Synaptic Stripping Research Articles

Clostridium perfringens epsilon toxin induces permanent neuronal degeneration and behavioral changes

Clostridium perfringens epsilon toxin (ETX), the most potent toxin produced by this bacteria, plays a key role in the pathogenesis of enterotoxaemia in ruminants, causing brain edema and encephalomalacia. Studies of animals suffering from ETX intoxication describe severe neurological disorders that are thought to be the result of vasogenic brain edemas and indirect neuronal toxicity, killing oligodendrocytes but not astrocytes, microglia, or neurons in vitro. In this study, by means of intravenous and intracerebroventricular delivery of sub-lethal concentrations of ETX, the histological and ultrastructural changes of the brain were studied in rats and mice. Histological analysis showed degenerative changes in neurons from the cortex, hippocampus, striatum and hypothalamus. Ultrastructurally, necrotic neurons and apoptotic cells were observed in these same areas, among axons with accumulation of neurofilaments and demyelination as well as synaptic stripping. Lesions observed in the brain after sub-lethal exposure to ETX, result in permanent behavioral changes in animals surviving ETX exposure, as observed individually in several animals and assessed in the Inclined Plane Test and the Wire Hang Test. Pharmacological studies showed that dexamethasone and reserpine but not ketamine or riluzole were able to reduce the brain lesions and the lethality of ETX. Cytotoxicity was not observed upon neuronal primary cultures in vitro. Therefore, we hypothesize that ETX can affect the brain of animals independently of death, producing changes on neurons or glia as the result of complex interactions, independently of ETX-BBB interactions.

Synaptic stripping and MHC class I expression in the facial motor nucleus of ALS mice

Pathogenetic mechanisms involved in the fatal, still incurable neurodegenerative disease amyotrophic lateral sclerosis (ALS), characterized by progressive motoneuron death, await full clarification, important for the development of new therapeutic approaches. In the ALS murine model provided by mutant SOD1(G93A) mice, we here investigated the presynaptic wiring of facial motoneurons in basal conditions and after facial nerve transection (a classical paradigm to examine the retrograde motoneuron response to injury), and major histocompatibility (MHC) class I antigen expression after axotomy. The study was based on fluorescent retrograde labeling of motoneurons, synaptophysin and MHC class I antigen immunostaining, electron microscopy. A significant decrease of excitatory axosomatic boutons was found in presymptomatic ALS mice compared to the wild-type (Wt) counterpart, indicating the occurrence of excitatory synapse detachment (presynaptic stripping) in mutant motoneurons. Synaptic stripping, which seems to represent a protective mechanism preserving the inhibitory input, became more marked in facial motoneurons of symptomatic ALS mice. After axotomy, synaptic stripping was consistently enhanced in ALS mice. In the axotomized facial motoneurons of Wt mice synaptic stripping was accompanied by induction of MHC class I antigens, immune molecules implicated in activity-dependent changes in synaptic connectivity and regeneration after injury. MHC class I antigen induction was instead decreased in the axotomized facial nucleus of presymptomatic ALS mice, and was very low, occurring only in glial cells, in symptomatic ALS mice. The findings demonstrate enhanced loss of excitatory presynaptic terminals, as well as a dissociation between this process and MHC class I antigen expression after injury, in motoneurons which carry a mutation committing them to death. The findings also implicate MHC class I antigen induction in glial cells surrounding ALS motoneurons in this intercellular crosstalk.

