VEGF is an essential retrograde trophic factor for motoneurons.

VEGF was initially discovered due to its angiogenic activity and therefore named "vascular endothelial growth factor." However, its more recently discovered neurotrophic activity may be evolutionarily more ancient. Our previous work showed that all the changes produced by axotomy on the firing activity and synaptic inputs of abducens motoneurons were completely restored after VEGF administration. Therefore, we hypothesized that the lack of VEGF delivered by retrograde transport from the periphery should also affect the physiology of otherwise intact abducens motoneurons. For VEGF retrograde blockade, we chronically applied a neutralizing VEGF antibody to the lateral rectus muscle. Recordings of extracellular single-unit activity and eye movements were made in alert cats before and after the application of the neutralizing antibody. Our data revealed that intact, noninjured abducens motoneurons retrogradely deprived of VEGF exhibited noticeable changes in their firing pattern. There is a general decrease in firing rate and a significant reduction in eye position and eye velocity sensitivity (i.e., a decrease in the tonic and phasic components of their discharge, respectively). Moreover, by means of confocal immunocytochemistry, motoneurons under VEGF blockade showed a marked reduction in the density of afferent synaptic terminals contacting with their cell bodies. Altogether, the present findings demonstrate that the lack of retrogradely delivered VEGF renders abducens motoneurons into an axotomy-like state. This indicates that VEGF is an essential retrograde factor for motoneuronal synaptic drive and discharge activity.

Microglia orchestrate synaptic and neuronal stripping: Implication in neuropsychiatric lupus.

Systemic lupus erythematosus (SLE), a multifactorial autoimmune disease, can affect the brain and cause neuropsychiatric dysfunction, also named neuropsychiatric lupus (NPSLE). Microglial activation is observed in NPSLE patients. However, the mechanisms regulating microglia-mediated neurotoxicity in NPSLE remain elusive. Here, we showed that M1-like proinflammatory cytokine levels were increased in the cerebrospinal fluid (CSF) of SLE patients, especially those with neuropsychiatric symptoms. We also demonstrated that MRL/lpr lupus mice developed anxiety-like behaviours and cognitive deficits in the early and active phases of lupus, respectively. An increase in microglial number was associated with upregulation of proinflammatory cytokines in the MRL/lpr mouse brain. RNA sequencing revealed that genes associated with phagocytosis and M1 polarization were upregulated in microglia from lupus mice. Functionally, activated microglia induced synaptic stripping in vivo and promoted neuronal death in vitro. Finally, tofacitinib ameliorated neuropsychiatric disorders in MRL/lpr mice, as evidenced by reductions in microglial number and synaptic/neuronal loss and alleviation of behavioural abnormalities. Thus, our results indicated that classically activated (M1) microglia play a crucial role in NPSLE pathogenesis. Minocycline and tofacitinib were found to alleviate NPSLE by inhibiting micrglial activation, providing a promising therapeutic strategy.

Microglia induce auditory dysfunction after status epilepticus in mice.

Auditory dysfunction and increased neuronal activity in the auditory pathways have been reported in patients with temporal lobe epilepsy, but the cellular mechanisms involved are unknown. Here, we report that microglia play a role in the disinhibition of auditory pathways after status epilepticus in mice. We found that neuronal activity in the auditory pathways, including the primary auditory cortex and the medial geniculate body (MGB), was increased and auditory discrimination was impaired after status epilepticus. We further demonstrated that microglia reduced inhibitory synapses on MGB relay neurons over an 8-week period after status epilepticus, resulting in auditory pathway hyperactivity. In addition, we found that local removal of microglia from the MGB attenuated the increase in c-Fos+ relay neurons and improved auditory discrimination. These findings reveal that thalamic microglia are involved in auditory dysfunction in epilepsy.

Microglia and neuroprotection.

Microglia were first identified over a century ago, but our knowledge about their ontogeny and functions has significantly expanded only recently. Microglia colonize the central nervous system (CNS) in utero and play essential roles in brain development. Once neural development is completed, microglia function as the resident innate immune cells of the CNS by surveying their microenvironment and becoming activated when the CNS is challenged by infection, injury, or disease. Despite the traditional view of microglia as being destructive in neurological diseases, recent studies have shown that microglia maintain CNS homeostasis and protect the CNS under various pathological conditions. Microglia can be prophylactically activated by modeling infection with systemic lipopolysaccharide injections and these activated microglia can protect the brain from traumatic injury through modulation of neuronal synapses. Microglia can also protect the CNS by promoting neurogenesis, clearing debris, and suppressing inflammation in diseases such as stroke, autism, and Alzheimer's. Microglia are the resident innate immune cells of the CNS. Despite the traditional view of microglia as being destructive in neurological diseases, recent studies have shown that they maintain tissue homeostasis and protect the CNS under various pathological conditions. They achieve so by clearing debris, promoting neurogenesis, suppressing inflammation and stripping inhibitory synapses. This review summarizes recent advances of our understanding on the multi-dimensional neuroprotective roles of microglia.

Celsr2 Knockout Alleviates Inhibitory Synaptic Stripping and Benefits Motoneuron Survival and Axon Regeneration After Branchial Plexus Avulsion.

