated to facilitate the enormous dynamics of and capacity for reconstruction within the nervous system. In this paper, we summarize some basic data on glial neuronal signaling to provide insight into synaptic modulation and reconstruction in physiology and protection and repair after damage

  3. Li Y, Liu L, Barger SW, Griffin WS (2003) Interleukin-1 mediates pathological effects of Microglia on tau phosphorylation and on synaptophysin synthesis in cortical neurons through a p38-MAPK pathway. J.Neurosci. 23:1605-1611
    Abstract: The presence of tangles of abnormally phosphorylated tau is a characteristic of Alzheimer's disease (AD), and the loss of synapses correlates with the degree of dementia. In addition, the overexpression of interleukin-1 (IL-1) has been implicated in tangle formation in AD. As a direct test of the requirement for IL-1 in tau phosphorylation and synaptophysin expression, IL-1 actions in neuron-Microglia cocultures were manipulated. Activation of Microglia with secreted beta-amyloid precursor protein or lipopolysaccharide elevated their expression of IL-1alpha, IL-1beta, and tumor necrosis factor alpha (TNFalpha) mRNA. When such activated Microglia were placed in coculture with primary neocortical neurons, a significant increase in the phosphorylation of neuronal tau was accompanied by a decline in synaptophysin levels. Similar effects were evoked by treatment of neurons with recombinant IL-1beta. IL-1 receptor antagonist (IL-1ra) as well as anti-IL-1beta antibody attenuated the influence of activated Microglia on neuronal tau and synaptophysin, but anti-TNFalpha antibody was ineffective. Some effects of Microglial activation on neurons appear to be mediated by activation of p38 mitogen-activated protein kinase (p38-MAPK), because activated Microglia stimulated p38-MAPK phosphorylation in neurons, and an inhibitor of p38-MAPK reversed the influence of IL-1beta on tau phosphorylation and synaptophysin levels. Our results, together with previous observations, suggest that activated Microglia may contribute to neurofibrillary pathology in AD through their production of IL-1, activation of neuronal p38-MAPK, and resultant changes in neuronal cytoskeletal and synaptic elements

  4. Liu PH, Wang YJ, Tseng GF (2003) Close axonal injury of rubrospinal neurons induced transient perineuronal astrocytic and Microglial reaction that coincided with their massive degeneration. Exp.Neurol. 179:111-126
    Abstract: To learn more about the pathophysiology of axonal injury and the significance of axon collaterals on the survival of axotomized cord-projection central neurons, we studied the survival rate, surrounding astrocytic and Microglial reactions, and bouton coverage on rat rubrospinal cell bodies following their axonal lesion at the brain stem and upper cervical level. The brain stem lesion disconnected most rubrospinal neurons from all their targets, while the upper cervical lesion spared their supraspinal collaterals. Much higher cell loss accompanied by robust astrocytic and Microglial reaction was found following brain stem than upper cervical lesion starting 4 days postaxotomy. The reaction of astrocytes had subsided while Microglial reaction remained relatively robust by 10 weeks postaxotomy when the cell loss had slowed down. Ultrastructural observation revealed that reactive astrocytes covered 40%, an increase from the 20% of control, of brain stem-axotomized rubrospinal cell body surface at 4 days and 2 weeks and returned to normal levels by 10 weeks postlesion. An increase of apposition by axons and dendrites and a moderate decrease of round and flattened vesicle-containing bouton contacts at 4 days and 2 weeks and returning to normal levels at 10 weeks postaxotomy accompanied this. It appears that although axotomy induced robust astrocytic reaction around cord-projection central neurons, this, unlike their periphery-projection counterparts, failed to effectively strip their somatic synapses. In effect, this might in part determine neuronal fate following axonal injury

  5. Tripanichkul W, Stanic D, Drago J, Finkelstein DI, Horne MK (2003) D2 Dopamine receptor blockade results in sprouting of DA axons in the intact animal but prevents sprouting following nigral lesions. Eur.J.Neurosci. 17:1033-1045
    Abstract: Recently it was demonstrated that sprouting of dopaminergic neurons and a Microglial and astrocyte response follows both partial lesions of the substantia nigra pars compacta and blockade of the D2 dopamine receptor. We therefore studied the effects of the combination of these two treatments (lesioning and D2 dopamine receptor blockade). Haloperidol administration caused a 57% increase in dopaminergic terminal tree size (measured as terminal density per substantia nigra pars compacta neuron) and an increase of glia in the striatum. Following small to medium nigral lesions (less than 60%), terminal tree size increased by 51% on average and returned density of dopaminergic terminals to normal. In contrast, administration of haloperidol for 16 weeks following lesioning resulted in reduced dopaminergic terminal density and terminal tree size (13%), consistent with absent or impaired sprouting. Glial cell numbers increased but were less than with lesions alone. When haloperidol was administered after the striatum had been reinnervated through sprouting (16-32 weeks after lesioning), terminal tree size increased up to 150%, similar to the effect of haloperidol in normal animals. By examining the effect of administering haloperidol at varying times following a lesion, we concluded that a switch in the effect of D2 dopamine receptor blockade occurred after dopaminergic synapses began to form in the striatum. We postulate that when synapses are present, D2 dopamine receptor blockade results in increased terminal density, whereas prior to synapse formation D2 dopamine receptor blockade causes attenuation of a sprouting response. We speculate that D2 dopamine receptors located on growth cones 'push' neurites toward their targets, and blockade of these receptors could lead to attenuation of sprouting

  6. Tropea D, Caleo M, Maffei L (2003) Synergistic effects of brain-derived neurotrophic factor and chondroitinase ABC on retinal fiber sprouting after denervation of the superior colliculus in adult rats. J.Neurosci. 23:7034-7044
    Abstract: Damage to the adult CNS often causes devastating and permanent deficits because of the limited capacity of the brain for anatomical reorganization. The finding that collateral sprouting of uninjured fiber tracts mediates recovery of function prompts the search for experimental strategies that stimulate axonal plasticity after CNS trauma. Here we characterize treatments that promote the sprouting of undamaged retinal afferents into the denervated superior colliculus (SC) after a partial retinal lesion in the adult rat. Delivery of brain-derived neurotrophic factor (BDNF) was performed to enhance the intrinsic potential of retinal ganglion cells to reelongate their axons. Reduction of the neurite growth-inhibitory properties of the adult SC was accomplished via treatment with chondroitinase ABC (C-ABC), which degrades chondroitin sulfate proteoglycans. Retinal axons were labeled via intraocular injections of fluorescently tagged cholera toxin B subunit, and fiber sprouting within the denervated SC was measured by quantitative laser-scanning confocal microscopy 1 week after the retinal lesion. We found that both the administration of BDNF and the injection of C-ABC induce significant sprouting of retinal afferents into the collicular scotoma. Remarkably, the combined treatment with BDNF and C-ABC showed synergistic effects on axon growth. Colocalization analysis with anti-synapsin antibodies demonstrated synapse formation by the sprouting axons. These results suggest that the combined treatment with BDNF and C-ABC can be relevant in therapies for the repair of the damaged adult CNS

  7. Andreoletti O, Berthon P, Levavasseur E, Marc D, Lantier F, Monks E, Elsen JM, Schelcher F (2002) Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry. J.Histochem.Cytochem. 50:1357-1370
    Abstract: Transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by amyloid deposition of protein-prion (PrPsc), the pathogenic isoform of the host cellular protein PrPc, in the immune and central nervous systems. In the absence of definitive data on the nature of the infectious agent, PrPsc immunohistochemistry (IHC) constitutes one of the main methodologies for pathogenesis studies of these diseases. In situ PrPsc immunolabeling requires formalin fixation and paraffin embedding of tissues, followed by post-embedding antigen retrieval steps such as formic acid and hydrated autoclaving treatments. These procedures result in poor cellular antigen preservation, precluding the phenotyping of cells involved in scrapie pathogenesis. Until now, PrPsc-positive cell phenotyping relied mainly on morphological criteria. To identify these cells under the PrPsc IHC conditions, a new, rapid, and highly sensitive PrPsc double-labeling technique was developed, using a panel of screened antibodies that allow specific labeling of most of the cell subsets and structures using paraffin-embedded lymphoid and neural tissues from sheep, leading to an accurate identification of ovine PrPsc-accumulating cells. This technique constitutes a useful tool for IHC investigation of scrapie pathogenesis and may be applicable to the study of other ovine infectious diseases

  8. Bi X, Gall CM, Zhou J, Lynch G (2002) Uptake and pathogenic effects of amyloid beta peptide 1-42 are enhanced by integrin antagonists and blocked by NMDA receptor antagonists. Neuroscience 112:827-840
    Abstract: Many synapses contain two types of receptors - integrins and N-methyl-D-aspartate (NMDA) receptors - that have been implicated in peptide internalization. The present studies tested if either class is involved in the uptake of the 42-residue form of amyloid beta peptide (Abeta1-42), an event hypothesized to be of importance in the development of Alzheimer's disease. Cultured hippocampal slices were exposed to Abeta1-42 for 6 days in the presence or absence of soluble Gly-Arg-Gly-Asp-Ser-Pro, a peptide antagonist of Arg-Gly-Asp (RGD)-binding integrins, or the disintegrin echistatin. Abeta uptake, as assessed with immunocytochemistry, occurred in 42% of the slices incubated with Abeta peptide alone but in more than 80% of the slices co-treated with integrin antagonists. Uptake was also found in a broader range of hippocampal subfields in RGD-treated slices. Increased sequestration was accompanied by two characteristics of early stage Alzheimer's disease: elevated concentrations of cathepsin D immunoreactivity and activation of Microglia. The selective NMDA receptor antagonist D-(-)-2-amino-5-phosphonovalerate completely blocked internalization of Abeta, up-regulation of cathepsin D, and activation of Microglia. Our results identify two classes of receptors that cooperatively regulate the internalization of Abeta1-42 and support the hypothesis that characteristic pathologies of Alzheimer's disease occur once critical intraneuronal Abeta concentrations are reached

  9. Ferrer I (2002) Synaptic pathology and cell death in the cerebellum in Creutzfeldt-Jakob disease. Cerebellum. 1:213-222
    Abstract: Prion protein (PrP(c)) is a cell membrane glycoprotein particularly abundant in the synapses. Prion diseases are characterized by the replacement of the normal PrPc by a protease-resistant, sheet-containing isoform (PrP(CJD), PrP(Sc), PrP(BSE)) that is pathogenic. Creutzfeldt-Jakob disease (CJD) in humans, scrapie (Sc) in sheep and goats, and bovine spongiform encephalopathy (BSE) in cattle are typical prion diseases. Classical CJD can be presented as sporadic, infectious or familial, whereas the new variant of CJD (nvCJD) is considered a BSE-derived human disease. Spongiform degeneration, glial proliferation, involving astrocytes and Microglia, neuron loss and abnormal PrP deposition are the main neuopathological findings in most human and animal prion diseases. Yet recent data point to synapses as principal targets of abnormal PrP deposition. Loss of synapses is an early abnormality in experimental scrapie. Decreased expression of crucial proteins linked to exocytosis and neurotransmission, covering synaptophysin, synaptosomal-associated protein of 25,000 mol wt (SNAP-25), synapsins, syntaxins and Rab3a occurs in the cerebral cortex and cerebellum in sporadic CJD. Moreover, impairment of glomerular synapses and attenuation of parallel fiber pre-synaptic terminals on Purkinje cell dendrites is a cardinal consequence of abnormal PrP metabolism in CJD. Accumulation of synaptic proteins in the soma and axonal torpedoes of Purkinje cells suggests additional impairment of axonal transport. Increase in nuclear DNA vulnerability leading to augmented numbers of cells bearing nuclear DNA fragments is a common feature in the brains of humans affected by prion diseases examined at post-mortem, but also in archival biopsy samples processed with the method of in situ end-labeling of nuclear DNA fragmentation. This form of cell death is reminiscent of apoptosis found in experimental scrapie in rodents. It is not clear that all forms of cell death in human and animal prion diseases are due to apoptosis. Yet new observations have shown cleaved (active) caspase-3 (17 kDa), a main executioner of apoptosis, expressed in scattered cells in the brains of mice with experimental scrapie and in the cerebellum of patients with sporadic CJD. Together, these data suggest activation of the caspase pathway of apoptosis in human and animal prion diseases

  10. Fields RD, Stevens-Graham B (2002) New insights into neuron-glia communication. Science 298:556-562
    Abstract: Two-way communication between neurons and nonneural cells called glia is essential for axonal conduction, synaptic transmission, and information processing and thus is required for normal functioning of the nervous system during development and throughout adult life. The signals between neurons and glia include ion fluxes, neurotransmitters, cell adhesion molecules, and specialized signaling molecules released from synaptic and nonsynaptic regions of the neuron. In contrast to the serial flow of information along chains of neurons, glia communicate with other glial cells through intracellular waves of calcium and via intercellular diffusion of chemical messengers. By releasing neurotransmitters and other extracellular signaling molecules, glia can affect neuronal excitability and synaptic transmission and perhaps coordinate activity across networks of neurons

  11. Hauss-Wegrzyniak B, Lynch MA, Vraniak PD, Wenk GL (2002) Chronic brain inflammation results in cell loss in the entorhinal cortex and impaired LTP in perforant path-granule cell synapses. Exp.Neurol. 176:336-341
    Abstract: Alzheimer's disease (AD) is characterized by chronic neuroinflammation, significant temporal lobe cell loss, and dementia. We investigated the influence of chronic neuroinflammation produced by chronic infusion of lipopolysaccharide (LPS) into the fourth ventricle for 4 weeks upon the induction and maintenance of long-term potentiation (LTP) in the dentate gyrus of the hippocampus, a well-characterized model of cellular synaptic plasticity. We also examined for pyramidal cell loss within the entorhinal cortex an area of the brain that contains the cell bodies of the perforant path. The results demonstrate that chronic neuroinflammation results in the loss of pyramidal cells within layers II and III of the entorhinal cortex and a significant attenuation of LTP within the dentate gyrus. Similar changes may underlie the temporal lobe pathology and dementia associated with AD

  12. Peters A, Sethares C (2002) The effects of age on the cells in layer 1 of primate cerebral cortex. Cereb.Cortex 12:27-36
    Abstract: Although there is significant thinning of layer 1 with age in both occipital area 17 and prefrontal area 46 of the rhesus monkey, there are no significant age-related changes in the numbers of neurons, astrocytes, or Microglia and oligodendrocytes in this layer. A few profiles of degenerating neurons have been encountered in old monkeys, but they are uncommon. Some astrocytes undergo hypertrophy with age, as evidenced by the increased thickness of the glial limiting membrane, and throughout layer 1 the amount of filaments in the cytoplasm of both their cell bodies and processes increases. The astrocytes also come to contain phagocytic material in the old monkeys, as do the Microglial cells. We have previously shown that in both areas 17 and 46 there is an age-related loss of synapses from layer 1 and a concomitant loss of dendritic branches from the apical tufts of pyramidal cells from layer 1. These may be the sources of the material phagocytosed by the astrocytes and Microglial cells

  13. Peters A (2002) Structural changes that occur during normal aging of primate cerebral hemispheres. Neurosci.Biobehav.Rev. 26:733-741
    Abstract: Human and non-human primates show cognitive decline during normal aging. Originally, the decline was attributed to a loss of cortical neurons, but recent studies have shown there is no significant cortical neuronal loss with age. Neurons acquire pigment, but the only other obvious changes are in layer 1 of neocortex. Layer 1 becomes thinner as apical tufts of pyramidal cells lose branches, as well as synapses, and at the same time the glial limiting membrane thickens. How dendrites and synapses in deep layers are affected by age is uncertain, but there are decreases in the levels of some neurotransmitters and receptors. Throughout the brain myelin sheaths show signs of breakdown. This may contribute to cognitive decline because it would cause a slowing of conduction along nerve fibers, disrupting the timing in neuronal circuits. Concomitantly, the myelin-forming oligodendrocytes develop swellings along their processes and gain dense inclusions. Microglial cells and astrocytes accumulate large amounts of phagocytosed material with age, although the origins of this material are not known

  14. Peters A (2002) Structural changes in the normally aging cerebral cortex of primates. Prog.Brain Res. 136:455-465
    Abstract: During normal aging humans exhibit some cognitive decline, but it is difficult to determine the underlying causes of this decline, because information about cognitive status is rarely available and preservation of the brain is usually inadequate for detailed cytological examination. One solution to this problem is to use a nonhuman primate model, such as the rhesus monkey, which exhibits age-related cognitive decline similar to humans, and can be cognitively tested before the brains are preserved for detailed examination. It is now known that cognitive decline in human and nonhuman primates is not due to loss of cortical neurons and there is no correlation between the frequency of senile plaques and cognitive status. Indeed apart from layer 1, neurons of cerebral cortex show few signs of aging, although there may be some loss of synapses throughout cortex. In contrast, both Microglia and astrocytes come to contain phagocytosed material, but its origin is unknown. There is also loss of white matter, which is accompanied by some breakdown of myelin sheaths and alterations in oligodendrocytes. It is suggested that the myelin changes alter conduction velocities along axons. This would alter timing in neuronal circuits, contributing to cognitive decline

  15. Siso S, Puig B, Varea R, Vidal E, Acin C, Prinz M, Montrasio F, Badiola J, Aguzzi A, Pumarola M, Ferrer I (2002) Abnormal synaptic protein expression and cell death in murine scrapie. Acta Neuropathol.(Berl) 103:615-626
    Abstract: Reduced expression of synaptophysin p38, synaptic-associated protein of molecular weight 25,000 (SNAP-25), syntaxin-1, synapsin-1, and alpha- and beta-synuclein, matching the distribution of spongiform degeneration, was found in the neurological phase of scrapie-infected mice. In addition, synaptophysin and SNAP-25 were accumulated in isolated neurons, mainly in the thalamus, midbrain and pons, and granular deposits of alpha- and beta-synuclein were present in the neuropil of the same areas. No modifications in the steady state levels of Bcl-2, Bax, Fas and Fas ligand were observed following infection. Yet antibodies against the c-Jun N-terminal peptide, which cross-react with products emerging after caspase-mediate proteolysis, recognize coarse granular deposits in the cytoplasm of reactive Microglia. In situ end-labeling of nuclear DNA fragmentation showed positive nuclei with extreme chromatin condensation in the thalamus, pons, hippocampus and, in particular, the granular layer of the cerebellum. More importantly, expression of cleaved caspase-3, a major executioner of apoptosis, was seen in a few cells in the same regions, thus indicating that cell death by apoptosis in scrapie-infected mice is associated with caspase-3 activation. The present findings support the concept that synaptic pathology is a major substrate of neurological impairment and that caspase-3 activation may play a pivotal role in apoptosis in experimental scrapie. However, there is no correlation between decreased synaptic protein expression and caspase-3-associated apoptosis, which suggests that in addition to abnormal prion protein deposition, there may be other factors that distinctively influence synaptic vulnerability and cell death in murine scrapie