Endogenous modulation of TrkB signaling by treadmill exercise after peripheral nerve injury

After peripheral nerve injury, transected fibers distal to the lesion are disconnected from the neuronal body. This results in target denervation but also massive stripping of the central synapses of axotomized motoneurons, disrupting spinal circuits. Even when axonal regeneration is successful, the non-specific target reinnervation and the limited rebuilding of spinal circuits impair functional recovery. Therefore, strategies aimed to preserve spinal circuits after nerve lesions may improve the functional outcome. Activity-dependent therapy in the form of early treadmill running reduces synaptic stripping, mainly of excitatory synapses, and the disorganization of perineuronal nets (PNNs) on axotomized motoneurons. The mechanism underlying these effects remains unknown, although the benefits of exercise are often attributed to an increase in the neurotrophin brain-derived neurotrophic factor (BDNF). In this study, tropomyosin-related kinase (TrkB) agonist and antagonist were administered to rats subjected to sciatic nerve injury in order to shed light on the role of BDNF. The maintenance of synapses on axotomized motoneurons induced by treadmill running was partially dependent on TrkB activation. Treatment with the TrkB agonist at a low dose, but not at a high dose, prevented the decrease of excitatory glutamatergic synapses, and both doses increased the density of inhibitory synapses. TrkB inactivation counteracted only some of the positive effects exerted by exercise after nerve injury, such as maintenance of excitatory synapses surrounding motoneurons. Therefore, specific regimes of physical exercise are a better strategy to attenuate the alterations that motoneurons suffer after axotomy than pharmacological modulation of the TrkB pathway.

Astroglioma conditioned medium increases synaptic elimination and correlates with major histocompatibility complex of class I (MHC I) upregulation in PC12Cells

Astrocytes are multifunctional glial cells that actively participate in synaptic plasticity in health and disease. Little is known about molecular interactions between neurons and glial cells that result in synaptic stability or elimination. In this sense, the main histocompatibility complex of class I (MHC I) has been shown to play a role in the synaptic plasticity process during development and after lesion of the CNS. MHC I levels in neurons appear to be influenced by astrocyte secreted molecules, which may generate endoplasmic reticulum stress. In vitro studies are of relevance since cell contact can be avoided by the use of astrocyte conditioned medium, allowing investigation of soluble factors isolated from cell direct interaction. Thus, we investigated synaptic preservation by synaptophysin and MHC I immunolabeling in PC12 neuron-like cells exposed to NG97 astroglioma conditioned medium (CM). For that, PC12 cells were cultured and differentiated into neuron-like profile with nerve growth factor. MHC I was induced with interferon beta treatment (IFN), and the effects were compared to PC12 exposure to NG97 CM. Overall, the results show that NG97 CM increases, more than IFN alone, the expression of MHC I, negatively influencing synaptic stability. This indicates that glial soluble factors influence synapse elimination, compatible to in vivo synaptic stripping process, in a cell contact independent fashion. In turn, our results indicate that deleterious effects of astroglioma are not only restricted to rapid growth ratio of the tumor, but also correlated with secretion of stress-related molecules that directly affect neuronal networks.

The microglial cell response to sterile nerve injury in the brain is regulated by IRF8

Abstract Microglia, the resident immune cells of the central nervous system (CNS), respond vigorously to insults such as nerve injury. We have shown previously in the normal CNS the properties of microglia are dramatically altered in the absence of IRF8. Here we examined whether IRF8 is involved in the microglial cell response to sterile nerve injury. Facial nerve axotomy (FNA) was performed in wild type (WT) and IRF8−/− (KO) mice and the brain removed at different days post-injury (dpi). In brains from IRF8 KO mice, nucleoside diphosphatase (NDPase) histochemistry revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied but were present in comparable numbers with WT. In WT mice, a progressive increase in microglial cell activation was observed peaking at 7 dpi and was accompanied by dense staining for lectin, NDPase and CD11b. By contrast, in IRF8 KO mice, the microglial cell activation was grossly attenuated with the density of staining for lectin, NDPase and CD11b reduced significantly. The attenuated microglial cell activation in IRF8 KO mice was paralleled by a significant decrease in microglial cell proliferation at 7 and 14 dpi. The wrapping of individual motor neuron cell bodies in the axotomised facial nucleus by microglia involved in synaptic stripping and phagocytosis was incomplete in IRF8 KO mice and was accompanied by an increase in degenerated neurons. These findings show that in addition to maintenance of homeostatic properties, IRF8 also has an important role in regulating a number of key processes in the microglial cell response to neuronal injury.