Axotomy-induced synaptic stripping modulates survival and axon regeneration of injured motoneurons. Celsr2 is supposed to mediate homophilic interactions of neighboring cells during development, and its role in synaptic stripping remains unknow. In a model of brachial plexus avulsion, Celsr2 knockout improved functional recovery, motoneuron survival, and axon regeneration. Celsr2 was indicated to express in spinal motoneurons, excitatory and inhibitory interneurons, astrocytes, and a subset of oligodendrocytes using Celsr2LacZ mice. Double immunostaining showed that the coverage of inhibitory and excitatory vesicles on injured motoneurons were remarkably reduced after injury, whereas more inhibitory vesicles were maintained in Celsr2-/- mutants than control mice. In the ultrastructure, the density of inhibitory F-boutons on injured motoneurons was higher in Celsr2-/- mutants than controls. Conditional knockout of Celsr2 in astrocytes or oligodendrocytes showed the similar axotomy-induced synaptic withdrawal to the control. RNAseq of injured spinal samples identified 12 MHC I molecules with significant changes between Celsr2-/- and control mice. After injury, expression of MHC I surrounding injured motoneurons was increased, particularly high in Celsr2-/- mutants. In conclusion, Celsr2 knockout enhances MHC I signaling, alleviates inhibitory synaptic stripping cell-autonomously, and contributes to motoneuron survival and regeneration, and Celsr2 is a potential target for neural repair.

Extraocular Motoneurons and Neurotrophism.

Extraocular motoneurons are located in three brainstem nuclei: the abducens, trochlear and oculomotor. They control all types of eye movements by innervating three pairs of agonistic/antagonistic extraocular muscles. They exhibit a tonic-phasic discharge pattern, demonstrating sensitivity to eye position and sensitivity to eye velocity. According to their innervation pattern, extraocular muscle fibers can be classified as singly innervated muscle fiber (SIF), or the peculiar multiply innervated muscle fiber (MIF). SIF motoneurons show anatomical and physiological differences with MIF motoneurons. The latter are smaller and display lower eye position and velocity sensitivities as compared with SIF motoneurons.

A Single Intraventricular Injection of VEGF Leads to Long-Term Neurotrophic Effects in Axotomized Motoneurons.

Vascular endothelial growth factor (VEGF) has been recently demonstrated to induce neuroprotective and synaptotrophic effects on lesioned neurons. Hitherto, the administration of VEGF in different animal models of lesion or disease has been conducted following a chronic protocol of administration. We questioned whether a single dose of VEGF, administered intraventricularly, could induce long-term neurotrophic effects on injured motoneurons. For this purpose, we performed in cats the axotomy of abducens motoneurons and the injection of VEGF into the fourth ventricle in the same surgical session and investigated the discharge characteristics of axotomized and treated motoneurons by single-unit extracellular recordings in the chronic alert preparation. We found that injured motoneurons treated with a single VEGF application discharged with normal characteristics, showing neuronal eye position (EP) and velocity sensitivities similar to control, thereby preventing the axotomy-induced alterations. These effects were present for a prolonged period of time (50 d) after VEGF administration. By confocal immunofluorescence we also showed that the synaptic stripping that ensues lesion was not present, rather motoneurons showed a normal synaptic coverage. Moreover, we demonstrated that VEGF did not lead to any angiogenic response pointing to a direct action of the factor on neurons. In summary, a single dose of VEFG administered just after motoneuron axotomy is able to prevent for a long time the axotomy-induced firing and synaptic alterations without any associated vascular sprouting. We consider that these data are of great relevance due to the potentiality of VEGF as a therapeutic agent in neuronal lesions and diseases.

Microglia: The Neural Cells of Nonneural Origin.

Microglia are neural cells of nonneural origin; they originate from fetal macrophages that invade neural tube early in embryogenesis and undergo the most idiosyncratic metamorphosis which coverts them into elements of neural circuitry. Microglia appeared early in evolution with neural immune cells being operative in leeches and mollusks. Microglial cells acquire specific morphology characterized by small cell bodies and long motile processes which are packed with receptors sensing both physiological and pathological stimuli. Microglial cells actively sculpture neuronal networks through synaptic stripping and phagocytosis of redundant neurons; microglia also secrete neuroactive factors regulating synaptic transmission. Novel techniques emerging in recent decade allowed an in-depth understanding of physiological and pathophysiological functions of microglia.

Synaptic loss and firing alterations in Axotomized Motoneurons are restored by vascular endothelial growth factor (VEGF) and VEGF-B.