  16. Barger SW, Basile AS (2001) Activation of Microglia by secreted amyloid precursor protein evokes release of glutamate by cystine exchange and attenuates synaptic function. J.Neurochem. 76:846-854
    Abstract: Microglial activation as part of a chronic inflammatory response is a prominent component of Alzheimer's disease. Secreted forms of the beta-amyloid precursor protein (sAPP) previously were found to activate Microglia, elevating their neurotoxic potential. To explore neurotoxic mechanisms, we analyzed Microglia-conditioned medium for agents that could activate glutamate receptors. Conditioned medium from primary rat Microglia activated by sAPP caused a calcium elevation in hippocampal neurons, whereas medium from untreated Microglia did not. This response was sensitive to the NMDA receptor antagonist, aminophosphonovaleric acid. Analysis of Microglia-conditioned by HPLC revealed dramatically higher concentrations of glutamate in cultures exposed to sAPP. Indeed, the glutamate levels in sAPP-treated cultures were substantially higher than those in cultures treated with amyloid beta-peptide. This sAPP-evoked glutamate release was completely blocked by inhibition of the cystine-glutamate antiporter by alpha-aminoadipate or use of cystine-free medium. Furthermore, a sublethal concentration of sAPP compromised synaptic density in Microglia-neuron cocultures, as evidenced by neuronal connectivity assay. Finally, the neurotoxicity evoked by sAPP in Microglia-neuron cocultures was attenuated by inhibitors of either the neuronal nitric oxide synthase (N(G)-propyl-L-arginine) or inducible nitric oxide synthase (1400 W). Together, these data indicate a scenario by which Microglia activated by sAPP release excitotoxic levels of glutamate, probably as a consequence of autoprotective antioxidant glutathione production within the Microglia, ultimately causing synaptic degeneration and neuronal death

  17. DeFelipe J, Segura T, Arellano JI, Merchan A, DeFelipe-Oroquieta J, Martin P, Maestu F, Cajal S, Sanchez A, Sola RG (2001) Neuropathological findings in a patient with epilepsy and the Parry-Romberg syndrome. Epilepsia 42:1198-1203
    Abstract: PURPOSE: The Parry-Romberg syndrome is an unusual disorder frequently associated with epilepsy. The origin of this disease, and the cause of epilepsy, are unknown. This study is the first reported case of the Parry-Romberg syndrome, with intractable temporal lobe epilepsy, in which detailed microanatomic analyses have been performed on resected brain tissue obtained after surgical intervention. METHODS: Standard histopathologic methods and correlative light and electron microscopy, combined with immunocytochemical techniques, were used to study in detail the synaptic microorganization of the resected hippocampal formation. RESULTS: After surgery, the patient was seizure free (follow-up period of 4 years and 7 months). The resected temporal lobe showed a variety of dramatic microanatomic alterations (small groups of ectopic cells, neuronal loss, gliosis, and activated Microglial cells) in mesial structures, including the entorhinal cortex, subiculum, and dentate gyrus. At the electron-microscopic level, we found that in the dentate gyrus, the number of synapses in the cell-sparse region adjacent to the ectopic mass of neurons was almost twice that found in the molecular and polymorph cell layers, indicating the intrusion of neuritic processes and synapse formation. In addition, the symmetrical axosomatic synapses characteristically found on granule cells, which are likely derived from gamma-aminobutyric acid (GABA)ergic inhibitory basket cells, were not observed. CONCLUSION: The complete seizure relief after surgery suggests that the pacemaker region(s) of seizure activity were within the resected tissue. However, we do not know which of the multiple neuropathologic findings reported here were the primary cause of seizure activity. Nevertheless, the changes found in the dentate gyrus circuitry appear to be among the most important alterations that would lead to epilepsy

  18. Gerecke KM, Wyss JM, Karavanova I, Buonanno A, Carroll SL (2001) ErbB transmembrane tyrosine kinase receptors are differentially expressed throughout the adult rat central nervous system. J.Comp Neurol. 433:86-100
    Abstract: The neuregulin (NRG) family of growth and differentiation factors and their erbB receptors contribute importantly to the development of the nervous system, but their distribution and function in the adult brain are poorly understood. The present study showed that erbB2, erbB3, and erbB4 transcripts and protein are distributed throughout all areas of adult rat brain. These three receptors were differentially expressed in neurons and glia. Some neurons expressed only a subset of erbB kinases, whereas other neurons expressed all three erbB receptors but sequestered each of these polypeptides into distinct cellular compartments. In synapse-rich regions, erbB immunoreactivity appeared as punctate-, axon-, and/or dendrite-associated staining, suggesting that NRGs are involved in the formation and maintenance of synapses in adult brain. ErbB labeling also was present in neuronal soma, indicating that NRGs act at sites in addition to the synapse. Glia in adult brain also differentially expressed erbB3 and erbB4. Approximately half of the erbB3 labeling in white matter was associated with S100beta+/glial fibrillary acidic protein negative macroglia (i.e., oligodendrocytes or glial fibrillary acidic protein negative astrocytes). In contrast, macroglia in gray matter did not express erbB3. The remaining erbB3 immunoreactivity in white matter and erbB4 glial staining seemed to be associated with Microglia. These results showed that erbB receptors are expressed widely in adult rat brain and that each erbB receptor subtype has a distinct distribution. The differential distributions of erbB receptors in neurons and glia and the known functional differences between these kinases suggest that NRGs have distinct effects on these cells. The continued expression of NRGs and their erbB receptors in mature brain also implies that these molecules perform important functions in the brain throughout life

  19. James G, Butt AM (2001) Changes in P2Y and P2X purinoceptors in reactive glia following axonal degeneration in the rat optic nerve. Neurosci.Lett. 312:33-36
    Abstract: Purinoceptors have been shown to be important in mediating Ca(2+) signalling in glial cells and it has been proposed that they may have a role in their response to injury. To investigate this, the glial response to adenosine 5'triphosphate (ATP) was measured in situ, in optic nerves from juvenile rats that were enucleated at postnatal day (P) 1; age-matched normal nerves were used as controls. The optic nerve is a typical central nervous system (CNS) white matter tract containing axons and glial cells, but not neurones or synapses. Following neonatal enucleation, axons degenerate and oligodendrocytes do not develop, so that the optic nerve is populated predominantly by reactive astrocytes, with a minor population of activated Microglia. Application of 1 mM ATP evoked a large and rapid increase in glial [Ca(2+)](i) in fura-2 ratiometric whole nerve recordings from normal and gliotic axon-free nerves. Significantly, the response to ATP had a prolonged duration in gliotic axon-free nerves and there was as shift in the agonist rank order of potency from ATP = ADP > UTP >> alpha,beta-metATP to ATP > ADP = UTP = alpha,beta-metATP. The results indicate an in situ role for ATP signalling in reactive astrocytes, via metabotropic P2Y(1) and P2Y(2/4) purinoceptors and ionotropic P2X purinoreceptors. The change in the purinoceptor profile following axon degeneration suggests a special role for P2X purinoceptors in mediating the glial reaction to CNS injury

  20. Kalla R, Liu Z, Xu S, Koppius A, Imai Y, Kloss CU, Kohsaka S, Gschwendtner A, Moller JC, Werner A, Raivich G (2001) Microglia and the early phase of immune surveillance in the axotomized facial motor nucleus: impaired Microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage-colony stimulating factor-deficient mice. J.Comp Neurol. 436:182-201
    Abstract: Activation of Microglia is among the first cellular changes in the injured CNS. However, little is known about their specific contribution to secondary damage or repair processes in neighboring neurons and nonneuronal cells or to the immune surveillance of the damaged tissue. Animal models with defective Microglial response such as osteopetrosis provide an approach to explore these effects. Osteopetrosis (op) is an autosomal recessive mutation with a complete deficiency of the macrophage-colony stimulating factor (MCSF; CSF-1), an important mitogen for brain Microglia. In the current study we examined the effects of this MCSF deficiency on the Microglial reaction and the overall cellular response to nerve injury in the mouse axotomized facial motor nucleus. In the brain, MCSF receptor immunoreactivity was found only on Microglia and was strongly up-regulated following injury. MCSF deficiency led to a failure of Microglia to show a normal increase in early activation markers (thrombospondin, MCSF receptor, alpha M beta 2- and alpha 5 beta 1-integrins), to spread on the surface of axotomized motoneurons, and to proliferate after injury. Early recruitment of CD3(+) T-lymphocytes to the facial nucleus 24 hours after injury was reduced by 60%. In contrast, the neuronal and astrocyte response was not affected. There was a normal increase in the neuropeptides calcitonin gene-related peptide and galanin, neuronal c-JUN, and NADPH-diaphorase and a decrease in choline acetyltransferase and acetylcholinesterase. Astrocyte glial fibrillary acidic protein immunoreactivity also showed a normal increase. There was a normal influx of macrophages and granulocytes into the injured facial nerve. Synaptic stripping, neuronal survival, and speed of axonal regeneration were also not affected. The current results show a strong, selective effect of MCSF on the early activation of Microglia and, indirectly, on lymphocyte recruitment. This early phase of Microglial activation appears not to be involved in the process of repair following peripheral nerve injury. However, it is important in the initiation of inflammatory changes in the brain and in the interaction with the immune system

  21. Nomura H, Furuta A, Suzuki SO, Iwaki T (2001) Dorsal horn lesion resulting from spinal root avulsion leads to the accumulation of stress-responsive proteins. Brain Res. 893:84-94
    Abstract: The aim of this study was to demonstrate acute to subacute molecular episodes in the dorsal horn following root avulsion using immunohistochemical methods with the markers for synapses, astrocytes and such stress-responsive molecules as heat shock proteins (Hsps) and p38 MAP kinase (p38). Among them, Hsp27 was accumulated selectively in the injured substantia gelatinosa 24 h after avulsion injury. The localization of Hsp27 in astrocytes within the substantia gelatinosa was confirmed by the double immunofluorescence method using anti-Hsp27 antibody and either anti-synaptophysin antibody or anti-glutamine synthetase antibody and by immunoelectron microscopy for Hsp27. The pattern of Hsp27 expression subsequently changed from glial pattern to punctate pattern by 7 days. Immunoelectron microscopy revealed that the punctate pattern in the subacute stage corresponded to distal parts of the astrocytic processes. Hsp27 immunoreaction was decreased 21 days after root avulsion. In the distal axotomy model, Hsp27 was accumulated later in the ipsilateral dorsal horn in a punctate pattern from 7 days after the axotomy. Phosphorylation of p38 was detected in Microglia in the dorsal horn following both avulsion and axotomy. Substance P was slightly decreased in the injured substantia gelatinosa in both the avulsion and axotomy models around 14-21 days. We conclude that Hsp27 is a useful marker for demonstrating dorsal horn lesions following avulsion injury and that avulsion injury may induce Hsp27 in the dorsal horn more rapidly than distal axotomy

  22. Wegiel J, Wang KC, Imaki H, Rubenstein R, Wronska A, Osuchowski M, Lipinski WJ, Walker LC, LeVine H (2001) The role of Microglial cells and astrocytes in fibrillar plaque evolution in transgenic APP(SW) mice. Neurobiol.Aging 22:49-61
    Abstract: Ultrastructural reconstruction of 27 fibrillar plaques in different stages of formation and maturation was undertaken to characterize the development of fibrillar plaques in the brains of human APP(SW) transgenic mice (Tg2576). The study suggests that Microglial cells are not engaged in Abeta removal and plaque degradation, but in contrast, are a driving force in plaque formation and development. Fibrillar Abeta deposition at the amyloid pole of Microglial cells appears to initiate three types of neuropil response: degeneration of neurons, protective activation of astrocytes, and attraction and activation of Microglial cells sustaining plaque growth. Enlargement of neuronal processes and synapses with accumulation of degenerated mitochondria, dense bodies, and Hirano-type bodies is the marker of toxic injury of neurons by fibrillar Abeta. Separation of amyloid cores from neurons and degradation of amyloid cores by cytoplasmic processes of hypertrophic astrocytes suggest the protective and defensive character of astrocytic response to fibrillar Abeta. The growth of cored plaque from a small plaque with one Microglial cell with an amyloid star and a few dystrophic neurites to a large plaque formed by several dozen Microglial cells seen in old mice is the effect of attraction and activation of Microglial cells residing outside of the plaque perimeter. This mechanism of growth of plaques appears to be characteristic of cored plaques in transgenic mice. Other features in mouse Microglial cells that are absent in human brain are clusters of vacuoles, probably of lysosomal origin. They evolve into circular cisternae and finally into large vacuoles filled with osmiophilic, amorphous material and bundles of fibrils that are poorly labeled with antibody to Abeta. Microglial cells appear to release large amounts of fibrillar Abeta and accumulate traces of fibrillar Abeta in a lysosomal pathway

  23. Bechmann I, Nitsch R (2000) Involvement of non-neuronal cells in entorhinal-hippocampal reorganization following lesions. Ann.N.Y.Acad.Sci. 911:192-206
    Abstract: Entorhinal lesion leads to anterograde degeneration of perforant path fibers in their main hippocampal termination zones. Subsequently, remaining fibers sprout and form new synapses on the denervated dendrites. This degeneration and reorganization is accompanied by sequential changes in glial morphology and function. Within a few hours following the lesion, amoeboid Microglia migrate into the zone of denervation. Some hours later, signs of activation can be seen on astrocytes in the zone of denervation, where both cell types proliferate and remain in an activated state for more than two weeks. These activated glial cells might be involved in lesion-induced plasticity in at least two ways: (1) by releasing cytokines and growth factors which regulate layer-specific sprouting and (2) by phagocytosis of axonal debris, because myelin sheaths act as obstacles for sprouting fibers in the central nervous system. Whereas direct evidence for the former is still missing, the latter was investigated using phagocytosis-dependent labeling techniques. Both Microglial cells and astrocytes incorporate axonal debris. Phagocytosing Microglial cells develop the immune phenotype of antigen-presenting cells, whereas astrocytes strongly express FasL (CD95L), which induces apoptosis of activated lymphocytes. Thus, the interaction of glial cells with immune cells might be another, previously underestimated, aspect of reorganization following entorhinal lesion

  24. Gilbert DL (2000) Fifty years of radical ideas. Ann.N.Y.Acad.Sci. 899:1-14
    Abstract: My role in the free radical theory of oxygen toxicity is discussed. Rebeca Gerschman and I published several papers on this subject. This sparked my interest in geochemistry and I developed the idea that oxygen was the best qualified biological potential energy source for the following reasons: great abundance, easily accessible, possession of a high thermodynamic potential, and its slow reaction rate. Ionization radiation can be viewed as a catalyst for reactive oxygen species since a killing dose imparts an infinitesimal small amount of energy. Next, Carol A. Colton and I showed that in the mammalian brain that stimulated Microglia produce the superoxide radical anion and its implications in Alzheimer's disease is discussed. More recently, I have become interested in the role of sulfhydryl groups in transcription factors

  25. Gonatas JO, Stieber A, Gonatas NK, Messing A (2000) The golgi apparatus is present in perisynaptic, subependymal and perivascular processes of astrocytes and in processes of retinal Muller glia. Brain Res. 855:23-31
    Abstract: The Golgi apparatus (GA) of innervated rat and chicken skeletal muscle is present in a typical perinuclear location, and in subsynaptic areas where it disperses after denervation. It was suggested that the subsynaptic segments of the GA are linked with functions involved in the maturation and targeting of synaptic proteins. Similarly, the GA of rat myocardium is found in a perinuclear location and between myofibrils, adjacent to the T system of tubules. These findings raise the question whether the GA of polarized cells is present in a typical perinuclear location, for the performance of general "housekeeping" functions, and in distal areas, for the mediation of specialized functions. Astrocytes may contain GA within their long cytoplasmic processes which are difficult to identify in thin sections. To ensure the astrocytic origin of GA in otherwise unidentifiable small processes, we used transgenic mice expressing the rat MG160 medial Golgi sialoglycoprotein only in the GA of astrocytes, and visualized the GA with monoclonal antibody 10A8 (mAb10A8) which reacts only with rat MG160. Thus, we identified cisternae of the GA in distal perisynaptic and subependymal processes, in perivascular foot plates of cerebral astrocytes, and in processes of the Muller glia in the retina. A similar strategy may be adopted in future investigations aiming at the detection of elements of the GA in distal processes of neurons and oligodendrocytes. The functional implications of GA in perisynaptic astrocytic processes and other processes are unknown. However, the isolation and molecular characterization of the perisynaptic subset of astrocytic Golgi may be feasible, since others have purified the astrocytic glutamate transporter 1 (GLT1) from crude synaptosomal fractions in which astrocytic processes are probably unavoidable contaminants

  26. Lopez-Redondo F, Nakajima K, Honda S, Kohsaka S (2000) Glutamate transporter GLT-1 is highly expressed in activated Microglia following facial nerve axotomy. Brain Res.Mol.Brain Res. 76:429-435
    Abstract: Glutamate transporters play an important role in the re-uptake of glutamate after its release from glutamatergic synapses. So far five of such transporters subtypes have been cloned from rodent and human brains. The densities of glutamate transporters are recognised to be developmentally regulated, but the role of glutamate transporters in the mechanisms underlying the occurrence of neuronal traumatic injury has not been widely studied. In the present study quantitative Western blotting and immunohistochemical technique were employed to study the expression of GLT-1/EAAT2 in the facial nuclei of adult rats following unilateral facial nerve axotomy. The total content of GLT-1 protein decreased in the ipsilateral axotomised rat facial nucleus. However, activated Microglia surrounding motoneurons showed high expression of GLT-1 after facial nerve axotomy. Parallel studies revealed that primary cultured Microglial cells also showed GLT-1-immunoreactivity. To our knowledge, this is the first direct demonstration of the expression of GLT-1 protein in activated Microglial cells, suggesting a neuroprotective role of Microglia against glutamate excitotoxicity following nerve axotomy