FGF-2 is required to prevent astrogliosis in the facial nucleus after facial nerve injury and mechanical stimulation of denervated vibrissal muscles.

Recently, we have shown that manual stimulation of paralyzed vibrissal muscles after facial-facial anastomosis reduced the poly-innervation of neuromuscular junctions and restored vibrissal whisking. Using gene knock outs, we found a differential dependence of manual stimulation effects on growth factors. Thus, insulin-like growth factor-1 and brain-derived neurotrophic factor are required to underpin manual stimulation-mediated improvements, whereas FGF-2 is not. The lack of dependence on FGF-2 in mediating these peripheral effects prompted us to look centrally, i.e. within the facial nucleus where increased astrogliosis after facial-facial anastomosis follows "synaptic stripping". We measured the intensity of Cy3-fluorescence after immunostaining for glial fibrillary acidic protein (GFAP) as an indirect indicator of synaptic coverage of axotomized neurons in the facial nucleus of mice lacking FGF-2 (FGF-2-/- mice). There was no difference in GFAP-Cy3-fluorescence (pixel number, gray value range 17–103) between intact wildtype mice (2.12± 0.37×107) and their intact FGF-2-/- counterparts (2.12± 0.27×107) nor after facial-facial anastomosis +handling (wildtype: 4.06± 0.32×107; FGF-2-/-: 4.39±0.17×107). However, after facial-facial anastomosis, GFAP-Cy3-fluorescence remained elevated in FGF-2-/--animals (4.54±0.12×107), whereas manual stimulation reduced the intensity of GFAP-immunofluorescence in wild type mice to values that were not significantly different from intact mice (2.63± 0.39×10 ). We conclude that FGF-2 is not required to underpin the beneficial effects of manual stimulation at the neuro-muscular junction, but it is required to minimize astrogliosis in the brainstem and, by implication, restore synaptic coverage of recovering facial motoneurons.

Dietary obesity reversibly induces synaptic stripping by microglia and impairs hippocampal plasticity

Obesity increases risk of age-related cognitive decline and is accompanied by peripheral inflammation. Studies in rodent models of obesity have demonstrated that impaired hippocampal function correlates with microglial activation, but the possibility that neuron/microglia interactions might be perturbed in obesity has never been directly examined. The goal of this study was to determine whether high fat diet-induced obesity promotes synaptic stripping by microglia, and whether any potential changes might be reversible by a return to low-fat diet (LFD). Time course experiments revealed that hippocampal inflammatory cytokine induction and loss of synaptic protein expression were detectable after three months of HFD, therefore subsequent groups of mice were maintained on HFD for three months before being switched to LFD for an additional two months on LFD (HFD/LFD). Additional HFD mice continued to receive HFD during this period (HFD/HFD), while another group of mice were maintained on LFD throughout the experiment (LFD/LFD). Dietary obesity impaired hippocampus-dependent memory, reduced long-term potentiation (LTP), and induced expression of the activation marker major histocompatibility complex II (MHCII) in hippocampal microglia. Diet reversal only partially attenuated increases in adiposity in HFD/LFD mice, but plasticity deficits and MHCII induction were normalized to within the range of LFD/LFD mice. Microglial activation and deficits in hippocampal function were accompanied by perturbation of spatial relationships between microglial processes and synaptic puncta. Analysis of primary microglia isolated from HFD/HFD mice revealed selective increases in internalization of synaptosomes labeled with a pH-sensitive fluorophore. Taken together, these findings indicate that dietary obesity reversibly impairs hippocampal function, and that deficits may be attributable to synaptic stripping by microglia.

Neuronal BDNF signaling is necessary for the effects of treadmill exercise on synaptic stripping of axotomized motoneurons.