Vascular endothelial growth factor (VEGF), also known as VEGF-A, was discovered due to its vasculogenic and angiogenic activity, but a neuroprotective role for VEGF was later proven for lesions and disorders. In different models of motoneuronal degeneration, VEGF administration leads to a significant reduction of motoneuronal death. However, there is no information about the physiological state of spared motoneurons. We examined the trophic role of VEGF on axotomized motoneurons with recordings in alert animals using the oculomotor system as the experimental model, complemented with a synaptic study at the confocal microscopy level. Axotomy leads to drastic alterations in the discharge characteristics of abducens motoneurons, as well as to a substantial loss of their synaptic inputs. Retrograde delivery of VEGF completely restored the discharge activity and synaptically-driven signals in injured motoneurons, as demonstrated by correlating motoneuronal firing rate with motor performance. Moreover, VEGF-treated motoneurons recovered a normal density of synaptic boutons around motoneuronal somata and in the neuropil, in contrast to the low levels of synaptic terminals found after axotomy. VEGF also reduced the astrogliosis induced by axotomy in the abducens nucleus to control values. The administration of VEGF-B produced results similar to those of VEGF. This is the first work demonstrating that VEGF and VEGF-B restore the normal operating mode and synaptic inputs on injured motoneurons. Altogether these data indicate that these molecules are relevant synaptotrophic factors for motoneurons and support their clinical potential for the treatment of motoneuronal disorders.

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.

Effects of acoustic trauma on the auditory system of the rat: The role of microglia.

Exposure to loud, prolonged sounds (acoustic trauma, AT) leads to the death of both inner and outer hair cells (IHCs and OHCs), death of neurons of the spiral ganglion and degeneration of the auditory nerve. The auditory nerve (8cn) projects to the three subdivisions of the cochlear nuclei (CN), the dorsal cochlear nucleus (DC) and the anterior (VCA) and posterior (VCP) subdivisions of the ventral cochlear nucleus (VCN). There is both anatomical and physiological evidence for plastic reorganization in the denervated CN after AT. Anatomical findings show axonal sprouting and synaptogenesis; physiologically there is an increase in spontaneous activity suggesting reorganization of circuitry. The mechanisms underlying this plasticity are not understood. Recent data suggest that activated microglia may have a role in facilitating plastic reorganization in addition to removing trauma-induced debris. In order to investigate the roles of activated microglia in the CN subsequent to AT we exposed animals to bilateral noise sufficient to cause massive hair cell death. We studied four groups of animals at different survival times: 30 days, 60 days, 6 months and 9 months. We used silver staining to examine the time course and pattern of auditory nerve degeneration, and immunohistochemistry to label activated microglia in the denervated CN. We found both degenerating auditory nerve fibers and activated microglia in the CN at 30 and 60 days and 6 months after AT. There was close geographic overlap between the degenerating fibers and activated microglia, consistent with a scavenger role for activated microglia. At the longest survival time, there were still silver-stained fibers but very little staining of activated microglia in overlapping regions. There were, however, activated microglia in the surrounding brainstem and cerebellar white matter.

Retrograde response in axotomized motoneurons: nitric oxide as a key player in triggering reversion toward a dedifferentiated phenotype.

The adult brain retains a considerable capacity to functionally reorganize its circuits, which mainly relies on the prevalence of three basic processes that confer plastic potential: synaptic plasticity, plastic changes in intrinsic excitability and, in certain central nervous system (CNS) regions, also neurogenesis. Experimental models of peripheral nerve injury have provided a useful paradigm for studying injury-induced mechanisms of central plasticity. In particular, axotomy of somatic motoneurons triggers a robust retrograde reaction in the CNS, characterized by the expression of plastic changes affecting motoneurons, their synaptic inputs and surrounding glia. Axotomized motoneurons undergo a reprograming of their gene expression and biosynthetic machineries which produce cell components required for axonal regrowth and lead them to resume a functionally dedifferentiated phenotype characterized by the removal of afferent synaptic contacts, atrophy of dendritic arbors and an enhanced somato-dendritic excitability. Although experimental research has provided valuable clues to unravel many basic aspects of this central response, we are still lacking detailed information on the cellular/molecular mechanisms underlying its expression. It becomes clear, however, that the state-switch must be orchestrated by motoneuron-derived signals produced under the direction of the re-activated growth program. Our group has identified the highly reactive gas nitric oxide (NO) as one of these signals, by providing robust evidence for its key role to induce synapse elimination and increases in intrinsic excitability following motor axon damage. We have elucidated operational principles of the NO-triggered downstream transduction pathways mediating each of these changes. Our findings further demonstrate that de novo NO synthesis is not only "necessary" but also "sufficient" to promote the expression of at least some of the features that reflect reversion toward a dedifferentiated state in axotomized adult motoneurons.

The role of microglia in synaptic stripping and synaptic degeneration: a revised perspective.

Chronic neurodegenerative diseases of the CNS (central nervous system) are characterized by the loss of neurons. There is, however, growing evidence to show that an early stage of this process involves degeneration of presynaptic terminals prior to the loss of the cell body. Synaptic plasticity in CNS pathology has been associated with microglia and the phenomenon of synaptic stripping. We review here the evidence for the involvement of microglia in synaptic stripping and synapse degeneration and we conclude that this is a case of guilt by association. In disease models of chronic neurodegeneration, there is no evidence that microglia play an active role in either synaptic stripping or synapse degeneration, but the degeneration of the synapse and the envelopment of a degenerating terminal appears to be a neuron autonomous event. We highlight here some of the gaps in our understanding of synapse degeneration in chronic neurodegenerative disease.