  27. McNamara RK, Lenox RH (2000) Differential regulation of primary protein kinase C substrate (MARCKS, MLP, GAP-43, RC3) mRNAs in the hippocampus during kainic acid-induced seizures and synaptic reorganization. J.Neurosci.Res. 62:416-426
    Abstract: In the mature hippocampus, kainic acid seizures lead to excitotoxic cell death and synaptic reorganization in which granule cell axons (mossy fibers) form ectopic synapses on granule cell dendrites. In the present study, we examined the expression of four major, developmentally regulated protein kinase C (PKC) substrates (MARCKS, MLP, GAP-43, RC3), which have different subcellular and regional localizations in the hippocampus at several time points (6 hr, 12 hr, 18 hr, 24 hr, 48 hr, 5 days, or 15 days) following kainic acid seizures using in situ hybridization. Consistent with previous reports, following kainate seizures, GAP-43 mRNA expression exhibited a delayed and protracted elevation in the granule cell layer, which peaked at 24 hr, whereas expression in fields CA1 and CA3 remained relatively unchanged. Conversely, RC3 mRNA expression exhibited a delayed reduction in the granule cell layer that was maximal at 18 hr, as well as a reduction CA1 at 48 hr, whereas CA3 levels did not change. MARCKS mRNA expression in the granule cell layer and CA1 remained stable following kainate, although an elevation was observed in subfield CA3c at 12 hr. Similarly, MLP mRNA expression did not change in the granule cell layer or CA1 following kainate but exhibited a protracted elevation in subfields CA3b,c beginning at 6 hr post-kainate. Collectively these data demonstrate that different PKC substrate mRNAs exhibit unique expression profiles and regulation in the different cell fields of the mature hippocampus following kainic acid seizures and during subsequent synaptic reorganization. The expression profiles following kainate seizures bear resemblance to those observed during postnatal hippocampal development, which may indicate the recruitment of common regulatory mechanisms

  28. Novikov LN, Novikova LN, Holmberg P, Kellerth J (2000) Exogenous brain-derived neurotrophic factor regulates the synaptic composition of axonally lesioned and normal adult rat motoneurons. Neuroscience 100:171-181
    Abstract: Brain-derived neurotrophic factor has previously been shown to promote survival and axonal regeneration in injured spinal motoneurons and, also, to modulate synaptic transmission and regulate the density of synaptic innervation in a variety of neurons. The present light and electron microscopic study demonstrates synaptotrophic effects of exogenously applied brain-derived neurotrophic factor on the synaptic composition of both normal and axonally lesioned adult rat spinal motoneurons. After L5-L6 ventral root avulsion, a massive loss of all types of boutons occurred on the somata of the lesioned motoneurons which persisted for at least 12 weeks postoperatively. We found that (i) intrathecal infusion of brain-derived neurotrophic factor during the first postoperative week did not prevent the synaptic detachment and activation of glial cells; (ii) prolonged treatment for four weeks restored synaptic covering and significantly reduced Microglial reaction; (iii) the synaptotrophic effect remained significant for at least eight weeks after cessation of the treatment; (iv) brain-derived neurotrophic factor mainly supported F-type boutons with presumably inhibitory function, while it had little effect on S-type boutons associated with excitatory action; and (v) in normal unlesioned motoneurons, four weeks of treatment with brain-derived neurotrophic factor induced sprouting of F-type boutons, a loss of S-type boutons and motoneuron atrophy.The present data show that exogenous neurotrophins not only help to restore synaptic circuitry in axonally injured motoneurons, but also strongly influence the synaptic composition in normal motoneurons

  29. Terry RD (2000) Cell death or synaptic loss in Alzheimer disease. J.Neuropathol.Exp.Neurol. 59:1118-1119
    Abstract: It is an erroneous but common assumption that loss of neuronal perikarya causes the cognitive change in Alzheimer disease. Neither are senile plaques nor neurofibrillary tangles primarily to blame. In fact, it is the loss of synaptic contact that leads directly to the personal devastation. The death of neocortical synapses in the neuropil between plaques is probably the factor that activates the Microglia

  30. Wegiel J, Wang KC, Tarnawski M, Lach B (2000) Microglia cells are the driving force in fibrillar plaque formation, whereas astrocytes are a leading factor in plague degradation. Acta Neuropathol.(Berl) 100:356-364
    Abstract: Ultrastructural three-dimensional reconstruction of human classical plaques in different stages of development shows that Microglial cells are the major factor driving plaque formation by fibrillar amyloid-beta (Abeta) deposition. The amount of fibrillar Abeta released by Microglial cells and the area of direct contact between amyloid and neuron determine the extent of dystrophic changes in neuronal processes and synapses. The volume of hypertrophic astrocytic processes separating fibrillar amyloid from neuron is a measure of the protective activation of astrocytes. On the bases of the volume of amyloid star, Microglial cells, dystrophic neurites, and hypertrophic astrocytic processes, and spatial relationships between plaque components, three stages in classical plaque development have been distinguished: early, mature, and late. In early plaque, the leading pathology is fibrillar Abeta deposition by Microglial cells with amyloid star formation. The mature plaque is characterized by a balance between amyloid production, neuronal dystrophy, and astrocyte hypertrophy. In late classical plaque, Microglial cells retract and expose neuropil on direct contact with amyloid star, enhancing both dystrophic changes in neurons and hypertrophic changes in astrocytes. In late plaques, activation of astrocytes predominates. They degrade amyloid star and peripheral amyloid wisps. The effect of these changes is classical plaque degradation to fibrillar primitive and finally to nonfibrillar, diffuse-like plaques

  31. Aldskogius H, Liu L, Svensson M (1999) Glial responses to synaptic damage and plasticity. J.Neurosci.Res. 58:33-41
    Abstract: We review three principally different forms of injury-induced synaptic alterations. (1) Displacement of presynaptic terminals from perikarya and dendrites of axotomized neurons, (2) central changes in primary afferent terminals of peripherally axotomized sensory ganglion cells, and (3) anterograde Wallerian-type degeneration following interruption of central axonal pathways. All these instances rapidly activate astrocytes and Microglia in the vicinity of the affected synaptic terminals. The evidence suggests that activated astrocytes play important and direct roles in synapse elimination and in the processes mediating collateral reinnervation. The roles of Microglia are enigmatic. They undergo activation close to axotomized motoneuron perikarya, where synapse displacement occurs, but not adjacent to axotomized intrinsic central nervous system neurons, where synapse displacement also occurs. Microglia are also rapidly activated around central primary sensory terminals of peripherally axotomized sensory ganglion cells. Occasional phagocytosis of degenerating axon terminals by Microglia occur in the latter situation. However, the role of Microglia may be more oriented toward the general tissue conditions rather than specifically toward synaptic terminals

  32. Bruce-Keller AJ (1999) Microglial-neuronal interactions in synaptic damage and recovery. J.Neurosci.Res. 58:191-201
    Abstract: An understanding of the role of Microglial cells in synaptic signaling is still elusive, but the neuron-Microglia relationship may have important ramifications for brain plasticity and injury. This review summarizes current knowledge and theories concerning Microglial-neuronal signaling, both in terms of neuron-to-Microglia signals that cause activation and Microglia-to-neuron signals that affect neuronal response to injury. Microglial activation in the brain involves a stereotypical pattern of changes including proliferation and migration to sites of neuronal activity or injury, increased or de novo expression of immunomodulators including cytokines and growth factors, and the full transformation into brain-resident phagocytes capable of clearing damaged cells and debris. The factors released from neurons that elicit such phenotypical and functional alterations are not well known but may include cytokines, oxidized lipids, and/or neurotransmitters. Once activated, Microglia can promote neuronal injury through the release of low-molecular-weight neurotoxins and support neuronal recovery through the release of growth factors and the isolation/removal of damaged neurons and myelin debris. Because Microglia respond quickly to neuronal damage and have robust effects on neurons, astrocytes, and oligodendrocytes, Microglial cells could play potentially key roles in orchestrating the multicell cascade that follows synaptic plasticity and damage

  33. Hegg CC, Thayer SA (1999) Monocytic cells secrete factors that evoke excitatory synaptic activity in rat hippocampal cultures. Eur.J.Pharmacol. 385:231-237
    Abstract: Microglia- and macrophage-induced neuronal death may underlie a number of neurodegenerative diseases. The effects of factors secreted by monocytic cells were studied on glutamatergic synaptic transmission between cultured rat hippocampal neurons. Conditioned media from differentiated human U937 cells was collected after 24 h and applied to neurons (0.5%-30% dilution). Unactivated U937 cells spontaneously released factors that when applied to neuronal cultures evoked bursts of action potentials and elicited neuronal death (29+/-4%). Conditioned media collected from U937 cells evoked intracellular calcium ([Ca(2+)](i)) spiking (0.5%-2% dilution) and at higher concentrations evoked sustained increases in intracellular calcium (3%-30% dilution), as measured by indo-1-based photometry in single neurons. Activation of the U937 cells with zymosin A (500 microg/ml) enhanced the potency of the conditioned media to increase intraneuronal [Ca(2+)](i) as indicated by a leftward shift in the concentration-response curve. Selective antagonists to voltage-gated Na(+) and Ca(2+) channels and NMDA-gated channels (tetrodotoxin, nimodipine, and (+/-)-2-amino-5-phosphonopentanoic acid, respectively) blocked the calcium transients elicited by unactivated and zymosin -A-treated conditioned media. This pharmacologic profile is consistent with U937-released factors that excite the synaptic network that forms between cultured hippocampal neurons

  34. Love S (1999) Oxidative stress in brain ischemia. Brain Pathol. 9:119-131
    Abstract: Brain ischemia initiates a complex cascade of metabolic events, several of which involve the generation of nitrogen and oxygen free radicals. These free radicals and related reactive chemical species mediate much of damage that occurs after transient brain ischemia, and in the penumbral region of infarcts caused by permanent ischemia. Nitric oxide, a water- and lipid-soluble free radical, is generated by the action of nitric oxide synthases. Ischemia causes a surge in nitric oxide synthase 1 (NOS 1) activity in neurons and, possibly, glia, increased NOS 3 activity in vascular endothelium, and later an increase in NOS 2 activity in a range of cells including infiltrating neutrophils and macrophages, activated Microglia and astrocytes. The effects of ischemia on the activity of NOS 1, a Ca2+-dependent enzyme, are thought to be secondary to reversal of glutamate reuptake at synapses, activation of NMDA receptors, and resulting elevation of intracellular Ca2+. The up-regulation of NOS 2 activity is mediated by transcriptional inducers. In the context of brain ischemia, the activity of NOS 1 and NOS 2 is broadly deleterious, and their inhibition or inactivation is neuroprotective. However, the production of nitric oxide in blood vessels by NOS 3, which, like NOS 1, is Ca2+-dependent, causes vasodilatation and improves blood flow in the penumbral region of brain infarcts. In addition to causing the synthesis of nitric oxide, brain ischemia leads to the generation of superoxide, through the action of nitric oxide synthases, xanthine oxidase, leakage from the mitochondrial electron transport chain, and other mechanisms. Nitric oxide and superoxide are themselves highly reactive but can also combine to form a highly toxic anion, peroxynitrite. The toxicity of the free radicals and peroxynitrite results from their modification of macromolecules, especially DNA, and from the resulting induction of apoptotic and necrotic pathways. The mode of cell death that prevails probably depends on the severity and precise nature of the ischemic injury. Recent studies have emphasized the role of peroxynitrite in causing single-strand breaks in DNA, which activate the DNA repair protein poly(ADP-ribose) polymerase (PARP). This catalyzes the cleavage and thereby the consumption of NAD+, the source of energy for many vital cellular processes. Over-activation of PARP, with resulting depletion of NAD+, has been shown to make a major contribution to brain damage after transient focal ischemia in experimental animals. Neuronal accumulation of poly(ADP-ribose), the end-product of PARP activity has been demonstrated after brain ischemia in man. Several therapeutic strategies have been used to try to prevent oxidative damage and its consequences after brain ischemia in man. Although some of the drugs used in early studies were ineffective or had unacceptable side effects, other trials with antioxidant drugs have proven highly encouraging. The findings in recent animal studies are likely to lead to a range of further pharmacological strategies to limit brain injury in stroke patients

  35. Nacher J, Ramirez C, Palop JJ, Molowny A, Luis de la Iglesia JA, Lopez-Garcia C (1999) Radial glia and cell debris removal during lesion-regeneration of the lizard medial cortex. Histol.Histopathol. 14:89-101
    Abstract: Intraperitoneal injection of the neurotoxin 3-acetylpyridine (3AP) induces a rapid degeneration of the medial cerebral cortex (lizard fascia dentata) granular layer and of its zinc enriched axonal projection (lizard mossy fibres). After 6-8 weeks post-lesion the cell debris have been removed and the granular layer is repopulated by neurons generated in the subjacent ependyma. Both processes, neuron incorporation and debris removal, seem to be crucial for successful regeneration. Scavenging processes in the lesioned mammalian CNS are usually carried out by Microglia and/or astrocytes. In the lizard cerebral cortex there are no free astrocytes and the only glial fibrillary acid (GFAP) immunoreactive cells are radial glia-ependymocytes, similar to those present during mammalian CNS development. Ependymocytes, in addition to their help in vertical migrations of just generated immature neurons, built the cortical glial scaffold, insulate the blood capillaries, form the outer glial limiting membrane, thus playing an essential role in the lizard cortical blood-brain barrier. In this study, by means of GFAP-immunocytochemistry and electron microscopy, we have shown that radial glial cells participate actively in the removal/phagocytosis of cellular debris generated in the lesion process: mainly degenerated synapses, but interestingly, also some neuronal somata. Cell debris taken up by ependymocyte lateral processes seem to be progressively transported to either distal (pial) or proximal (ventricular) poles of the cell, where they result in lipofuscin accumulations. The hypothetical subsequent exchange of debris from ependymoglia by Microglia/macrophages and Kolmer cells is discussed

  36. Ong WY, Levine JM (1999) A light and electron microscopic study of NG2 chondroitin sulfate proteoglycan-positive oligodendrocyte precursor cells in the normal and kainate-lesioned rat hippocampus. Neuroscience 92:83-95
    Abstract: The adult brain contains a large population of oligodendrocyte precursor cells that can be identified using antibodies against the NG2 chondroitin sulfate proteoglycan. The functions of this newly recognized class of glial cells in the normal or pathological brain are not well understood. To begin to elucidate these functions, we have examined the morphology and distribution of oligodendrocyte precursor cells in the hippocampus and neocortex of normal and kainate-lesioned rats by anti-NG2 immunocytochemistry using light and electron microscopy. Large numbers of oligodendrocyte precursor cells were present in all layers of the neocortex and hippocampus. These cells differed in their morphology from astrocytes, oligodendrocytes and Microglia. The processes of these cells often surrounded unlabeled areas of clear cytoplasm. At the electron microscopic level, some of the profiles that were enclosed by oligodendrocyte precursor cell processes contained synaptic vesicles. Other enclosed profiles were dendrites or dendritic spines. NG2-immunopositive processes were also observed to interpose between axon terminals containing round vesicles and dendrites with thick postsynaptic densities. After kainate injection, the NG2-positive oligodendrocyte precursor cells in the hippocampus displayed reactive changes characterized by swollen cell bodies, an increased number of small, filopodial-like processes, and higher levels of immunodetectable NG2. Both viable and degenerating oligodendrocyte precursor cells were observed with electron microscopy. These observations emphasize the dynamic nature of the oligodendrocyte precursor cell and suggest that, in addition to participating in the glial reactions to excitotoxic damage, oligodendrocyte precursor cells may regulate the stability, structure and function of synapses in the normal central nervous system

  37. Pasinetti GM, Hassler M, Stone D, Finch CE (1999) Glial gene expression during aging in rat striatum and in long-term responses to 6-OHDA lesions. synapse 31:278-284
    Abstract: The male rat striatum was examined for age-related changes in mRNAs expressed in astrocytes and Microglia in two rat genotypes that differ by 35% in mean and maximum life spans: F344 and the longer-lived F1 (BN x F344) hybrid. The findings extend the established age-related increases in GFAP (glial fibrillary acidic protein) to other glial mRNAs: two lipoprotein mRNAs that are predominantly expressed in striatal astrocytes, apoE (apolipoprotein E) and apoJ (apolipoprotein J, clusterin, CLI, or SGP-2), and two mRNAs expressed in striatal Microglia, TGF-beta1 and complement C1qB. By Northern blot hybridization, both genotypes showed progressive increases of GFAP mRNA to > 2.5-fold by the lifespan. Although the rat strains differed 35% in life span, the slope of the GFAP mRNA regression on age did not differ. Relative to GFAP, the increases of apoE, apoJ, C1q, and TGF-beta1 mRNAs were smaller, < or = 1.5-fold. Because prior studies showed that acute damage to striatal afferents induced astrocyte gene expression increases resembling those that also occur during aging, we examined long-term effects of damage to substantia nigra neurons on striatal astrocyte changes during aging. Young F344 rats were given 6-OHDA lesions that cause striatal dopamine deficits and induce GFAP. When examined 15 months later at age 18 months, there was no effect during prior lesions on the age-related elevation of GFAP mRNA. We conclude that aging changes in striatal GFAP mRNAs do not interact with loss of dopaminergic output to the striatum from 6-OHDA lesions and may be independent of the relatively modest dopaminergic losses during normal aging