The withdrawal of synaptic inputs from the somata and proximal dendrites of spinal motoneurons following peripheral nerve injury could contribute to poor functional recovery. Decreased availability of neurotrophins to afferent terminals on axotomized motoneurons has been implicated as one cause of the withdrawal. No reduction in contacts made by synaptic inputs immunoreactive to the vesicular glutamate transporter 1 and glutamic acid decarboxylase 67 is noted on axotomized motoneurons if modest treadmill exercise, which stimulates the production of neurotrophins by spinal motoneurons, is applied after nerve injury. In conditional, neuron-specific brain-derived neurotrophic factor (BDNF) knockout mice, a reduction in synaptic contacts onto motoneurons was noted in intact animals which was similar in magnitude to that observed after nerve transection in wild-type controls. No further reduction in coverage was found if nerves were cut in knockout mice. Two weeks of moderate daily treadmill exercise following nerve injury in these BDNF knockout mice did not affect synaptic inputs onto motoneurons. Treadmill exercise has a profound effect on synaptic inputs to motoneurons after peripheral nerve injury which requires BDNF production by those postsynaptic cells.

Activity dependent therapies modulate the spinal changes that motoneurons suffer after a peripheral nerve injury

Injury of a peripheral nerve not only leads to target denervation, but also induces massive stripping of spinal synapses on axotomized motoneurons, with disruption of spinal circuits. Even when regeneration is successful, unspecific reinnervation and the limited reconnection of the spinal circuits impair functional recovery. The aim of this study was to describe the changes that axotomized motoneurons suffer after peripheral nerve injury and how activity-dependent therapies and neurotrophic factors can modulate these events. We observed a marked decrease in glutamatergic synapses, with a maximum peak at two weeks post-axotomy, which was only partially reversed with time. This decrease was accompanied by an increase in gephyrin immunoreactivity and a disintegration of perineuronal nets (PNNs) surrounding the motoneurons. Direct application of neurotrophins at the proximal stump was not able to reverse these effects. In contrast, activity-dependent treatment, in the form of treadmill running, reduced the observed destructuring of perineuronal nets and the loss of glutamatergic synapses two weeks after injury. These changes were proportional to the intensity of the exercise protocol. Blockade of sensory inputs from the homolateral hindlimb also reduced PNN immunoreactivity around intact motoneurons, and in that case treadmill running did not reverse that loss, suggesting that the effects of exercise on motoneuron PNN depend on increased sensory activity. Preservation of motoneuron PNN and reduction of synaptic stripping by exercise could facilitate the maintenance of the spinal circuitry and benefit functional recovery after peripheral nerve injury.

202: Interferon regulator factor (IRF) 8 regulates the microglial response to neuronal injury

The transcription factor interferon regulatory factor (IRF) 8 has a key role in the cellular response to IFN-γ and is involved in myeloid cell differentiation. We have previously identified IRF8 to be a constitutive and IFN-γ-stimulated nuclear factor that regulates the homeostatic properties of microglia. Our study aimed to determine the role of IRF8 in the microglial response to sterile neuronal injury. Facial nerve axotomy (FNA) was performed in wildtype (WT) and IRF8 −/− (KO) mice and the brains removed at different times post-lesion. A subset of mice was injected with bromodeoxyuridine (BrdU) prior to retrieval of the brain. Changes in the facial nucleus (FN) were examined by immunohistochemistry and histochemistry. In brains from IRF8KO mice, nucleoside diphosphatase (NDPase) hisotchemistry revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied. After FNA in WT mice, a progressive increase in microglial activation was observed in the lesioned FN peaking at day 7 and was accompanied by dense staining for Iba1, lectin, NDPase and CD11b. By contrast, in IRF8KO mice, the microglial response was markedly attenuated with little staining for Iba1, while the density of staining for lectin and NDPase was reduced significantly. The attenuated microglial response in IRF8KO mice was paralleled by a significant decrease at day 3 post-lesion in proliferation as monitored by BrdU, phosphohistone 3 and Ki67-positive cells. Furthermore, a decrease in PU.1-positive cells was observed in the FN of IRF8KO mice compared with WT. The wrapping of individual motor neuron cell bodies by microglia involved in synaptic stripping and phagocytosis was incomplete in the absences of IRF8 in the axotomised FN. Quantitative analysis showed that in IRF8KO mice, the degeneration of axotomised motor neurons was significantly increased. These studies extend on our previous finding that IRF8 is a key homeostatic transcription regulator of microglial cell function in the healthy brain, and moreover, has a crucial role in the regulating the response to microglia to neuronal injury.

Up-regulation of matrix metallopeptidase 12 in motor neurons undergoing synaptic stripping

Axotomy of the rodent facial nerve represents a well-established model of synaptic plasticity. Post-traumatic “synaptic stripping” was originally discovered in this system. We report upregulation of matrix metalloproteinase MMP12 in regenerating motor neurons of the mouse and rat facial nucleus. Matrix metalloproteinases (matrix metallopeptidases, MMPs) are zinc-binding proteases capable of degrading components of the extracellular matrix and of regulating extracellular signaling networks including within synapses. MMP12 protein expression in facial motor neurons was enhanced following axotomy and peaked at day 3 after the operation. The peak of neuronal MMP12 expression preceded the peak of experimentally induced synaptic plasticity. At the same time, MMP12 redistributed intracellularly and became predominantly localized beneath the neuronal somatic cytoplasmic membrane. Both findings point to a role of MMP12 in the neuronal initiation of the synaptic stripping process. MMP12 is the first candidate molecule for such a trigger function and has potential as a therapeutic target. Moreover, since statins have been shown to increase the expression of MMP12, interference with synaptic stability may represent one mechanism by which these widely used drugs exert their side effects on higher CNS functions.

The effects of exercise on synaptic stripping require androgen receptor signaling.

Following peripheral nerve injury, synapses are withdrawn from axotomized motoneurons. Moderate daily treadmill exercise, which promotes axon regeneration of cut peripheral nerves, also influences this synaptic stripping. Different exercise protocols are required to promote axon regeneration in male and female animals, but the sex requirements for an effect of exercise on synaptic stripping are unknown. In male and female C57BL/6 mice, the sciatic nerve was transected in the mid-thigh. Mice were then exercised five days per week for two weeks, beginning on the third post-transection day. Half of the exercised mice were trained by walking slowly (10 M/min) on a level treadmill for one hour per day (continuous training). Other mice were interval trained; four short (two min) sprints at 20 M/min separated by five minute rest periods. A third group was untrained. The extent of synaptic contacts made by structures immunoreactive to vesicular glutamate transporter 1 and glutamic acid decarboxylase 67 onto axotomized motoneurons was studied in confocal images of retrogradely labeled cells. Both types of presumed synaptic contacts were reduced markedly in unexercised mice following nerve transection, relative to intact mice. No significant reduction was found in continuous trained males or interval trained females. Reductions in these contacts in interval trained males and continuous trained females were identical to that observed in untrained mice. Treatments with the anti-androgen, flutamide, blocked the effect of sex-appropriate exercise on synaptic contacts in both males and females. Moderate daily exercise has a potent effect on synaptic inputs to axotomized motoneurons. Successful effects of exercise have different requirements in males and females, but require androgen receptor signaling in both sexes.

Microglia: dismantling and rebuilding circuits after acute neurological injury.

The brain is comprised of neurons and its support system including astrocytes, glial cells and microglia, thereby forming neurovascular units. Neurons require support from glial cells to establish and maintain functional circuits, but microglia are often overlooked. Microglia function as the immune cell of the central nervous system, acting to monitor the microenvironment for changes in signaling, pathogens and injury. More recently, other functional roles for microglia within the healthy brain have been identified, including regulating synapse formation, elimination and function. This review aims to highlight and discuss these alternate microglial roles in the healthy and in contrast, diseased brain with a focus on two acute neurological diseases, traumatic brain injury and epilepsy. In these conditions, microglial roles in synaptic stripping and stabilization as part of neuronal:glial interactions may position them as mediators of the transition between injury-induced circuit dismantling and subsequent reorganization. Increased understanding of microglia roles could identify therapeutic targets to mitigate the consequences of neurological disease.