  38. Rinaman L, Roesch MR, Card JP (1999) Retrograde transynaptic pseudorabies virus infection of central autonomic circuits in neonatal rats. Brain Res.Dev.Brain Res. 114:207-216
    Abstract: Pseudorabies virus (PRV) is widely used to map synaptically-linked neural circuits in adult animals. The present study sought to determine whether PRV has similar utility in neonatal rats, and whether central PRV infection in neonates elicits astrocytic and Microglia/macrophage responses similar to those that contribute to specific transynaptic neuronal infection in adult rats. Retrograde transneuronal infection of autonomic circuits was examined 24-64 h after injection of an attenuated strain of PRV (PRV-Bartha) into the ventral stomach wall of 1-day-old rats. Brain and spinal cord sections were processed for immunocytochemical detection of PRV. Alternate sections were processed for immunolocalization of glial fibrillary acidic protein (GFAP) to identify fibrous astrocytes, or for an antigen associated with the complement C3bi receptor (OX42) to identify Microglia. As in adult rats, the number and distribution of infected CNS neurons in neonatal rats increased progressively with advancing post-inoculation survival. Infected CNS neurons initially were restricted to the thoracic intermediolateral cell column and the dorsal motor nucleus of the vagus. Longer survival times led to retrograde transynaptic infection of additional neurons in the thoracic spinal cord, nucleus of the solitary tract, ventrolateral medulla, and caudal raphe nuclei. At the longest post-inoculation intervals, infected neurons also were observed in the area postrema and in certain autonomic-related regions of the rostral brainstem, hypothalamus, and amygdala. Quantitative analysis of immunolabeling in the dorsal vagal complex demonstrated that regions containing neurons at early stages of viral infection displayed increased astrocytic GFAP immunostaining; conversely, areas containing neurons at later stages of infection were characterized by a significant loss of GFAP staining and a parallel increase of OX42 Microglia/macrophage immunolabeling. We conclude that PRV is effectively transported through synaptically-linked CNS circuits in neonatal rats, and that spatiotemporally-ordered responses by non-neuronal cells may contribute to the synaptic specificity of transneuronal viral transport

  39. Schiefer J, Kampe K, Dodt HU, Zieglgansberger W, Kreutzberg GW (1999) Microglial motility in the rat facial nucleus following peripheral axotomy. J.Neurocytol. 28:439-453
    Abstract: 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

  40. Buttini M, Westland CE, Masliah E, Yafeh AM, Wyss-Coray T, Mucke L (1998) Novel role of human CD4 molecule identified in neurodegeneration. Nat.Med. 4:441-446
    Abstract: The human CD4 molecule (hCD4) is expressed on T lymphocytes and macrophages and acts as a key component of the cellular receptor for HIV. At baseline, hCD4 transgenic mice expressed hCD4 on Microglia, the resident mononuclear phagocytes of the brain, and showed no neuronal damage. Activation of brain Microglia by peripheral immune challenges elicited neurodegeneration in hCD4 mice but not in nontransgenic controls. In post-mortem brain tissues from AIDS patients with opportunistic infections, but without typical HIV encephalitis, hCD4 expression correlated with neurodegeneration. We conclude that hCD4 may function as an important mediator of indirect neuronal damage in infectious and immune-mediated diseases of the central nervous system

  41. Cotman CW, Hailer NP, Pfister KK, Soltesz I, Schachner M (1998) Cell adhesion molecules in neural plasticity and pathology: similar mechanisms, distinct organizations? Prog.Neurobiol. 55:659-669
    Abstract: Brain plasticity and the mechanisms controlling plasticity are central to learning and memory as well as the recovery of function after brain injury. While it is clear that neurotrophic factors are one of the molecular classes that continue to regulate brain plasticity in the adult central nervous system (CNS), less appreciated but equally profound is the role of cell adhesion molecules (CAMs) in plasticity mechanisms such as long term potentiation, preservation of neurons and regeneration. Ironically, however, CAMs can also reorganize the extra-cellular space and cause disturbances that drive the development of brain pathology in conditions such as Alzheimer's disease and multiple sclerosis. Candidate molecules include the amyloid precursor protein which shares many properties of a classical CAM and beta-amyloid which can masquerade as a pseudo CAM. Beta-Amyloid serves as a nidus for the formation of senile plaques in Alzheimer's disease and like CAMs provides an environment for organizing neurotrophic factors and other CAMs. Inflammatory responses evolve in this environment and can initiate a vicious cycle of perpetuated neuronal damage that is medicated by Microglia, complement and other factors. Certain CAMs may converge on common signal transduction pathways involving focal adhesion kinases. Thus a breakdown in the organization of key CAMs and activation of their signal transduction mechanisms may serve as a new principle for the generation of brain pathology

  42. Kluge A, Hailer NP, Horvath TL, Bechmann I, Nitsch R (1998) Tracing of the entorhinal-hippocampal pathway in vitro. Hippocampus 8:57-68
    Abstract: In vitro tract tracing allowing for continuous observation of the perforant path is a crucial prerequisite for experimental studies on the entorhinal-hippocampal interaction in an organotypic slice culture containing the entorhinal cortex, the perforant path, and the dentate gyrus (OEHSC). We prepared horizontal slices of the temporal entorhinal-hippocampal region of the rat on a vibratome, and the perforant path axons were traced by application of the fluorescent tracer Mini Ruby on the entorhinal cortex. After 2 days in vitro (div), the perforant path became visible in most cultures. Entorhinal neurons and single perforant fibers could be followed to the outer molecular layers of the dentate gyrus by in vitro fluorescence microscopy and it was possible to monitor the perforant path directly over a period of 25 div. Moreover, ultrastructural analysis proved the existence of traced perforant path boutons forming synapses with spines and dendritic shafts in the outer molecular layers of the dentate gyrus. Transsection of the prelabelled perforant path in vitro resulted in anterograde degeneration and subsequent phagocytosis of axonal material by activated Microglial cells in the zone of denervation. In conclusion, in vitro tracing demonstrates the maintenance of the entorhinal-hippocampal pathway in OEHSCs and permits monitoring of dynamic changes in the prelabeled perforant path after various lesion paradigms, e.g., transsection or neurotoxin treatment. This approach permits further studies on the efficacy of neuroprotectants, cytokines, and growth factors in the treatment of lesion-induced neuronal degeneration

  43. Nishimura I, Uetsuki T, Dani SU, Ohsawa Y, Saito I, Okamura H, Uchiyama Y, Yoshikawa K (1998) Degeneration in vivo of rat hippocampal neurons by wild-type Alzheimer amyloid precursor protein overexpressed by adenovirus-mediated gene transfer. J.Neurosci. 18:2387-2398
    Abstract: In an attempt to elucidate the pathological implications of intracellular accumulation of the amyloid precursor protein (APP) in postmitotic neurons in vivo, we transferred APP695 cDNA into rat hippocampal neurons by using a replication-defective adenovirus vector. We first improved the efficiency of adenovirus-mediated gene transfer into neurons in vivo by using hypertonic mannitol. When a beta-galactosidase-expressing recombinant adenovirus suspended in 1 M mannitol was injected into a dorsal hippocampal region, a number of neurons in remote areas were positively stained, presumably owing to increased retrograde transport of the virus. When an APP695-expressing adenovirus was injected into the same site, part of the infected neurons in the hippocampal formation underwent severe degeneration in a few days, whereas astrocytes near the injection site showed no apparent degeneration. These degenerating neurons accumulated different epitopes of APP, and beta/A4 protein (Abeta)-immunoreactive materials were undetected in the extracellular space. A small number of degenerating neurons showed nuclear DNA fragmentation. Electron microscopic examinations demonstrated that degenerating neurons had shrunken perikarya along with synaptic abnormalities. Microglial cells/macrophages were often found in close proximity to degenerating neurons, and in some cases they phagocytosed these neurons. These results suggest that intracellular accumulation of wild-type APP695 causes a specific type of neuronal degeneration in vivo in the absence of extracellular Abeta deposition

  44. Riehl B, Schlue WR (1998) Morphological organization of neuropile glial cells in the central nervous system of the medicinal leech (Hirudo medicinalis). Tissue Cell 30:177-186
    Abstract: Neuropile glial (NPG) cells in the central nervous system of the medicinal leech, Hirudo medicinalis, were studied by histological, histochemical and immunocytochemical techniques. The NPG cells are often surrounded by electron-dense Microglial cells. The central cytoplasm of NPG cells shows a significant zonation. The zone around the nucleus contains mitochondria, glycogen and vesicles. The cytoplasm also contains many ribosomes, a few dictyosomes and distinct inclusions up to 2 microns in diameter. A second zone around the perinuclear region is marked by the occurrence of bundles of intermediate filaments that correspond in thickness to glial filaments of vertebrates. We found a positive reaction with polyclonal antibodies against human glial fibrillary acidic protein (GFAP), and the areas of intense fluorescence correspond to the regions where intermediate filaments were found to be abundant. The peripheral zone contains numerous membrane stacks that could not be contrasted by lanthane nitrate or tannic acid. Therefore, the membrane stacks could be part of an extensive smooth endoplasmic reticulum, which is characteristic of cells with active lipid metabolism

  45. Shafer OT, Chen A, Kumar SM, Muller KJ, Sahley CL (1998) Injury-induced expression of endothelial nitric oxide synthase by glial and Microglial cells in the leech central nervous system within minutes after injury. Proc.R.Soc.Lond B Biol.Sci. 265:2171-2175
    Abstract: It is known that nitric oxide (NO) is produced by injured tissues of the mammalian central nervous system (CNS) within days of injury. The aim of the present experiments was to determine the cellular synthesis of NO in the CNS immediately after injury, using the CNS of the leech which is capable of synapse regeneration, as a step towards understanding the role of NO in nerve repair. We report that within minutes after crushing the nerve cord of the leech, the region of damage stained histochemically for NADPH diaphorase, which is indicative of nitric oxide synthase (NOS) activity, and was immunoreactive for endothelial NOS (eNOS). On immunoblots of leech CNS extract, the same antibody detected a band with a relative molecular mass of 140,000, which is approximately the size of vertebrate eNOS. Cells expressing eNOS immunoreactivity as a result of injury were identified after freezing nerve cords, a procedure that produced less tissue distortion than mechanical crushing. Immunoreactive cells included connective glia and some Microglia. Calmodulin was necessary for the eNOS immunoreactivity: it was blocked by calmodulin antagonist W7 (25 microM), but not by similar concentrations of the less potent calmodulin antagonist W12. Thus in the leech CNS, in which axon and synapse regeneration is successful, an increase in NOS activity at lesions appears to be among the earliest responses to injury and may be important for repair of axons

  46. Vikman K, Robertson B, Grant G, Liljeborg A, Kristensson K (1998) Interferon-gamma receptors are expressed at synapses in the rat superficial dorsal horn and lateral spinal nucleus. J.Neurocytol. 27:749-759
    Abstract: Interferon-gamma can facilitate the spinal nociceptive flexor reflex and may elicit neuropathic pain-related behavior in rats and mice. Immunoreactivity for the interferon-gamma receptor (IFN-gamma R) occurs in the superficial layers of the dorsal horn and the lateral spinal nucleus in the rat and mouse spinal cord, as well as in subsets of neurons in the dorsal root ganglia. The aim of the present study was to examine the cellular localization and origin of the IFN-gamma R in the spinal cord. As viewed by confocal microscopy, the immunopositivity for the IFN-gamma R was co-localized with that of the presynaptic marker synaptophysin and with neuronal nitric oxide synthase in the lateral spinal nucleus, whereas only a minor overlap with these molecules was observed in laminae I and II of the dorsal horn. There was no co-localization of the IFN-gamma R with markers for astrocytes and Microglial cells. Ultrastructurally, the IFN-gamma R was found predominantly in axon terminals in the lateral spinal nucleus, but at postsynaptic sites in dendrites in laminae I and II. The IFN-gamma R expressed in neurons in dorsal root ganglia was transported in axons both centrally and peripherally. Hemisection of the spinal cord caused no reduction in immunolabelling of the IFN-gamma R in the dorsal horn or the lateral spinal nucleus. Since rhizotomy does not affect the immunolabelling in the lateral spinal nucleus, our observation indicates that the presynaptic receptors in this nucleus are derived from intrinsic neurons. The localization of the IFN-gamma R in the spinal cord differed from that of the AMPA glutamate receptor subunits 2 and 3 and the substance P receptor (NK1). Our results, showing localization of IFN-gamma R to pre- and postsynaptic sites in the dorsal horn and lateral spinal nucleus indicate that IFN-gamma can modulate nociception at the spinal cord level

  47. Wu YP, Ling EA (1998) Transsynaptic changes of neurons and associated Microglial reaction in the spinal cord of rats following middle cerebral artery occlusion. Neurosci.Lett. 256:41-44
    Abstract: This study investigated transsynaptic neuronal damage and Microglial reaction in the spinal cord contralateral to focal cerebral ischaemia in rats induced by permanent occlusion of the right middle cerebral artery (MCA). Three and five days after MCA occlusion, some neurons in the dorsal horn of lumbar spinal cord underwent degeneration and they appeared to be engulfed by reactive Microglia; on the other hand, the ventral horn neurons remained ultrastructurally intact. It is suggested that the neuronal degeneration in the dorsal horn was attributed to deafferentation of the local neurons following ischaemic lesion of the corticospinal neurons which are the main source of afferent inputs

  48. Appel NM, Rapoport SI, O'Callaghan JP (1997) Sequelae of parenteral domoic acid administration in rats: comparison of effects on different anatomical markers in brain. synapse 25:350-358
    Abstract: Brain damage following administration of domoic acid, a structural analog of the excitatory amino acids glutamic acid and kainic acid, was compared using different anatomic markers in adult rats. Seven days after administration of domoic acid (2.25 mg/kg i.p.) or vehicle, brains were collected and sectioned and stained to visualize Nissl substance using thionin, argyrophilia using a cupric silver staining method, astroglia using immunohistochemistry to detect glial fibrillary acidic protein-like immunoreactivity (GFAP-ir), and activated Microglia using lectin histochemistry to detect Griffonia simplicifolia I-B4 isolectin (GSI-B4) binding in adjacent sections. In approximately 60% of rats to which it was administered, domoic acid caused stereotyped behavior within 60 min, followed by convulsions within 2-3 h. Brains of domoic acid-administered rats that did not manifest stereotyped behavior or convulsions did not differ from brains from vehicle-administered controls. In animals that had manifested stereotyped behavior and convulsions, Nissl staining was mostly unremarkable in brain sections. In contrast, there was intense argyrophilia in anterior olfactory nucleus, CA1 hippocampus, lateral septum, parietal (layer IV), piriform, and entorhinal cortices, ventral posterolateral thalamus, and amygdala. This pattern was reminiscent of that seen in postmortem specimens from humans who consumed domoic acid-tainted mussels and in experimental animals after kainic acid administration. Adjacent sections displayed astrogliosis, evidenced by increased GFAP-ir, which was more diffuse than the argyrophilic reaction. Activated Microglia were revealed using GSI-B4 histochemistry. These data suggest activation of discrete brain circuits in rats that convulse following domoic acid administration and subsequent pathological alterations. The data strongly suggest that neuropathology following domoic acid occurs only in animals manifesting domoic acid-induced sterotypy and convulsions. The data do not rule out more insidious damage in behaviorally normal rats that receive domoic acid

  49. Dal Canto MC (1997) Mechanisms of HIV infection of the central nervous system and pathogenesis of AIDS-dementia complex. Neuroimaging Clin.N.Am. 7:231-241
    Abstract: In many patients with AIDS, severe neurologic deficits develop that have been designated the cf2HIV-associated cognitive-motor complex. cf1 Pathologically, these symptoms correlate with a low-grade inflammatory condition, referred to as cf2HIV encephalitis,cf1 in which the most characteristic change is the presence of multinucleated giant cells. Cortical changes include neuronal loss and alterations of dendrites and synapses. There is pallor of white matter generally associated with the mononuclear inflammatory infiltrates. The only cells that seem to be directly infected by HIV are the Microglia/monocyte and the giant cells derived from fusion of monocytes. It is hypothesized, therefore, that cortical and white matter alterations in patients with this syndrome depend on the production of injurious soluble factors liberated by these cells and by astrocytes under the influence of many of these same factors. This article reviews recent advances in the understanding of these secondary effects and discusses pathogenetic mechanisms of tissue injury

  50. Kotecha SA, Eley DW, Turner RW (1997) Tissue printed cells from teleost electrosensory and cerebellar structures. J.Comp Neurol. 386:277-292
    Abstract: A modification of the tissue printing technique was used to acutely isolate and culture cells from the electrosensory lateral line lobe (ELL), corpus cerebelli (CCb), and eminentia granularis pars posterior (EGp) of the adult weakly electric fish, Apteronotus leptorhynchus. Cells were isolated without the use of proteolytic enzymes and tissue printed as a monolayer onto glass coverslips through centrifugation in the presence of a medium designed to preserve cell structure. Tissue printed cells were reliably distributed in an organotypic fashion that allowed for the identification of anatomical boundaries between the ELL and cerebellar regions, distinct sensory maps in the ELL, and specific cell laminae. Many cells were isolated with an excellent preservation of soma-dendritic structure, permitting direct identification of all electrosensory cell classes according to morphological or immunocytochemical criteria. Several classes of glial cells were isolated, including small diameter Microglia and the complex arborizations of oligodendrocytes. A plexus of fine processes were often isolated in conjunction with cell somata and dendrites, potentially preserving synaptic contacts in vitro. In particular, immunolabel for gamma-aminobutyric acid (GABA) revealed a previously unrecognized network of GABAergic axonal processes in the CCb and EGp granule cell body and molecular layers. Tissue printed cells were readily maintained with an organotypic distribution of glial and neuronal elements for up to 27 days in culture. This procedure will allow for the isolation of electrosensory cells from adult central nervous system for electrophysiological analyses of membrane properties or synaptic interactions between identified cells

  51. Kretzschmar HA, Giese A, Brown DR, Herms J, Keller B, Schmidt B, Groschup M (1997) Cell death in prion disease. J.Neural Transm.Suppl 50:191-210
    Abstract: Prion diseases are neurodegenerative transmissible diseases. The infectious agent, termed prion, is thought to consist of an altered host-encoded protein. The pathogenesis of these diseases which typically in a very short time lead to rampant nerve cell death and astrocytic gliosis is poorly understood. Investigations using the in situ endlabeling technique and electron microscopy in a scrapie model in the mouse (79A strain) show that nerve cell death is due to apoptosis. A cell culture model using a synthetic peptide of the prion protein (PrP106-126) shows that this peptide is toxic only to normal neurons whereas nerve cells derived from PrP knock-out (PrP0/0) mice are unaffected by this neurotoxic effect. In addition, Microglia play a crucial part in this process by secreting reactive oxygen species. Experiments in animals will have to show whether these cell culture findings adequately reflect the in vivo pathogenesis

  52. O'Hearn E, Molliver ME (1997) The olivocerebellar projection mediates ibogaine-induced degeneration of Purkinje cells: a model of indirect, trans-synaptic excitotoxicity. J.Neurosci. 17:8828-8841
    Abstract: Ibogaine, an indole alkaloid that causes hallucinations, tremor, and ataxia, produces cerebellar neurotoxicity in rats, manifested by degeneration of Purkinje cells aligned in narrow parasagittal bands that are coextensive with activated glial cells. Harmaline, a closely related alkaloid that excites inferior olivary neurons, causes the same pattern of Purkinje cell degeneration, providing a clue to the mechanism of toxicity. We have proposed that ibogaine, like harmaline, excites neurons in the inferior olive, leading to sustained release of glutamate at climbing fiber synapses on Purkinje cells. The objective of this study was to test the hypothesis that increased climbing fiber activity induced by ibogaine mediates excitotoxic Purkinje cell degeneration. The inferior olive was pharmacologically ablated in rats by a neurotoxic drug regimen using 3-acetylpyridine, and cerebellar damage attributed to subsequent administration of ibogaine was analyzed using immunocytochemical markers for neurons and glial cells. The results show that ibogaine administered after inferior olive ablation produced little or no Purkinje cell degeneration or glial activation. That a lesion of the inferior olive almost completely prevents the neurotoxicity demonstrates that ibogaine is not directly toxic to Purkinje cells, but that the toxicity is indirect and dependent on integrity of the olivocerebellar projection. We postulate that ibogaine-induced activation of inferior olivary neurons leads to release of glutamate simultaneously at hundreds of climbing fiber terminals distributed widely over the surface of each Purkinje cell. The unique circuitry of the olivocerebellar projection provides this system with maximum synaptic security, a feature that confers on Purkinje cells a high degree of vulnerability to excitotoxic injury

  53. Pfrieger FW, Barres BA (1997) Synaptic efficacy enhanced by glial cells in vitro. Science 277:1684-1687
    Abstract: In the developing nervous system, glial cells guide axons to their target areas, but it is unknown whether they help neurons to establish functional synaptic connections. The role of glial cells in synapse formation and function was studied in cultures of purified neurons from the rat central nervous system. In glia-free cultures, retinal ganglion cells formed synapses with normal ultrastructure but displayed little spontaneous synaptic activity and high failure rates in evoked synaptic transmission. In cocultures with neuroglia, the frequency and amplitude of spontaneous postsynaptic currents were potentiated by 70-fold and 5-fold, respectively, and fewer transmission failures occurred. Glial cells increased the action potential-independent quantal release by 12-fold without affecting neuronal survival. Thus, developing neurons in culture form inefficient synapses that require glial signals to become fully functional

  54. Schoen SW, Kreutzberg GW (1997) 5'-nucleotidase enzyme cytochemistry as a tool for revealing activated glial cells and malleable synapses in CNS development and regeneration. Brain Res.Brain Res.Protoc. 1:33-43
    Abstract: The demonstration of 5'-nucleotidase in neural tissue is achieved at both the light and electron microscopic levels by means of an enzyme cytochemical lead method, which is specific, sensitive and fast. By its activity this adenosine-producing ecto-enzyme (EC 3.1.3.5) outlines cellular surface membranes at the ultrastructural level. It is classically known as a marker of myelin and of astrocytes as well as (activated) Microglial cells in the mature nervous system. In recent years, we discovered that 5'-nucleotidase is transiently active within synaptic clefts under conditions of development and regeneration. The enzyme is also seen at terminals in the mature retina and olfactory bulb, where spontaneous synaptic turnover occurs at adulthood. Thus, 5'-nucleotidase cytochemistry is useful in revealing sites of glial reactions and synaptic plasticity in CNS development and repair. It is assumed that the molecule affects terminal formation and cell motility due to dual functions in adenosine production and cell adhesion. Finally, at the light microscopic level, 5'-nucleotidase activity displays a dense neuropil staining which identifies topographic sub-units of certain parts of the nervous system, such as the striosomes of the basal ganglia, ocular dominance columns of the visual cortex and parasagittal bands of the cerebellum

  55. Thanos S (1997) Neurobiology of the regenerating retina and its functional reconnection with the brain by means of peripheral nerve transplants in adult rats. Surv.Ophthalmol. 42 Suppl 1:S5-26
    Abstract: Axotomy-induced degradation of retinal ganglion cells (RGC) can be delayed if the destructive features of activated Microglial cells are pharmacologically neutralized, and prevented if the axons are permitted to regrow into transplanted autologous peripheral nerve (PN) pieces. Axotomized central nervous system neurons, whose regenerating axons are guided to their natural target areas in the brain with the aid of PN grafts, are capable of establishing synaptic contacts with normal morphological and electrophysiological properties. This study was undertaken to 1) morphometrically characterize and classify the regenerating rat RGC, 2) examine target-dependent effects on survival of subsets of neurons, and 3) investigate whether reconnected neurons are capable of restoring visual functions. In analogy to the normal rat retina, as a first step, the retrogradely labeled, regenerating RGC were categorized into five classes which are morphologically distinct and reminiscent of normal RGC correlates (called types RI, RII, RIII, Rdelta-cells, and displaced RGC). It appeared that all types of ganglion cells contributed proportionally to regeneration of axons. Transplantation of a PN graft which was not reconnected with a central target (blind-ending group) and monitoring of the extant neurons showed a progressive disappearance of the regenerating RGC, such that 6 months after surgery predominantly few, large cells survived. When the retinas were treated with macrophage/Microglia inhibiting factor (MIF), and the regenerating axons were guided into the pretectum, predominantly large RGC of type RI survived. Guidance of the axons into their major natural target, the superior colliculus (SC), resulted in selective survival of many small, RII-like RGC. Calculation of the dendritic coverage factors for the major types of RGC revealed that dendrites of the most abundant, small cells of type RII overlapped uniformly and covered the retinal surface completely, whereas cells of types RI and RIII did not suffice for surface coverage. The results of this first part of the work suggest that combined suppression of axotomy-induced Microglial activation and guidance of regenerating axons with a PN graft into central targets is a suitable technique to produce sufficient numbers of regenerating axons which may retrieve some functional properties. Target-specific neuronal contacts are likely involved in morphological stabilization and better survival of regenerating neurons. The second goal of this study was to analyze the functional significance of the reestablished synaptic contacts made by regenerated retinocollicular neurons. Adult rats were trained in a T- or Y-maze to obtain a food reward with the aid of visual cues. One of their optic nerves was transected and the regenerating axons were guided into the optic tract with a PN graft, to enable them to reinnervate the SC and thalamus. Postoperative testing of the animals showed a drastic improvement of visual perception. The protocol of denervation of the SC (prior to, simultaneous with, or with a delay with respect to fiber arrival) determined the performance of the animals. Rats belonging to the first two groups performed almost as well as they had before the transplantation. The functional integrity of the retina was assessed by electroretinography, which revealed typical rod spectral sensitivity at 380 and 500 nm but reduced responsiveness to illumination. In accordance, neuroanatomical assessment of the functionally relevant RGC revealed intact morphologies and multiple synaptic contacts both within the retina and within the SC. Neuroanatomical tracing of small contingents of axons throughout the regenerative pathway revealed a rough retinotopic arrangement within the graft and the area of termination. Thus, animals could discriminate between simplified vertical versus horizontal stripes, and visual evoked potentials were positive after grafting. (ABSTRACT TRUNCATED)

  56. Fine SM, Angel RA, Perry SW, Epstein LG, Rothstein JD, Dewhurst S, Gelbard HA (1996) Tumor necrosis factor alpha inhibits glutamate uptake by primary human astrocytes. Implications for pathogenesis of HIV-1 dementia. J.Biol.Chem. 271:15303-15306
    Abstract: Human immunodeficiency virus (HIV) infection is commonly associated with neurological disease that occurs in the apparent absence of extensive infection of brain cells by HIV, suggesting that indirect mechanisms account for neuropathogenesis in the CNS, perhaps including changes in the normal neuroprotective functions of astrocytes. To test this hypothesis, we examined the effect of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFalpha), produced by HIV-1-infected macrophages and Microglia, on glutamate transport by primary human fetal astrocytes (PHFAs). A dose-dependent inhibition of high affinity glutamate uptake sites was observed 12-24 h after addition of exogenous recombinant human TNFalpha to PHFAs. This effect was specific since it was blocked by a neutralizing monoclonal antibody directed against TNFalpha. Furthermore, the inhibitory effect was reproduced by a monoclonal antibody that is an agonist at the 55-kDa TNF receptor. These results suggest that the neurotoxic effects of TNFalpha may be due in part to its ability to inhibit glutamate uptake by astrocytes, which in turn may result in excitotoxic concentrations of glutamate in synapses

  57. Hull M, Strauss S, Berger M, Volk B, Bauer J (1996) Inflammatory mechanisms in Alzheimer's disease. Eur.Arch.Psychiatry Clin.Neurosci. 246:124-128
    Abstract: In recent years many studies have indicated an involvement of inflammatory mechanisms in Alzheimer's disease (AD). Acute-phase proteins such as alpha 1-antichymotrypsin and c-reactive protein, elements of the complement system, and activated Microglial and astroglial cells are consistently found in brains of AD patients. Most importantly, also cytokines such as interleukin-6 (IL-6) have been detected in the cortices of AD patients, indicating a local activation of components of the unspecific inflammatory system. Up to now it has remained unclear whether inflammatory mechanisms represent a primary event or only an unspecific reaction to brain tissue damage. Therefore, we investigated whether IL-6 immunoreactivity could be found in plaques prior to the onset of neuritic changes, or whether the presence of this cytokine is restricted to later stages of plaque pathology. We confirmed our previous observation that IL-6 is detectable in a significant proportion of plaques in the brains of demented patients. In AD patients IL-6 was found in diffuse plaques in a significant higher ratio as would have been expected from a random distribution of IL-6 among all plaque types. This observation suggests that IL-6 may precede neuritic changes, and that immunological mechanism may be involved both in the transformation from diffuse to neuritic plaques in AD and in the development of dementia

  58. Mason A, Muller KJ (1996) Accurate synapse regeneration despite ablation of the distal axon segment. Eur.J.Neurosci. 8:11-20
    Abstract: In each body ganglion of the leech Hirudo medicinalis there is a single S-cell. After an S-cell axon is severed, it regenerates along its surviving distal segment and reconnects with its synaptic target, the axon of the neighbouring S-cell. In approximately half the cases the regenerating axon forms a temporary electrical synapse specifically with the distal segment, which remains active and connected to the target, thereby functioning as a splice until regeneration is complete. To determine whether the distal axon segment is required for successful regeneration, distal segments of severed S-cell axons were ablated by intracellular injection of bacterial protease. Fifty-seven preparations were examined from 2 to 212 days after injection of the axon segment. The extent of S-cell axon regeneration was assessed electrophysiologically by intracellular and extracellular recording, and anatomically by intracellular injection of markers followed by light microscopy and electron microscopy. The S-cell axons regenerated successfully in almost 90% of animals examined after 2 weeks or more. In a further four animals the target S-cell was ablated in addition to the distal axon segment, permanently disrupting conduction along the S-cell pathway. Nevertheless, the regenerating axon grew along its usual pathway and there was no evidence that alternative connections were formed. It is concluded that, although the distal axon segment can provide a means for rapid functional repair, the segment is not required for reliable regeneration of the axon along its usual pathway and accurate formation of an electrical synapse

  59. Mennerick S, Benz A, Zorumski CF (1996) Components of glial responses to exogenous and synaptic glutamate in rat hippocampal microcultures. J.Neurosci. 16:55-64
    Abstract: Although glia are known to be sensitive to exogenously applied neurotransmitter substances, little is known about how glia respond to neuronal activity on the millisecond time scale of individual synaptic events. We have explored the electrophysiological effects of excitatory neuronal signaling on glial cells in rat hippocampal microcultures. Exogenous applications of glutamate analogs to islands of glia revealed a large ionotropic AMPA receptor-mediated current and a smaller current mediated by electrogenic glutamate uptake. Glia demonstrated no evidence of NMDA or metabotropic receptor-mediated currents or membrane conductance changes. Despite the dominant contribution of AMPA receptors to exogenous glutamate applications in glia, AMPA receptor currents contributed only approximately 20% to the response of glia to endogenous glutamate release from solitary excitatory neurons. Electrogenic glutamate uptake contributed strongly to glial responses to neuronal stimulation. In addition, some glia exhibited a residual current is response to neuronal stimulation that was not attributable to calcium-dependent transmitter release or to neuronal potassium efflux. These results help provide a context for understanding the role of glial transporters and receptors in nervous system signaling

  60. Oster-Granite ML, McPhie DL, Greenan J, Neve RL (1996) Age-dependent neuronal and synaptic degeneration in mice transgenic for the C terminus of the amyloid precursor protein. J.Neurosci. 16:6732-6741
    Abstract: The molecular basis for the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature in Alzheimer's disease (AD) is incompletely understood. We have proposed that one molecule common to these abnormal processes is a fragment of the Alzheimer amyloid precursor protein (APP) comprising the C-terminal 100 amino acids of this molecule (APP-C100). We tested this hypothesis by creating transgenic mice expressing APP-C100 in the brain. We report here that aging (18-28 month) APP-C100 transgenic mice exhibit profound degeneration of neurons and synapses in Ammon's horn and the dentate gyrus of the hippocampal formation. Of the 106 transgenic mice between 8 and 28 months of age that were examined, all of those older than 18 months displayed severe hippocampal degeneration. The numerous degenerating axonal profiles contained increased numbers of neurofilaments, whorls of membrane, and accumulations of debris resembling secondary lysosomes near the cell body. The dendrites of degenerating granule and pyramidal cells contained disorganized, wavy microtubules. Cerebral blood vessels had thickened refractile basal laminae, and Microglia laden with debris lay adjacent to larger venous vessels. Mice transgenic for Flag-APP-C100 (in which the hydrophilic Flag tag was fused to the N terminus of APP-C100) showed a similar degree of neurodegeneration in the hippocampal formation as early as 12 months of age. The 45 control mice displayed only occasional necrotic cells and no extensive cell degeneration in the same brain regions. These findings show that APP-C100 is capable of causing some of the neuropathological features of AD

  61. Nacimiento W, Sappok T, Brook GA, Toth L, Schoen SW, Noth J, Kreutzberg GW (1995) Structural changes of anterior horn neurons and their synaptic input caudal to a low thoracic spinal cord hemisection in the adult rat: a light and electron microscopic study. Acta Neuropathol.(Berl) 90:552-564
    Abstract: Structural changes in lumbosacral ventral horn neurons and their synaptic input were studied at 3, 10, 21, 42, and 90 days following low thoracic cord hemisection in adult rats by light microscopic examination of synaptophysin immunoreactivity (SYN-IR) and by electron microscopy. There was an ipsilateral transient decrease in SYN-IR at the somal and proximal dendritic surfaces of anterior horn neurons which extended caudally from the site of injury over a postoperative (p.o.) period of 42 days. Concomitantly, at 21 days p.o., perineuronal SYN-IR started to recover in upper lumbar segments. By 90 days p.o., a normal staining pattern of SYN was noted in upper and mid lumbar segments, but the perineuronal SYN-IR was still slightly below normal levels in low lumbar and sacral segments. Electron microscopy revealed ultrastructural changes coincident with the alterations in SYN-IR. At 3 days p.o., phagocytosis of degenerating axon terminals by activated Microglial cells was observed at the somal and proximal dendritic surfaces of ventral horn neurons. These changes were most prominent up to two segments caudal to the lesion. At 10 days p.o., advanced stages of bouton phagocytosis were still detectable in all lumbosacral motor nuclei. Additionally, abnormal axon terminals, with a few dispersed synaptic vesicles and accumulations of large mitochondria, appeared at the scalloped somal surfaces of anterior horn neurons. At 21 days p.o., several large lumbosacral motoneurons had developed chromatolysis-like ultrastructural alterations and motoneuronal cell bodies had become partially covered by astrocytic lamellae. At 42 days p.o., there was a transient appearance of polyribosomes in some M-type boutons. In addition, at 42 and 90 days p.o., a few degenerating motoneurons were detected in all lumbosacral segments, but most displayed normal neuronal cell bodies contacted by numerous intact synapses as well as by astrocytic processes. In contrast to these striking alterations of synaptic input at somal and proximal dendritic surfaces of motoneurons, relatively few degenerating boutons were detected in the neuropil of motor nuclei at all the p.o. times studied. We suggest that the preferential disturbance of the predominantly inhibitory axosomatic synapses on ventral horn neurons may be involved in the mechanisms which influence the well-established increase in motoneuronal excitability after spinal cord injury

  62. von Bernhardi R, Muller KJ (1995) Repair of the central nervous system: lessons from lesions in leeches. J.Neurobiol. 27:353-366
    Abstract: In contrast to the limited repair observed in the mammalian central nervous system (CNS), injured neurons in the leech reliably regenerate synapses and restore function with remarkable accuracy at the level of individual neurons. New and recent results reveal important roles for Microglial cells and extracellular matrix components, including laminin, in repair. Tissue culture experiments have permitted isolation of neurons and manipulation of their environment, providing insights into the influence of substrate, electrical activity, and other cells, including Microglia, on axon growth and synapse formation. The results account for distinctive features of successful repair in the adult leech, where axonal sprouting and target selection can be influenced by unequal competition between neurons. Differences between the formation of connections during embryonic development and repair in the adult include dissimilarities in the roles of glia and Microglia in adults and embryos, suggesting that axon growth during regeneration in the CNS is not simply a recapitulation of processes observed during embryonic development. It may be possible in the future to improve mammalian CNS regeneration by recruiting cells whose counterparts in the leech have been identified as instrumental in repair