Axonal Regeneration after Sciatic Nerve Lesion Is Delayed but Complete in GFAP- and Vimentin-Deficient Mice

Peripheral axotomy of motoneurons triggers Wallerian degeneration of injured axons distal to the lesion, followed by axon regeneration. Centrally, axotomy induces loss of synapses (synaptic stripping) from the surface of lesioned motoneurons in the spinal cord. At the lesion site, reactive Schwann cells provide trophic support and guidance for outgrowing axons. The mechanisms of synaptic stripping remain elusive, but reactive astrocytes and microglia appear to be important in this process. We studied axonal regeneration and synaptic stripping of motoneurons after a sciatic nerve lesion in mice lacking the intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin, which are upregulated in reactive astrocytes and Schwann cells. Seven days after sciatic nerve transection, ultrastructural analysis of synaptic density on the somata of injured motoneurons revealed more remaining boutons covering injured somata in GFAP–/–Vim–/– mice. After sciatic nerve crush in GFAP–/–Vim–/– mice, the fraction of reinnervated motor endplates on muscle fibers of the gastrocnemius muscle was reduced 13 days after the injury, and axonal regeneration and functional recovery were delayed but complete. Thus, the absence of GFAP and vimentin in glial cells does not seem to affect the outcome after peripheral motoneuron injury but may have an important effect on the response dynamics.

285: Interferon regulatory factor 8 is essential for regulating the response of microglia to neuronal injury

The transcription factor interferon regulatory factor (IRF) 8 has a key role in the cellular response to IFN-gamma and is involved in myeloid cell differentiation. We have identified IRF8 to be a constitutive and IFN-gamma-stimulated nuclear factor in microglia. The objective of this study was to determine the role of IRF8 in the microglial response to sterile neuronal injury induced by axotomy. Facial nerve axotomy (FNA) was performed in wild type (WT) and IRF8 KO mice and the brain removed at different times post-lesion. A subset of mice was injected with bromodeoxyuridine (BrdU) every 2 h during the 24 h prior to retrieval of the brain. In brains from IRF8 KO mice, NDPase histochemistry revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied. Microglia from IRF8 KO mice also exhibited significant alterations in a number of key myeloid and immune markers. After FNA in WT mice, a progressive increase in microglial activation was observed in the lesioned facial nucleus that peaked at day 7 post-lesion. By contrast, in IRF8 KO mice, the microglial response appeared markedly attenuated and delayed. The attenuated microglial response to FNA in IRF8 KO mice was paralleled by a large decrease in proliferation as monitored by BrdU and phosphohistone 3 positive cells. The wrapping of individual motor neuron cell bodies by microglia involved in synaptic stripping and phagocytosis was incomplete in the absence of IRF8. Quantitative analysis showed that in IRF8 KO mice, the degeneration of axotomized motor neurons was slightly increased. These studies indicate that IRF8: (1) is a key homeostatic transcriptional regulator of microglial cell function in the healthy brain, and (2) has a crucial role in the functional response of microglia to neuronal injury. Supported by NHMRC project Grant APP1007757.

Interferon regulatory factor 8 deficiency causes phenotypic and functional changes in myeloid cells inside and outside the CNS (P4145)

Abstract We identified IRF8 as a constitutive and IFN-γ-stimulated nuclear factor in microglia of the CNS. IRF8 is a transcription factor with a key role in the cellular response to IFN-γ but is also pivotal in myeloid cell differentiation. Here we employed IRF8 KO mice to determine the role of IRF8 in microglial development and function in the healthy CNS and in the microglial response to injury (facial nerve axotomy [FNA]) or infection (West Nile Virus [WNV] encephalitis). Microglia were present in the CNS of IRF8 KO mice but showed altered morphology and significantly reduced surface area and had alterations in a number of key markers including reduced levels of Iba1, CCR2 and CCR5 but increased levels tomato lectin binding. Following FNA the microglial response was markedly delayed and attenuated in IRF8 KO mice and the wrapping of motor neuron cell bodies by microglia involved in synaptic stripping and phagocytosis was incomplete. Finally, IRF8 KO mice had increased susceptibility to WNV accompanied by a marked reduction in both microglial cell activation and leukocyte accumulation in the brain. In the periphery, ly6Cint monocytes were increased in number but exhibited defective trafficking to the infected CNS while ly6Chi monocytes were almost completely absent. In conclusion: (1) IRF8 is a major intrinsic determinant of the microglial phenotype in the healthy and the diseased CNS, and (2) IRF8 has a crucial role in the development and trafficking of inflammatory monocytes.

The Extent of Synaptic Stripping of Motoneurons after Axotomy Is Not Correlated to Activation of Surrounding Glia or Downregulation of Postsynaptic Adhesion Molecules

Synapse elimination in the adult central nervous system can be modelled by axotomy of spinal motoneurons which triggers removal of synapses from the cell surface of lesioned motoneurons by processes that remain elusive. Proposed candidate mechanisms are removal of synapses by reactive microglia and astrocytes, based on the remarkable activation of these cell types in the vicinity of motoneurons following axon lesion, and/or decreased expression of synaptic adhesion molecules in lesioned motoneurons. In the present study, we investigated glia activation and adhesion molecule expression in motoneurons in two mouse strains with deviant patterns of synapse elimination following axotomy. Mice deficient in complement protein C3 display a markedly reduced loss of synapses from axotomized motoneurons, whereas mice with impaired function of major histocompatibility complex (MHC) class Ia display an augmented degree of stripping after axotomy. Activation of microglia and astrocytes was assessed by semiquantative immunohistochemistry for Iba 1 (microglia) and GFAP (astrocytes), while expression of synaptic adhesion molecules was determined by in situ hybridization. In spite of the fact that the two mouse strains display very different degrees of synapse elimination, no differences in terms of glial activation or in the downregulation of the studied adhesion molecules (SynCAM1, neuroligin-2,-3 and netrin G-2 ligand) could be detected. We conclude that neither glia activation nor downregulation of synaptic adhesion molecules are correlated to the different extent of the synaptic stripping in the two studied strains. Instead the magnitude of the stripping event is most likely a consequence of a precise molecular signaling, which at least in part is mediated by immune molecules.

Early presymptomatic cholinergic dysfunction in a murine model of amyotrophic lateral sclerosis

Sporadic and familiar amyotrophic lateral sclerosis (ALS) cases presented lower cholinergic activity than in healthy individuals in their still preserved spinal motoneurons (MNs) suggesting that cholinergic reduction might occur before MN death. To unravel how and when cholinergic function is compromised, we have analyzed the spatiotemporal expression of choline acetyltransferase (ChAT) from early presymptomatic stages of the SOD1G93A ALS mouse model by confocal immunohistochemistry. The analysis showed an early reduction in ChAT content in soma and presynaptic boutons apposed onto MNs (to 76%) as well as in cholinergic interneurons in the lumbar spinal cord of the 30-day-old SOD1G93A mice. Cholinergic synaptic stripping occurred simultaneously to the presence of abundant surrounding major histocompatibility complex II (MHC-II)-positive microglia and the accumulation of nuclear Tdp-43 and the appearance of mild oxidative stress within MNs. Besides, there was a loss of neuronal MHC-I expression, which is necessary for balanced synaptic stripping after axotomy. These events occurred before the selective raise of markers of denervation such as ATF3. By the same time, alterations in postsynaptic cholinergic-related structures were also revealed with a loss of the presence of sigma-1 receptor, a Ca2+ buffering chaperone in the postsynaptic cisternae. By 2 months of age, ChAT seemed to accumulate in the soma of MNs, and thus efferences toward Renshaw interneurons were drastically diminished. In conclusion, cholinergic dysfunction in the local circuitry of the spinal cord may be one of the earliest events in ALS etiopathogenesis.