  63. Guntinas-Lichius O, Neiss WF, Gunkel A, Stennert E (1994) Differences in glial, synaptic and motoneuron responses in the facial nucleus of the rat brainstem following facial nerve resection and nerve suture reanastomosis. Eur.Arch.Otorhinolaryngol. 251:410-417
    Abstract: Transection and reanastomosis of the facial nerve with microsurgical sutures in rats (facial-facial anastomosis) results in the complete regeneration of the facial nucleus, whereas resection of a 10 mm length of the peripheral facial nerve leads to degeneration and loss of neurons in the nucleus. Nerve sutures or resections were performed in 84 female Wistar rats, and the time course and differences between regenerative and degenerative reactions in the facial nuclei were compared after survival times of 4-112 days. The volume of the facial nucleus, number of facial motoneurons and motoneuron density were estimated stereologically by the physical dissector method. Synaptic plasticity, activation of astroglia and Microglia were studied cytochemically with anti-synaptophysin, anti-glial fibrillary acidic protein and the isolectin Griffonia simplicifolia I-B4 (GSA I-B4). After facial-facial anastomosis the volume of the facial nucleus and its number of motoneurons remained constant, whereas resection of the facial nerve caused shrinkage of the facial nucleus and loss of one-third of facial motoneurons within 112 days post-operation. Synaptic stripping, activation of Microglia and astroglia occurred in the same sequence and were reversible after both operations, but these reactions were more severe and prolonged after resection, i.e. without suture of the facial nerve. It appears to be most important clinically that differences between de- and regeneration become clear within 7 days post-axotomy. Our results strongly support reconstruction of the facial nerve as early as possible after a nerve lesion

  64. Martin LJ, Pardo CA, Cork LC, Price DL (1994) Synaptic pathology and glial responses to neuronal injury precede the formation of senile plaques and amyloid deposits in the aging cerebral cortex. Am.J.Pathol. 145:1358-1381
    Abstract: The cerebral cortices of macaques (ranging in age from 10 to 37 years; n = 17) were analyzed by immunocytochemistry and electron microscopy to determine the cellular and subcellular localizations of the amyloid precursor protein and beta-amyloid protein, the cellular participants in the formation of senile plaques and parenchymal deposits of the beta-amyloid protein, and the temporal/spatial development of these lesions. Amyloid precursor protein was enriched within the cytoplasm of pyramidal and nonpyramidal neuronal cell bodies in young and old monkeys. In the neuropil, amyloid precursor protein was most abundant within dendrites and dendritic spines; few axons, axonal terminals, and resting astrocytes and Microglia contained the amyloid precursor protein. At synapses, amyloid precursor protein was found predominantly within postsynaptic elements and was enriched at postsynaptic densities of asymmetrical synapses. The earliest morphological change related to senile plaque formation was an age-related abnormality in the cortical neuropil characterized by the formation of dense bodies within presynaptic terminals and dendrites and an augmented localization of the amyloid precursor protein to astrocytes and Microglia. In most monkeys > 26 years of age, the neocortical parenchyma exhibited neuritic pathology and plaques characterized by swollen cytoplasmic processes, interspersed somata of neurons, and reactive glia within or at the periphery of senile plaques. Neurites and reactive astrocytes and Microglia within these plaques were enriched with the amyloid precursor protein. In diffuse plaques, nonfibrillar beta-amyloid protein immunoreactivity was visualized within cytoplasmic lysosomes of neuronal perikarya and dendrites and the cell bodies and processes of activated astrocytes and Microglia. In mature plaques, beta-amyloid protein immunoreactivity was associated with extracellular fibrils within the parenchyma; some cytoplasmic membranes of degenerating dendrites and somata as well as processes of activated glia showed diffuse intracellular beta-amyloid protein immunoreactivity. We conclude that morphological abnormalities at synapses (including changes in both pre- and postsynaptic elements) precede the accumulation of the amyloid precursor protein within neurites and activated astrocytes and Microglia as well as the deposition of extracellular fibrillar beta-amyloid protein; neuronal perikarya/dendrites and reactive glia containing the amyloid precursor protein are primary sources of the beta-amyloid protein within senile plaques; and nonfibrillar beta-amyloid protein exists intracellularly within neurons and nonneuronal cells prior to the appearance of extracellular deposits of the beta-amyloid protein and the formation of beta-pleated fibrils.(ABSTRACT TRUNCATED AT 400 WORDS)

  65. Pallera AM, Schweitzer JB, Book AA, Wiley RG (1994) 192 IgG-saporin causes a major loss of synaptic content in rat olfactory bulb. Exp.Neurol. 127:265-277
    Abstract: An immunotoxin composed of a monoclonal antibody that recognizes the p75 nerve growth factor (NGF) receptor disulfide-linked to the ribosome-inactivating protein saporin selectively eliminates p75-expressing cholinergic neurons in the basal forebrain, while sparing other neurons in the forebrain, both cholinergic and noncholinergic. We now report the effect that intraventricular administration of this immunotoxin has on the synaptic content of the olfactory bulb, one of the major terminal fields of the cholinergic basal forebrain system. Control substances or immunotoxin were given to rats followed by a 2-week survival. Unilateral transection of the olfactory tract and peduncle was also studied. Both qualitative and quantitative evaluation of olfactory bulbs processed for synaptophysin immunohistochemistry indicated dramatic loss of synapses in the four regions of neuropil evaluated (glomeruli, outer and inner halves of the external plexiform layer, and internal plexiform layer) compared with the administration of control substances. Surgical transection of the bulb produced a visually similar decrement, but quantitative studies showed synaptic loss to be consistently greater following tract transection. The effects of these two insults on the glial response were remarkably different. Transection produced an obvious hyperplasia and hypertrophy of both astrocyte and Microglial elements, while immunotoxin produced small, almost undetectable reactions by these two cell types. The results in the glomeruli strongly suggest an effect of the immunotoxin on either periglomerular cells or olfactory nerve terminals, whether directly by NGF receptor (+) structures or by trans-synaptic mechanisms. We conclude that the immunotoxin produces a specific and large loss of synapses that does not produce much glial response

  66. Schoen SW, Kreutzberg GW (1994) Synaptic 5'-nucleotidase activity reflects lesion-induced sprouting within the adult rat dentate gyrus. Exp.Neurol. 127:106-118
    Abstract: In development, the ectoenzyme 5'-nucleotidase marks maturing cerebellar and cortical synapses, but it is localized in glial cells in the normal, adult nervous system. With a histochemical lead technique, we have now investigated its localization during reactive synaptogenesis in the dentate gyrus of adult rats deprived of entorhinal afferents. A band of enhanced 5'-nucleotidase reaction product was present in the outer portions of the dentate molecular layer between 5 and 75 days after destruction of the ipsilateral entorhinal cortex. At the ultrastructural level, 5'-nucleotidase-positive Microglia and degenerating axon terminals were numerous within this band during the first postoperative week. Between Days 7 and 75, intact synapses were found that exhibited 5'-nucleotidase reaction product in their clefts. Astrocytic labeling was abundant. No enzyme-positive synapses and few labeled glial elements were present in the control molecular layer. Conspicuous 5'-nucleotidase activity within synaptic clefts of mossy fiber terminals was present between Postoperative Days 10 and 40 on the operated side, but the staining was sporadic on the control side. We conclude that 5'-nucleotidase is associated with lesion-induced synaptic remodeling in the dentate gyrus. The band of 5'-nucleotidase reaction product within the outer molecular layer corresponds to the zone where the lesioned entorhinal fibers degenerate and where other afferents sprout. Here, the transient appearance of 5'-nucleotidase within synaptic clefts parallels the time course of synaptic reinnervation. The enzyme is also indicative of the sprouting response of mossy fiber terminals. Functional properties of 5'-nucleotidase in purinergic neuromodulation and cellular adhesion may be relevant for the generation and plasticity of synaptic contacts

  67. Beyreuther K, Pollwein P, Multhaup G, Monning U, Konig G, Dyrks T, Schubert W, Masters CL (1993) Regulation and expression of the Alzheimer's beta/A4 amyloid protein precursor in health, disease, and Down's syndrome. Ann.N.Y.Acad.Sci. 695:91-102
    Abstract: A four- to fivefold overexpression of the gene for the Alzheimer beta/A4 amyloid precursor protein (APP) in individuals with Down's Syndrome (DS) appears to be responsible for the fifty year earlier onset of Alzheimer's disease (AD) pathology in DS compared to the normal population. It is therefore likely that a deregulated overexpression of the APP gene is a risk factor for the beta/A4 amyloid formation. To test this hypothesis and to get a better understanding of how APP expression is regulated, we studied the 5' control region of the human APP gene, alternative splicing of the 19 APP exons, and APP biogenesis, metabolism and function. The analysis of the APP promoter revealed its similarity with those of housekeeping genes by the presence of a GC-rich region around the transcription start site and the lack of a TATA box. Gene transfer experiments showed this GC-rich region to contain overlapping binding sites for different transcription factors whose binding is mutually excluded. An imbalance between these factors may cause APP overexpression and predispose to AD pathology. Another putative risk factor for AD is regulation of splicing of exon 7 in APP mRNA's which changes in brain during aging. This is relevant for APP processing since exon 7 codes for a Kunitz protease inhibitory domain. Investigation of further splicing adjacent to the beta/A4 exons 16 and 17 which might also interfere with APP processing led to the identification of the leukocyte-derived (L-APP) splice forms which lack exon 15. In brain this splicing occurs in activated astrocytes and Microglia. The localization of APP at synaptic sites in brain suggests that APP regulation and expression are critical determinants of a potential and early impairment of central synapses. This may be the case during pathological evolution of AD and DS when beta/A4 derived from synaptic APP is converted to beta/A4 amyloid by radical generation

  68. Bruckner G, Brauer K, Hartig W, Wolff JR, Rickmann MJ, Derouiche A, Delpech B, Girard N, Oertel WH, Reichenbach A (1993) Perineuronal nets provide a polyanionic, glia-associated form of microenvironment around certain neurons in many parts of the rat brain. Glia 8:183-200
    Abstract: The nature and function of previously described perineuronal nets are still obscure. In the present study their polyanionic components were demonstrated in the rat brain using colloidal iron hydroxide (CIH) staining. In subcortical regions, such as the red nucleus, cerebellar, and vestibular nuclei, most neurons were ensheathed by CIH-binding material. In the cerebral cortex perineuronal nets were seen around numerous nonpyramidal neurons. Biotinylated hyaluronectin revealed that hyaluronan occurs in perineuronal nets. Two plant lectins [Wisteria floribunda agglutinin (WFA) and Vicia villosa agglutinin (VVA)] with affinity for N-acetylgalactosamine visualized perineuronal nets similar to those rich in anionic components. Glutamic acid decarboxylase (GAD)-immunoreactive synaptic boutons were shown to occupy numerous meshes of perineuronal VVA-positive nets. Electron microscopically, VVA binding sites were scattered throughout perisynaptic profiles, but accumulated at membranes and in the extracellular space except not in synaptic clefts. To investigate the spatial relationship between glial cell processes and perineuronal nets, two astrocytic markers (S100-protein and glutamine synthetase) were visualized at the light and electron microscopic level. Two methods to detect Microglia by the use of Griffonia simplicifolia agglutinin (GSA I-B4) and the monoclonal antibody, OX-42, were also applied. Labelled structures forming perineuronal nets were observed with both astrocytic, but not with Microglial, markers. It is concluded that perineuronal nets are composed of a specialized type of glia-associated extracellular matrix rich in polyanionic groups and N-acetylgalactosamine. The net-like appearance is due to perisynaptic arrangement of the astrocytic processes and these extracellular components. Similar to the ensheathment of nodes of Ranvier, perineuronal nets may provide a special ion buffering capacity required around various, perhaps highly active, types of neurons

  69. Fix AS, Horn JW, Wightman KA, Johnson CA, Long GG, Storts RW, Farber N, Wozniak DF, Olney JW (1993) Neuronal vacuolization and necrosis induced by the noncompetitive N-methyl-D-aspartate (NMDA) antagonist MK(+)801 (dizocilpine maleate): a light and electron microscopic evaluation of the rat retrosplenial cortex. Exp.Neurol. 123:204-215
    Abstract: MK(+)801 (dizocilpine maleate) is a noncompetitive antagonist at the N-methyl-D-aspartate (NMDA) receptor, the major glutamate receptor at excitatory synapses in the central nervous system. Since NMDA antagonists are neuroprotective, there is interest in their development for treatment of cerebral ischemia. Unfortunately, many of these compounds also induce vacuole formation in neurons of the rat retrosplenial cortex (Olney et al., Science 244: 1360-1362, 1989). Although vacuolization was initially reported to be reversible with MK(+)801, preliminary data later suggested that higher doses might produce neuronal necrosis. To explore this issue, young male Sprague-Dawley rats were given a single subcutaneous dose of vehicle or 1, 5, or 10 mg/kg MK(+)801. At 4 h and 1, 2, 3, 4, 7, and 14 days postdose (DPD), the retrosplenial cortex was examined by light and electron microscopy. At 4 h, vacuoles occurred in neurons of retrosplenial cortical layers 3 and 4 in all rats given MK(+)801. Mitochondria and endoplasmic reticulum contributed to vacuole formation. At 1 DPD, vacuoles or necrotic neurons were rarely observed. At all subsequent time points, necrotic neurons were readily evident in rats given 5 or 10 mg/kg MK(+)801, but only rarely evident in rats given 1 mg/kg. Necrotic neurons were associated with reactive Microglial cells that contained electron-dense debris ultrastructurally. If similar dose-dependent neuronal necrosis proves to be a feature of other NMDA antagonists, such effects might raise concerns for the development and use of these compounds in human cerebrovascular diseases

  70. Gehrmann J, Gold R, Linington C, Lannes-Vieira J, Wekerle H, Kreutzberg GW (1993) Microglial involvement in experimental autoimmune inflammation of the central and peripheral nervous system. Glia 7:50-59
    Abstract: Microglial cells form a network of potential antigen presenting cells throughout the nervous system. Much progress has recently been made towards a better understanding of their immunological properties. This study examines their activation in 2 models of T cell-mediated autoimmune inflammation of the nervous system, experimental autoimmune encephalomyelitis (EAE) and its peripheral counterpart, experimental autoimmune neuritis (EAN), induced by the transfer of antigen-specific T cell lines. In both models Microglial activation occurs at early stages of the disease. Activated Microglial cells show an increased expression of MHC class I and II antigens. In EAE ultrastructural analysis revealed that MHC antigen expression is pronounced on perivascular Microglial cells, suggesting this cell population may be important for antigen presentation at a site close to the blood-brain barrier. In contrast to EAE, the Microglial reaction in EAN occurs at sites remote from the inflammatory response in the peripheral nerve, not only in the spinal cord but also in the terminal projection fields of primary sensory neurons in the lower brainstem. This early Microglial activation in EAN suggests that a rapid and remote signaling mechanism can operate following peripheral inflammation. Immuno-electron microscopy revealed that activated Microglial cells are also involved in the synaptic deafferentation of spinal cord motoneurons during autoimmune reactions. The rapid involvement of Microglial cells in experimental autoimmune inflammation of the nervous system further points to their role as the main intrinsic immuneffector cell population of the central nervous system

  71. Graeber MB, Bise K, Mehraein P (1993) Synaptic stripping in the human facial nucleus. Acta Neuropathol.(Berl) 86:179-181
    Abstract: An autopsy case of severe peripheral facial nerve paresis with disconnection of synapses from facial motor neurons is reported. A 77-year-old man presented with left-sided otitis media and subsequent development of facial nerve paresis. Three months later, the patient died of an acute gastrointestinal bleeding from a chronic duodenal ulcer. Gross inspection of the brain revealed non-stenosing arteriosclerotic vascular changes and a single small cystic lesion in the right putamen. Microscopically, marked chromatolytic changes were observed in the left facial nucleus. Immunocytochemistry for synaptophysin revealed a marked loss of afferent synaptic contacts from somatic and stem dendritic surface membranes of all chromatolytic motor neurons. Wrapping of a number of neurons by newly formed glial fibrillary acidic protein-positive astrocytic cell processes could be detected in the regenerating facial motor nucleus. In addition, expression of HLA-DR was increased on a small number of Microglia and perivascular cells. These changes were absent from the contralateral, normal-appearing facial nucleus. To our knowledge, this case provides the first evidence for disconnection of synapses following peripheral nerve lesioning in humans. Occurrence of synaptic stripping is likely to explain nuclear hyperexcitability and failure of recovery of complex fine motor movements that are commonly observed following peripheral injury to the facial nerve

  72. Mudrick-Donnon LA, Williams PJ, Pittman QJ, MacVicar BA (1993) Postsynaptic potentials mediated by GABA and dopamine evoked in stellate glial cells of the pituitary pars intermedia. J.Neurosci. 13:4660-4668
    Abstract: Studies have shown that many glial cells in the CNS possess receptors for neurotransmitters and that synapse-like contacts exist between glial cells and axonal terminals. Although synapse-like contacts are present between the glial cells (stellate cells) of the pituitary pars intermedia and the axons from the arcuate nucleus, it is not known whether these cells are under synaptic control. The objective of the present study was to determine whether transmitter-mediated postsynaptic potentials occurred in the stellate cells of the rat pituitary pars intermedia. Whole pituitaries were maintained in vitro, and a stimulating electrode was placed on the stalk to activate afferent fibers. Intracellular recordings were obtained with sharp microelectrodes. Stellate cells showed electrophysiological characteristics of macroglia including a resting potential more negative than -65 mV, low input resistance (< 50 M omega), and no detectable voltage-activated conductances. Single-pulse afferent nerve (stalk) stimulation evoked a [Ca2+]o-dependent postsynaptic response in the stellate cells consisting of a depolarization (< 500 msec) and a long-lasting hyperpolarization (45-75 sec). The depolarization was mimicked by GABA application and blocked by the GABAA antagonist bicuculline (100 microM). Repetitive stimulation of the stalk increased the amplitude and prolonged the GABA-mediated depolarization, during which a decrease in input resistance was observed. The hyperpolarization was mimicked by dopamine and blocked by the D2 antagonists sulpiride (2 microM) and domperidone (10 microM). Nipecotic acid (100 microM; an inhibitor of GABA uptake) or GBR 12909 (15 microM; an inhibitor of dopamine uptake) had minimal effects on the synaptic responses.(ABSTRACT TRUNCATED AT 250 WORDS)