Rod microglia: elongation, alignment, and coupling to form trains across the somatosensory cortex after experimental diffuse brain injury

BackgroundSince their discovery, the morphology of microglia has been interpreted to mirror their function, with ramified microglia constantly surveying the micro-environment and rapidly activating when changes occur. In 1899, Franz Nissl discovered what we now recognize as a distinct microglial activation state, microglial rod cells (Stäbchenzellen), which he observed adjacent to neurons. These rod-shaped microglia are typically found in human autopsy cases of paralysis of the insane, a disease of the pre-penicillin era, and best known today from HIV-1-infected brains. Microglial rod cells have been implicated in cortical ‘synaptic stripping’ but their exact role has remained unclear. This is due at least in part to a scarcity of experimental models. Now we have noted these rod microglia after experimental diffuse brain injury in brain regions that have an associated sensory sensitivity. Here, we describe the time course, location, and surrounding architecture associated with rod microglia following experimental diffuse traumatic brain injury (TBI).MethodsRats were subjected to a moderate midline fluid percussion injury (mFPI), which resulted in transient suppression of their righting reflex (6 to 10 min). Multiple immunohistochemistry protocols targeting microglia with Iba1 and other known microglia markers were undertaken to identify the morphological activation of microglia. Additionally, labeling with Iba1 and cell markers for neurons and astrocytes identified the architecture that surrounds these rod cells.ResultsWe identified an abundance of Iba1-positive microglia with rod morphology in the primary sensory barrel fields (S1BF). Although present for at least 4 weeks post mFPI, they developed over the first week, peaking at 7 days post-injury. In the absence of contusion, Iba1-positive microglia appear to elongate with their processes extending from the apical and basal ends. These cells then abut one another and lay adjacent to cytoarchitecture of dendrites and axons, with no alignment with astrocytes and oligodendrocytes. Iba1-positive rod microglial cells differentially express other known markers for reactive microglia including OX-6 and CD68.ConclusionDiffuse traumatic brain injury induces a distinct rod microglia morphology, unique phenotype, and novel association between cells; these observations entice further investigation for impact on neurological outcome.

Reduced removal of synaptic terminals from axotomized spinal motoneurons in the absence of complement C3

Complement proteins C1q and C3 play a critical role in synaptic elimination during development. Axotomy of spinal motoneurons triggers removal of synaptic terminals from the cell surface of motoneurons by largely unknown mechanisms. We therefore hypothesized that the complement system is involved also in synaptic stripping of injured motoneurons. In the sciatic motor pool of wild type (WT) mice, the immunoreactivity (IR) for both C1q and C3 was increased after sciatic nerve transection (SNT). Mice deficient in C3 (C3−/−) showed a reduced loss of synaptic terminals from injured motoneurons at one week after SNT, as assessed by immunoreactivity for synaptic markers and electron microscopy. In particular, the removal of putative inhibitory terminals, immunopositive for vesicular inhibitory amino acid transporter (VIAAT) and ultrastructurally identified as type F synapses, was reduced in C3−/− mice. In contrast, lesion-induced removal of nerve terminals in C1q−/− mice appeared similar to WT mice. Growth associated protein (GAP)-43 mRNA expression in lesioned motoneurons increased much more in C3−/− compared to WT mice after SNT. After sciatic nerve crush (SNC), the C3−/− mice showed a faster functional recovery, assessed as grip strength, compared to WT mice. No differences were detected regarding nerve inflammation at the site of injury or pattern of muscle reinnervation. These data indicate that a non-classical pathway of complement activation is involved in axotomy-induced adult synapse removal, and that its inhibition promotes functional recovery.