  73. Svensson M, Aldskogius H (1993) Synaptic density of axotomized hypoglossal motorneurons following pharmacological blockade of the Microglial cell proliferation. Exp.Neurol. 120:123-131
    Abstract: The purpose of the present study was to examine the possible role of reactive Microglia in the removal of presynaptic terminals following motor axon injury. Quantitative electron microscopy was used to examine synaptic numbers and total relative synaptic coverage on hypoglossal neuronal perikarya following hypoglossal nerve transection in the rat with or without pharmacological blockade of the axotomy-induced Microglial cell proliferation. In a previous study we have shown that the axotomy-induced Microglial cell proliferation is selectively inhibited by continuous infusion of cytosine-arabinoside (ARA-C) into the ventricular system of the adult rat brain. Adopting this procedure in the present study resulted in an almost complete elimination of reactive Microglia. There was a statistically significant decrease in the number of synapses and the relative synaptic coverage in untreated as well as ARA-C-treated animals 4 and 7 days after nerve transection. Immunocytochemical labeling of terminals in the hypoglossal nucleus using antibodies to synaptophysin showed a reduction in immunoreactivity around hypoglossal nerve cell bodies ipsilateral to nerve transection in both groups of animals. These results indicate that reactive Microglia are not responsible for detachment of presynaptic terminals following motor axon injury

  74. Neiss WF, Lichius OG, Angelov DN, Gunkel A, Stennert E (1992) The hypoglossal-facial anastomosis as model of neuronal plasticity in the rat. Anat.Anz. 174:419-433
    Abstract: Hypoglossal-facial cross anastomosis (HFA) causes regeneration with change of function, as the axotomized hypoglossal motoneurons sprout into the facial plexus and reinnervate the mimic musculature. Following HFA, hypoglossal-hypoglossal single anastomosis (HHA) and resection of 8-10 mm peripheral hypoglossal nerve in 190 female adult Wistar rats, we compared the axon reactions in the hypoglossal nucleus during 1) regeneration with change of function, 2) regeneration with restoration of original function and 3) degeneration of the nucleus. Following postoperative survival times of 1-16 weeks we estimated the volume of the hypoglossal nucleus and counted the number of hypoglossal neurons with the physical disector on both sides of the brainstem. Additional sections of the same animals were reacted with anti-synaptophysin, anti-GFAP and the isolectin Griffonia simplicifolia I-B4 (GSA I-B4) as cytochemical markers for presynaptic boutons, activated astroglia and Microglia. After HHA and HFA all hypoglossal neurons survive and the volume of the hypoglossal nucleus remains constant. Resection of the hypoglossal nerve leads to the loss of one third of the hypoglossal neurons and of one third of the volume of the hypoglossal nucleus within 16 weeks post operation. Hypoglossal-facial anastomosis and hypoglossal-hypoglossal anastomosis differ in postoperative swelling of the hypoglossal nucleus, Microglia and astroglia activation and the duration of synaptic stripping. All differences are limited to the acute growth phase during regeneration. It is concluded that hypoglossal-facial anastomosis provides more stimulation and facilitates faster recovery of the hypoglossal nucleus than does hypoglossal-hypoglossal anastomosis

  75. Tay SS, Wong WC (1992) Long-term effects of alloxan-induced diabetes on the nucleus ventralis posterolateralis in the thalamus of the rat. Acta Anat.(Basel) 144:51-58
    Abstract: The present paper describes the long-term ultrastructural changes in the nucleus ventralis posterolateralis of the thalamus of male Wistar rats after alloxan-induced diabetes. Degenerating dendrites were characterized by an electron-dense cytoplasm with scattered endoplasmic reticulum and ribosomes. Degenerating axon terminals were characterized by an electron-dense cytoplasm and clustering of small spherical agranular vesicles. Degenerating axon terminals formed axosomatic synapses with seemingly normal cell bodies and axodendritic synapses with normal as well as degenerating dendrites. Degenerating axons (both myelinated and unmyelinated) were readily encountered in the neuropil. Activated Microglial and astrocytic cells in the neuropil were in the process of phagocytosis or had residua in their cytoplasm

  76. Becker L, Mito T, Takashima S, Onodera K (1991) Growth and development of the brain in Down syndrome. Prog.Clin.Biol.Res. 373:133-152
    Abstract: The brain of a child with Down syndrome develops differently from a normal one, attaining a form reduced in size and altered in configuration. Directly related to the mental retardation are neuronal modifications manifest as alterations of cortical lamination, reduced dendritic ramifications, and diminished synaptic formation. However, selected cholinergic marker enzymes such as choline acetyl transferase and acetyl cholinesterase have shown no alterations in young children with Down syndrome. The pace of the neuronal transformations is related to stage of maturation. With early growth and development, the normal dendritic tree continuously expands. In Down syndrome, at 4 months of age, the neurons show a relatively expanded dendritic tree, but during the first year the dendrites stop growing and become atrophic relative to control neurons. Accompanying these neuronal irregularities are subtle alterations of other cell types: astrocyte, oligodendrogliocyte, Microglia, and endothelial cell. In early infancy, one of the astrocytic markers, GFAP, is not altered, but there is greater expression of S-100 protein in the temporal lobe in Down syndrome. Oligodendrogliocyte dysfunction is reflected in delayed myelination in pathways of frontal and temporal lobes. Microglia appear more prominent in Down syndrome. A minority of children with Down syndrome have vascular dysplasias and focal calcification of basal ganglia. In young children, expression of beta-amyloid in Down syndrome is no different than in normal children but disappears after age two, only to reappear in adults. As some of these studies suggest, the identification of genes on chromosome 21 and the determination of the gene product allow the production of specific antibodies and, through immunohistochemical techniques, the identification of the expression of these proteins in both normal development and Down syndrome. Specifically, the localization and appearance in development of proteins such as the beta-subunit of S-100, beta-amyloid (A4 protein), superoxide dismutase, and OK-2 are providing the means for better understanding the morphogenesis of the cellular and eventually molecular basis for the mental retardation in Down syndrome

  77. Kristt DA (1987) Morphological responses to local CNS trauma: sprouting and synaptogenesis within membranes implanted into mature cerebral cortex of the rat. J.Neuropathol.Exp.Neurol. 46:668-681
    Abstract: The purpose of this study was to document in vivo neuronal sprouting in response to local trauma of the mature cerebral neocortex. The experimental approach enabled direct and unequivocal ultrastructural identification of newly sprouted neuronal processes and their synaptic relationships. Morphologically detectable factors that influence neuronal sprouting were also assessed. Tiny fragments of porous, synthetic membrane were implanted into the mature neocortex of the rat. After one week, axons, dendrites, growth cone-like structures, synapses, and processes from astrocytes and Microglia were present deep within the interstices of the implant. Morphometric analysis of synapses within the implant, support the qualitative impression of active neuronal ingrowth and de novo synaptogenesis by these sprouted elements. Astrocytic processes appear to comprise the major barrier to neuronal invasion of the implant. Several observations suggested that glia sometimes may facilitate neuronal ingrowth. These findings provide direct morphological documentation of axonal and dendritic plasticity in the mature rat neocortex. The work demonstrates a strategy that could be useful for further evaluating the cellular mechanism of in vivo neuronal regeneration

  78. Scott T, Pesce C (1987) The ultrastructure of the nervous tissue in a benign teratoma. Acta Neuropathol.(Berl) 73:281-286
    Abstract: The ultrastructure of the nervous tissue in a benign ovarian teratoma is described. This tissue was organized into areas having both "meningeal" and "ependymal" surfaces, between which were found astrocytes, ependymal cells, neurones with synapses and Microglia. These cells all had ultrastructural similarities to their normal counterparts in the nervous system. In addition, some signs of degenerative change--due possibly to the abnormal location of the nervous tissue--were observed. Oligodendrocytes and myelin were absent, possibly because of vascular insufficiency

  79. Ling EA, Wong WC, Yick TY, Leong SK (1986) Ultrastructural changes in the dorsal motor nucleus of monkey following bilateral cervical vagotomy. J.Neurocytol. 15:1-15
    Abstract: The neurons of the dorsal motor nucleus (DMN) of the monkey (Macaca fascicularis) were of two main types: small (13 X 8 micron) and medium-sized (20 X 13 micron). The latter, which were the predominant form, contained a pale oval nucleus surrounded by organelle-rich cytoplasm. Between one and three long principal dendrites per section profile arose from each of the somata. Both axosomatic and axodendritic synapses were seen on these cells although the latter were more common. No structural changes were noted in the DMN 1-3 days after bilateral cervical vagotomy. Some of the dendrites of the medium-sized axotomized vagal neurons appeared darkened 5-10 days after the operation. With longer surviving intervals, i.e. 21 and 28 days after operation, darkened dendrites were more commonly seen and the cytoplasmic density of these dendrites was dramatically enhanced. Their mitochondria were pale and some of them also showed vesiculation. Both normal and degenerating axon terminals were seen to form synaptic contacts with the darkened dendrites. The degenerating axon terminals were characterized by the clumping of their round agranular vesicles. Both darkened dendrites and degenerating axon terminals were phagocytosed by hypertrophied astrocytes and activated Microglial cells. Blood elements infiltrating into the DMN were a possible source for some of the neural macrophages. It was concluded from the present study that the dendrites of the vagal neurons were the first structures to degenerate in axotomy and these were subsequently removed by glial elements. Degenerating axon terminals on the darkened dendrites could represent endings of the central processes of peripheral vagal ganglion cells that had undergone transganglionic degeneration after damage to their peripheral processes

  80. Karcsu S, Toth L, Laszlo FA (1985) [Ultrastructural changes in the supraoptica-neurohypophyseal system after induction of lesions in the pituitary stalk in rats]. Acta Histochem. 76:183-192
    Abstract: Fine structural changes occurring in the supraoptic nucleus and the neural lobe have been studied in rats following the electrolytic lesion of the hypophysial stalk. Supraoptic neurosecretory neurones undergo a typical chromatolytic reaction. In addition, proliferating Microglial cells disconnect axosomatic synapses and phagocytose degenerating neurones. Surviving neurones show signs of structural restitution 3 to 4 weeks after surgery. In the neurohypophysis degeneration of secretory nerve endings started with the disintegration of secretory vesicles 3 to 4 d postoperatively. Degenerated axon terminals were engulfed by pituicytes. Signs indicative of axonal regeneration were not observed in the neural lobe up to the end of the 4th postoperative week

  81. Leibnitz L, Kastner I, Bar B (1985) [Morphologic and histochemical changes in the secondary focus following cobalt-induced epileptogenic bioelectrical activity of the visual cortex in the adult rat]. J.Hirnforsch. 26:601-614
    Abstract: Implantation of cobalt-agar rods into the visual cortex of 16 adult rats induced in some of the animals epileptiform bioelectrical activity and provoked in all of them histological and histochemical changes in the region of the implantation (primary focus) as well as in some ipsilateral projection sites of the visual cortex (secondary foci). The changes within the secondary foci are demonstrated in the Corpus geniculatum laterale, pars dorsale (dLGN), by means of 18 histochemical and 5 histological methods. Together with the appearance of hyperactive and degenerating neurones combined with neuronophagy and diminution of the number of synapses a marked gliosis developed, especially an increase of Microglia. The destruction of the tissue induced a depression of energy and transmitter metabolism and intensified lytic processes. This is confirmed by the decreased activities of LDH, SDH, GPDH, G6PDH, NAD(P)H-TR, GABA-T and GDH and the increased activity of acid phosphatase in the neuropil of the secondary foci. Single hyperactive nerve and glial cells were accented by high activities of those enzymes which had a reduced activity in the neuropil. Since in our experiments agar-rods without cobalt never induced histological or histochemical changes in subcortical grisea of the visual system, the secondary foci seem to result from the direct influence of the cobalt, migrating in the corticothalamic projection pathway and identifiable in the dLGN by the TIMM technique

  82. Dellmann HD, Stahl SJ (1984) Fine structural cytology of the rat subfornical organ during ontogenesis. Brain Res.Bull. 13:135-145
    Abstract: The main developmental events in the subfornical organ take place between 17 fetal days (fd) and 5 days post natum (dpn) at which time it possesses most of its mature fine structural characteristics. The surface regional characteristics of ependymal cells differentiate primarily during this time as well, while the ependymal cellular fine structure, shape and relationship with neurons and the vascularity are well established prior to birth. Undifferentiated neurons contain glycogen prior to 19 fd and then differentiate by developing processes and organelles characteristic of neurons. By 5 dpn, the various types of neurons found in the mature subfornical organ are all present, except for giant vacuolated cells. synapses containing only electron-lucent vesicles are first present at 20 fd, those containing additional electron-dense vesicles at 3 dpn. Microglial cells are first identifiable at 17 fd, and the first protoplasmic astrocytes are recognizable at 21 fd, while fibrous astrocytes are not detectable prior to 7 dpn. By 5 dpn, the cytological elements of the subfornical organ are all in place, and further developmental changes leading to adult fine structural characteristics by 30 dpn are essentially quantitative in nature

  83. Garcia Verdugo JM, Berbel NP, Regidor GJ, Lopez GC (1984) Ultrastructure of neuronal cell bodies in the medial cortex of Lacerta galloti. J.Hirnforsch. 25:187-196
    Abstract: According to ultrastructure, size, and location within the medial cortex of Lacerta galloti, seven basic kinds of neuronal somata are described in this study. In the outer plexiform layer of the medial cortex only a few somata can be detected. There, moreover some oligodendroglia and Microglia cells a few neuronal somata showing conspicuous nuclear invaginations and spongious chromatin (A) can be seen. The granular layer of the medial cortex is formed by up to seven strata of tightly packed neuronal somata in a pattern which resembles that of the profound stratum of the fascia dentata of the mammalian hippocampus. In this layer four neuronal somata can be seen: somata with nuclear invaginations, generally located aside the outer plexiform layer (in the upper stratum) (B); very abundant little neuronal somata with chromatin clumps (C); vertically fusiform shaped neuronal somata with numerous polyribosomes (D); and big neuronal somata with spongious chromatin (E). In the inner plexiform layer, there are a variety of scarce neuronal somata intermingled with ependymal sprouts and fiber tracts running from other cortical areas; two main types are described: big horizontal somata which are almost covered by axonic endings making synapses (F) and somata with dense cytoplasmic matrix and some small nuclear invaginations (G). Location of soma, frequency, and biometric data of them have been used to make a comparison with neuronal types of the medial cortex of Lacerta galloti defined by Golgi impregnations

  84. Sterman AB, Sposito N (1984) Motoneuron axosomatic synapses are altered in axonopathy. J.Neuropathol.Exp.Neurol. 43:201-209
    Abstract: This study was designed to answer two questions: 1) are synapses on motoneuron cell bodies affected during toxic neuropathy and 2) what is the nature and extent of the changes? We describe synaptic alterations on motoneuron cell bodies during intoxication with 2,5-hexanedione, a prototype neurotoxin known to induce axonal degeneration. Ultrastructural study of lumbar motoneurons from experimental and control rats revealed an array of pathologic changes including: partial and, less frequently, complete detachment of synaptic boutons; synaptic degeneration in a minority of boutons; and involvement of both Microglia and astrocytes. Quantitatively, there was a significant decrease in the percent of neuronal membrane contacted by both F and S boutons and a significant increase in F boutons displaying degeneration. These results suggest that some neurotoxin-induced diseases of axons are associated with alterations of synaptic complexes that may have important implications for neuronal functioning

  85. Kiss A, Borsosova D (1982) Morphological study on the central subdivision of the rat hypothalamic ventromedial nucleus. Anat.Anz. 152:113-124
    Abstract: Central subdivision of the rat hypothalamic ventromedial nucleus (VMN) was examined with the transmission electron microscope. This portion of VMN consists of densely packed neuronal somata which are characterized by a large pale nucleus with a prominent nucleolus, with clear cytoplasm containing many free ribosomes and randomly distributed fragments of rough endoplasmic reticulum, with small mitochondria and homogeneous lysosomes, with well developed Golgi complex surrounded by a few clear and largely 2-3 dense-core vesicles of 90-110 nm in size and with nucleotid bodies. Besides neuronal somata and their processes also astrocytes, oligodendrocytes and Microglial cells and their processes are often seen in this portion of VMN. In the neuropil axo-somatic and axo-dendritici synapses, capillaries, myelinated nerve fibers, nerve profiles filled with dense-core vesicles and distal parts of ependymal tanycytic processes are usually found in the central subdivision of VMN

  86. Komuro T, Baluk P, Burnstock G (1982) An ultrastructural study of neurons and non-neuronal cells in the myenteric plexus of the rabbit colon. Neuroscience 7:1797-1806
    Abstract: The myenteric plexus of the rabbit colon showed a degree of structural organization that was unusually high for the peripheral nervous system, providing a basis for the complex integrative activity which is known to occur. It resembled central nervous tissue in several respects: a wide range of neuron types was present; the proportion of glial cells to neurons was about 2:1; and there was a densely packed, avascular neuropil, not penetrated by connective tissue. Most neurons had at least one surface exposed to the extraganglionic space. Clear evidence was obtained for spontaneous neuronal degeneration. Three types of non-neuronal (glial) cells were observed: Type 1, which was most common, contained many 10 nm 'gliofilaments' and resembled enteric glial cells or astrocytes in the central nervous system; Type 2, composing about 5% of the glial cells, had few filaments; Type 3 was seen only rarely, had a small dark nucleus, little cytoplasm, may have been of extraganglionic origin and resembled Microglia of the central nervous system. Fibroblast-like cells were also present in extraganglionic sites. Schwann cells could not be identified within the myenteric ganglia

  87. Leibnitz L, Bar B, Gunther L, Ludwig R, Hedlich A (1982) The glia types inthe visual system of adult rats, their shape variability, distribution patterns, and their lightoptically visible contacts to other tissue structures. J.Hirnforsch. 23:225-238
    Abstract: In silver impregnated coronal sections of adult rat brains the glia types of the Corpus geniculatum laterale, pars dorsalis (CGLd) an in area 17 were registered considering their form variants (FV), their intraareal distribution and their light optically demonstrable connexions to other tissue structures. Compared with the shape-determining light optically visible processes and perikarya, astrocytes show the lowest, Microglial cells the greatest from variability. Transitional forms between the three glia types were not detected, but between elongated astrocytes and fiber astrocytes. An accumulation of elongated astrocytes, fiber astrocytes, and oligodendrocytes was found in the lateral zone of the CGLd, of protoplasmic astrocytes in the lateral and intermediary zones and at the rostral and caudal poles of the griseum, too. In the cortex distribution differences in the single laminae became also evident. Astrocytes were most concentrated in L I, oligodendrocytes and Microglia, however, in L V. A few FV of the Microglia were found to show a predominant localisation in specific laminae. Neither the total glia number nor the single glia types correlate with the neuron packing density. The frequency distribution of the FV of oligodendroglia in the CGLd and the visual cortex is similar, that of Microglia, however, significantly deviating. Compared with the CGLd the oligodendrocytes of area 17 have smaller somata. Astrocytes, oligodendrocytes and Microglial cells reveal structural contacts to different parts of the neurons, to blood vessels and other glial cells, too. Contacts between oligodendrocytes and Microglial cells as well as astrocytes in satellite position could never be observed. In the cortex a few FV of Microglia show typical connexions

  88. Murabe Y, Sano Y (1982) Morphological studies on neuroglia. V. Microglial cells in the cerebral cortex of the rat, with special reference to their possible involvement in synaptic function. Cell Tissue Res. 223:493-506
    Abstract: Electron-microscopic survey of selectively stained Microglial cells in the cerebral cortex of the rat reveals that the processes of this cell type often encircle axo-dendritic synapses. Enzyme-histochemical methods for thiamine pyrophosphatase (TPPase) or nucleoside diphosphatase (NDPase) were used for the selective marking of the Microglial cells; TPPase and NDPase activities were observed in the plasma membrane of Microglial cells. The synapses encircled by Microglial processes displayed presynaptic structures containing round clear vesicles (50 nm in diameter) and a prominent thickening of the postsynaptic membrane. In vitro, the above-mentioned enzymatic activities were completely suppressed by neuroactive agents such as catecholamines and phenothiazine derivatives. Examination using enzyme-histochemical techniques suggests that a single enzyme may be responsible for both Microglial cells in the normal central nervous tissue is discussed

  89. Mize RR, Spencer RF, Sterling P (1981) Neurons and glia in cat superior colliculus accumulate [3H]gamma-aminobutyric acid (GABA). J.Comp Neurol. 202:385-396
    Abstract: We have examined by autoradiography the labeling pattern in the cat superior colliculus following injection of tritiated gamma-aminobutyric acid (GABA). Silver grains were heavily distributed within the zonal layer and the upper 200 micrometer of the superficial gray. Fewer grains were observed deeper within the superficial gray, and still fewer were found within the optic and intermediate gray layers. The accumulation of label was restricted to certain classes of neuron and glia. Densely labeled neurons were small (8-12 micrometer in diameter) and located primarily within the upper 200 micrometer. Dark oligodendrocytes and astrocytes showed a moderate accumulation of label while pale oligodendrocytes and Microglia were unlabeled. Label was also selectively accumulated over several other types of profile within the neuropil, including presynaptic dendrites, axons, and axon terminals

  90. Nadler JV, Perry BW, Gentry C, Cotman CW (1980) Degeneration of hippocampal CA3 pyramidal cells induced by intraventricular kainic acid. J.Comp Neurol. 192:333-359
    Abstract: Degeneration of hippocampal CA3 pyramidal cells was investigated by light and electron microscopy after intraventricular injection of the potent convulsant, kainic acid. Electron microscopy revealed evidence of pyramidal cell degeneration within one hour. The earliest degenerative changes were confined to the cell body and proximal dendritic shafts. These included an increased incidence of lysosomal structures, deformation of the perikaryal and nuclear outlines, some increase in background electron density, and dilation of the cisternae of the endoplasmic reticulum accompanied by detachment of polyribosomes. Within the next few hours the pyramidal cells atrophied and became electron dense. Then these cells became electron lucent once more as ribosomes disappeared and their membranes and organelles broke up and disintegrated. Light microscopic changes correlated with these ultrastructural observations. The dendritic spines and the initial portion of the dendritic shaft became electron dense within four hours and degenerated rapidly, whereas the intermediate segment of the dendrites swelled moderately and became more electron lucent. No degenerative changes were evident in pyramidal cell axons and boutons until one day after kainic acid treatment. Less than one hour after kainic acid administration, astrocytes in the CA3 area swelled, initially in the vicinity of the cell body and mossy fiber layers. It is suggested that the paroxysmal discharges initiated in CA3 pyramidal cells by kainic acid served as the stimulus for this response. Phagocytosis commenced between one and three days after kainic acid administration, but remained incomplete at survival times of 6-8 weeks. Astrocytes, Microglia, and probably oligodendroglia phagocytized the degenerating material. These results point to the pyramidal cell body and possibly also the dendritic spines as primary targets of kainic acid neurotoxicity. In conjunction with other data, they support the view that lesions made by intraventricular kainic acid can serve as models of epileptic brain damage

  91. Sumner BE (1979) Ultrastructural data, with special reference to bouton/glial relationships, from the hypoglossal nucleus after a second axotomy of the hypoglossal nerve. Exp.Brain Res. 36:107-118
    Abstract: The left hypoglossal nerve of adult male albino rats was prevented from regenerating to the tongue after a distal axotomy by implanting the proximal stump into normally innervated left sternomasoid muscle. Eighty-four days after implantation, the hypoglossal nerve was transected again and its regeneration to the tongue unimpeded. From 8 to 70 days after this second axotomy the left hypoglossal nuclei were processed for quantitative ultrastructural analysis. The first aim of this study was to compare regeneration success in the hypoglossal nucleus after second axotomy with that accompanying outgrowth of the hypoglossal nerve into denervated sternomastoid muscle. During quantitative analysis a second aim developed, of elucidating bouton/glial relationships. The second axotomy induced loss and return of subsurface cisterns, dispersal and reassembly of Nissl substance, increase and decrease of Microglial numbers, slight further loss and partial return of boutons with clear spherical vesicles and symmetrical synapses, slight increase and decrease of boutons with clear flat vesicles and symmetrical synapses, regrowth of retracted dendrites and restoration of their synapses, and gradual diminution of numbers of electron-dense neurones and dendrites. Astrocytes remained hypertrophied throughout. When compared with events in the hypoglossal nucleus accompanying innervation of denervated sternomastoid muscle by the hypoglossal nerve, the results suggest (1) that regeneration of the hypoglossal nerve to its own tongue muscle instead of to a foreign muscle caused no acceleration of recovery in the hypoglossal nucleus, and (2) that the Microglial response is dependent on nerve integrity and not on bouton behaviour

  92. Chen DH (1978) Qualitative and quantitative study of synaptic displacement in chromatolyzed spinal motoneurons of the cat. J.Comp Neurol. 177:635-664
    Abstract: Qualitative and quantitative cytological evidences show a reduction in number and percentage of coverage of terminals on chromatolyzed spinal motoneurons 4 to 90 days after severance of their axons in the ventral root. The reduction in number and percentage of boutons on the surface membrane is due to detachment of synapses. This detachment is associated with changes in specialized regions (synaptic complexes: Conradi, '69) of synapses which involve disappearance of both pre-and postsynaptic membrane thickenings and widening of the synaptic cleft (width of 400-1,400 A). The separation of terminals from surface membrane is also associated with the presence of reactive astrocytes and Microglia. Most areas of surface membrane bared of synaptic contact are occupied by astrocytic processes. The astroglial responses coincide with the loss of synapses. Microglial cells, although extending to neuronal surface membrane, cover only a small portion of it. An extensive separation of boutons by Microglia is not observed. Most Microglia are seen located at some distance from surface membrane and are frequently separated by sheets of astrocytic processes. Synaptic restoration occurs by about 90 days after axotomy. Some motoneurons show good recovery, while others show only a limited amount

  93. Kreutzberg GW, Barron KD, Schubert P (1978) Cytochemical localization of 5'-nucleotidase in glial plasma membranes. Brain Res. 158:247-257
    Abstract: By means of a cytochemical procedure 5'-nucleotidase activity is demonstrated electron microscopically. The enzyme is present in plasma membranes of astrocytes, oligodendroglial cells and Microglial cells, but not in neuronal plasma membranes

  94. Sturrock RR (1978) Development of the indusium griseum. II. A semithin light microscopic and electron microscopic study. J.Anat. 125:433-445
    Abstract: Neurogenesis and gliogenesis in the indusium griseum were studied in semithin sections and with the electron microscope. Immature neurons were present at birth, but differentiated rapidly until, by 15 days, they appeared to be fully differentiated. At birth 38% of glia were glioblasts, 47% were immature astrocytes and 15% were mature Microglia. Astrocyte differentiation appeared to occur earlier than in adjacent white matter. In the adult, 57% of glia were astrocytes, 21% were oligodendrocytes, and 22% were Microglia. The volume density of neuronal nuclei did not change with age, but the volume density of neuronal perikaryon decreased from 34% at birth to 13% at 15 days, while the neuropil increased from 24% to 58%. Very few immature synapses were present at birth, but by 5 days a number of axodendritic synapses were present, and these increased with age

  95. Sumner BE (1977) Responses in the hypoglossal nucleus to delayed regeneration of the transected hypoglossal nerve, a quantitative ultrastructural study. Exp.Brain Res. 29:219-231
    Abstract: Electron micrographs of the left hypoglossal nucleus of adult male albino rats were quantitatively analyzed from 9-70 days after allowing the transected left hypoglossal nerve to regenerate after an 84 day delay. Delay was achieved by implanting the proximal stump into already innervated sternomastoid muscle, where no regeneration occurred. Regeneration was then allowed by denervating the sternomastoid. During the regenerative period the initially high number of abnormally electron dense perikarya and dendrites decreased to almost normal values, but no cell removal was seen. This suggested that the degenerate appearance of many profiles after prolonged prevention of regeneration, was reversible. The neuropil bouton and dendrite counts, and the numbers of synapsing boutons per dendrite, increased steadily to normal values from the low values of suppressed regeneration. Somatic bouton frequencies, even though already low, decreased further at 32 days, and later increased but not to normal values. The decrease at 32 days coincided with the loss of many subsurface cisterns, and dispersion of Nissl substance, all suggestive of chromatolysis. Later the subsurface cisterns and Nissl substance returned. It was suggested that the delay of complete recovery of somatic bouton frequencies might be because of lack of sensory information from the denervated muscle into which the hypoglossal nerve was regenerating, or because of abnormally low starting values for the recovery phase. Astrocyte (or, occasionally Microglial) sheaths persisted along bouton-free perikaryal surfaces

  96. Sumner BE (1977) Ultrastructural responses of the hypoglossal nucleus to the presence in the tongue of botulinum toxin, a quantitative study. Exp.Brain Res. 30:313-321
    Abstract: The ultrastructural effects of local injection of botulinum toxin into the left half of the tongue of the rat, were studied quantitatively 35 days postoperatively in the left hypoglossal nucleus. The results showed (1) a decrease in somatic and neuropil bouton numbers because of loss of boutons with symmetrical synapses and clear spherical synaptic vesicles, (2) a decrease in the numbers of dendrite profiles in the neuropil, (3) an increase in the proportion of dendrites and boutons with unusual inclusions, suggestive of profile retraction, (4) an increase in the proportion of profiles which were unusually electron-dense, (5) an increase in the amount of astrocyte, and a growth of astrocyte sheaths around bouton-free neurone surfaces, (6) the presence of occasional Microglia, and (7) subastrocytic subsurface cisterns. Control rats injected with boiled toxin had no responses except (3) and (4) above, and then only to a modest extent, possibly due to mechanical damage of a few axons or terminals at the time of injection, or to insufficient inactivation of the toxin by boiling. The results were compared with those at 35 days after axotomy, and it was concluded that botulinum toxin, which interrupts neuromuscular transmission, elicits the same responses in the hypoglossal neurones, as does transection of the hypoglossal nerve, even though earlier studies had discovered no glial replication after botulinum toxin, in contrast to axotomy

  97. Cragg BG (1976) Ultrastructural features of human cerebral cortex. J.Anat. 121:331-362
    Abstract: Neuronal and glial elements have been examined in the temporal or frontal cortex of four brains with normal parenchyma. The identifying features recognized in animal brains have been found valid in human cortex for stellate and pyramidal neurons and their dendrites, spines, synapses, initial segments of axons, nodes of Ranvier and axon terminals; also for astrocytes, oligodendrocytes of various electron densities, pericytes, blood vessels and basement lamina, and for the relation of subarachnoid space to arterioles. However, most Microglial cells have unexpectedly sparse and pale cytoplasm with few organelles, and the distribution of glial nuclear chromatin is not entirely in accordance with that described in animal brains. Fusiform neurons have been identified, and their cytoplasm resembles that of stellate cells. Peculiarities of human cortex include a nuclear inclusion that is present in stellate neurons only. Two neuronal cell-body profiles were myelinated, and a blood capillary was seen to penetrate a neuron. Somatic spines were found occasionally with or without synapses. Ribosomes can be present in axons, espicially at nodes of Ranvier, and a small portion of axon terminals contain a variety of dense bodies of which some are derived from mitochondria. Endoplasmic reticulum can occur in a compressed stack in neurons, and an unusual vesicular organelle has been seen in dendrites, axons and somata. Astrocytes as well as oligodendrocytes and occasional Microglial cells act as satellites to neurons, and the exchange of a membrane-bound body between a neuron and asatellite glial cell is illustrated. Some dark pycnotic neurons were present, and it was remarkable that a closely apposed neuron could be entirely normal. These findings are compared with published descriptions of cortical ultrastructure in the brains of laboratory animals

  98. Lemkey-Johnston N, Butler V, Reynolds WA (1976) Glial changes in the progress of a chemical lesion. An electron microscopic study. J.Comp Neurol. 167:481-501
    Abstract: The response of non-neuronal cells in the brain to monosodium glutamate (MSG) was studied in the neonatal mouse brain. Neurons rapidly degenerate in this chemically-induced lesion, and the glia reflect and respond to their early pyknosis and death. Astrocytic glia exhibit the most profound responses in the form of degenerative nuclear and cytoplasmic changes within 15 to 20 minutes after ingestion of MSG. After 6 to 12 hours, a large glial cell population, containing neuronal and synaptic debris, can be seen. Recovery of severely damaged astrocytes is unlikely. Further, evidence of multiplication of uninjured cells to supply this observed repopulation was found. Microglia start to engulf debris at 6 to 12 hours and continue this process through at least 48 hours. From 3 to 48 hours, a third cell type, tentatively identified as a Gitter cell, is preminently involved in phagocytosis of neuronal elements. Nuclei of these cells enlarge and their parikarya become vastly expanded in a fashion typical of Gitter cells. These cells appear to represent the transformed Microglia. Phagocytosis following a chemical lesion induced by glutamic acid, a neurotransmitter substance, is exceedingly rapid in comparison to that elicited by other types of lesions. Further, endogenous glial cells cope with the cellular debris; no participation in this process by hematogenous cells was observed

  99. Sumner BE (1976) Quantitative ultrastructural observations on the inhibited recovery of the hypoglossal nucleus from the axotomy response when regeneration of the hypoglossal nerve is prevented. Exp.Brain Res. 26:141-150
    Abstract: Electron micrographs of the left hypoglossal nucleus were quantitatively analyzed in adult rats 52 to 98 days after transection of the left hypoglossal nerve and implantation of the proximal stump into the already innervated ipsilateral sternomastoid muscle, a procedure which prevented the transected nerve from regenerating. Many presynaptic boutons with clear spherical synaptic vesicles and symmetrical synapses were lost from the injured perikarya and dendrites. Some perikarya and dendrites (and, rarely, boutons) became electron dense, and astrocyte or Microglial sheaths partly surrounded them. Numbers of dendrite profiles in the neuropil decreased. These statistically significant effects persisted throughout the postoperative period, whereas after axotomy with unimpeded nerve regeneration, these features would have returned to normal by 84 days postoperatively. It was therefore suggested that their recovery depended upon successful regeneration and reconnection of the hypoglossal nerve with the tongue. Subsurface cisterns, and profiles containing unusual inclusions, were numerically normal 52 to 98 days postoperatively, so it was suggested that their early response and recovery after simple axotomy might be entirely dependent on nerve disconnection and not on nerve reconnection

  100. Davidoff M, Galabov G (1975) [The postnatal development of the hypoglossal nucleus in the rat]. Z.Mikrosk.Anat.Forsch. 89:615-631
    Abstract: Using light and electron microscopy the neurons, glial cells and capillaries in hypoglossal nucleus of the rats have been examined up to 20 days after birth. The neuronal nuclei are usually situated ecentrically. The mitochondria and extensively developed Golgi-zones occupy the perinuclear region. The microtubules and lysosomes become more numerous with aging. At the earliest periods rough endoplasmic reticulum (ER) occupies the neuronal periphery, whereas after 14th day it is extended to the perinuclear region also. The ER forms elongated and concentric lamellated bodies and subsurface cisternae. At this time nucleolus like bodies are also numerous in the cytoplasm. After 4th and 6th days the extensive growth of dendrites, containing many cell organelles, and axons rich in microtubules are observed. Only at the birthday do neurons contain glycogen deposit. After 1st day the glycogen leaves the pericaryon, but it persists a long time in the neuronal processes. The symmetrical and asymmetrical contacts are characteristic for the examined period. The axo-somatic and axo-dendritic synapses are more abundant, but "double synapses" are also established. More synaptic boutons possess besides synaptic vesicles dense-core vesicles at the earlier periods. The quantity of asymmetric synapses increases with differentiation. Extensive cell degeneration has been established between 8 and 18th days. At 4 and 6 days the glial cells penetrate from subependymal layer and they have satellite neuronal position. This is more pronounced between 14 and 18 days when the oligodendrocytes are more numerous and active. At the same time fibrous astrocyte like cells are appeared. Microglial cells were not observed. Capillary differentiation, expressed by changes of the endothelial cells, pericytes and connective tissue cells, continues after birth also

  101. Sumner BE, Sutherland FI (1973) Quantitative electron microscopy on the injured hypoglossal nucleus in the rat. J.Neurocytol. 2:315-328

  102. Blinzinger K, Kreutzberg G (1968) Displacement of synaptic terminals from regenerating motoneurons by Microglial cells. Z.Zellforsch.Mikrosk.Anat. 85:145-157