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Abstract Background Migraine affects a significant fraction of the world population, yet its etiology is not completely understood. In vitro results highlighted thalamocortical and intra-cortical glutamatergic synaptic gain-of-function associated with a monogenic form of migraine (familial-hemiplegic-migraine-type-1: FHM1). However, how these alterations reverberate on cortical activity remains unclear. As altered responsivity to visual stimuli and abnormal processing of visual sensory information are common hallmarks of migraine, herein we investigated the effects of FHM1-driven synaptic alterations in the visual cortex of awake mice. Methods We recorded extracellular field potentials from the primary visual cortex (V1) of head-fixed awake FHM1 knock-in (n = 12) and wild type (n = 12) mice in response to square-wave gratings with different visual contrasts. Additionally, we reproduced in silico the obtained experimental results with a novel spiking neurons network model of mouse V1, by implementing in the model both the synaptic alterations characterizing the FHM1 genetic mouse model adopted. Results FHM1 mice displayed similar amplitude but slower temporal evolution of visual evoked potentials. Visual contrast stimuli induced a lower increase of multi-unit activity in FHM1 mice, while the amount of information content about contrast level remained, however, similar to WT. Spectral analysis of the local field potentials revealed an increase in the β/low γ range of WT mice following the abrupt reversal of contrast gratings. Such frequency range transitioned to the high γ range in FHM1 mice. Despite this change in the encoding channel, these oscillations preserved the amount of information conveyed about visual contrast. The computational model showed how these network effects may arise from a combination of changes in thalamocortical and intra-cortical synaptic transmission, with the former inducing a lower cortical activity and the latter inducing the higher frequencies ɣ oscillations. Conclusions Contrast-driven ɣ modulation in V1 activity occurs at a much higher frequency in FHM1. This is likely to play a role in the altered processing of visual information. Computational studies suggest that this shift is specifically due to enhanced cortical excitatory transmission. Our network model can help to shed light on the relationship between cellular and network levels of migraine neural alterations. Graphical Abstract

Cephalic tetanus (CT) is a severe form of tetanus that follows head wounds and the intoxication of cranial nerves by tetanus neurotoxin (TeNT). Hallmarks of CT are cerebral palsy, which anticipates the spastic paralysis of tetanus, and rapid evolution of cardiorespiratory deficit even without generalized tetanus. How TeNT causes this unexpected flaccid paralysis, and how the canonical spasticity then rapidly evolves into cardiorespiratory defects, remain unresolved aspects of CT pathophysiology. Using electrophysiology and immunohistochemistry, we demonstrate that TeNT cleaves its substrate vesicle-associated membrane protein within facial neuromuscular junctions and causes a botulism-like paralysis overshadowing tetanus spasticity. Meanwhile, TeNT spreads among brainstem neuronal nuclei and, as shown by an assay measuring the ventilation ability of CT mice, harms essential functions like respiration. A partial axotomy of the facial nerve revealed a potentially new ability of TeNT to undergo intra-brainstem diffusion, which allows the toxin to spread to brainstem nuclei devoid of direct peripheral efferents. This mechanism is likely to be involved in the transition from local to generalized tetanus. Overall, the present findings suggest that patients with idiopathic facial nerve palsy should be immediately considered for CT and treated with antisera to block the potential progression to a life-threatening form of tetanus.

Abstract Krabbe disease (KD; or globoid cell leukodystrophy) is an autosomal recessive lysosomal storage disorder caused by deficiency of the galactosylceramidase (GALC) enzyme. No cure is currently available for KD. Clinical applied treatments are supportive only. Recently, we demonstrated that two differently acting autophagy inducers (lithium and rapamycin) can improve some KD hallmarks in‐vitro, laying the foundation for their in‐vivo pre‐clinical testing. Here, we test lithium carbonate in‐vivo, in the spontaneous mouse model for KD, the Twitcher (TWI) mouse. The drug is administered ad libitum via drinking water (600 mg/L) starting from post natal day 20. We longitudinally monitor the mouse motor performance through the grip strength, the hanging wire and the rotarod tests, and a set of biochemical parameters related to the KD pathogenesis [i.e., GALC enzymatic activity, psychosine (PSY) accumulation and astrogliosis]. Additionally, we investigate the expression of some crucial markers related to the two pathways that could be altered by lithium: the autophagy and the β‐catenin‐dependent pathways. Results demonstrate that lithium has not a significant rescue effect on the TWI phenotype, although it can slightly and transiently improves muscle strength. We also show that lithium, with this administration protocol, is unable to stimulate autophagy in the TWI mice central nervous system, whereas results suggest that it can restore the β‐catenin activation status in the TWI sciatic nerve. Overall, these data provide intriguing inputs for further evaluations of lithium treatment in TWI mice.

Experience-dependent neuronal changes and brain plasticity occur throughout life as animals adapt to their environment. Structural, morphological, and cellular modifications promoted by exposure to environmental enrichment (EE) have been reported to improve neuronal functions, increase hippocampal neurogenesis, ameliorate memory tasks and cognitive performance, and have beneficial effects on several brain diseases, including cancer. We specifically addressed the role of the EE in counteracting neuronal dysfunction in mice bearing glioma in the primary visual cortex. By recording spontaneous and evoked currents with patch clamp techniques in acute slices obtained from standard and enriched-housed mice, we found that the presence of glioma globally reduced the excitatory and inhibitory transmissions in the peritumoral area. The exposure to an enriched environment counteracts the tumor-mediated depression of both excitatory and inhibitory neuronal activities, with a more pronounced impact on evoked transmission. The effect of EE on glioma was also associated with reduced tumor cell proliferation. These results elucidate the impact of EE on excitatory and inhibitory neurotransmission of the primary visual cortex in control and glioma-bearing mice.

Abstract Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV’s) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV’s both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV’s isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1).

Introduction: Obesity and psychosocial stress (PS) co-exist in individuals of Western society. Nevertheless, how PS impacts cardiac and hippocampal phenotype in obese subjects is still unknown. Nor is it clear whether changes in local brain-derived neurotrophic factor (BDNF) account, at least in part, for myocardial and behavioral abnormalities in obese experiencing PS. Methods: In adult male WT mice, obesity was induced via a high-fat diet (HFD). The resident-intruder paradigm was superimposed to trigger PS. In vivo left ventricular (LV) performance was evaluated by echocardiography and pressure-volume loops. Behaviour was indagated by elevated plus maze (EPM) and Y-maze. LV myocardium was assayed for apoptosis, fibrosis, vessel density and oxidative stress. Hippocampus was analyzed for volume, neurogenesis, GABAergic markers and astrogliosis. Cardiac and hippocampal BDNF and TrkB levels were measured by ELISA and WB. We investigated the pathogenetic role played by BDNF signaling in additional cardiac-selective TrkB (cTrkB) KO mice. Findings: When combined, obesity and PS jeopardized LV performance, causing prominent apoptosis, fibrosis, oxidative stress and remodeling of the larger coronary branches, along with lower BDNF and TrkB levels. HFD/PS weakened LV function similarly in WT and cTrkB KO mice. The latter exhibited elevated LV ROS emission already at baseline. Obesity/PS augmented anxiety-like behaviour and impaired spatial memory. These changes were coupled to reduced hippocampal volume, neurogenesis, local BDNF and TrkB content and augmented astrogliosis. Interpretation: PS and obesity synergistically deteriorate myocardial structure and function by depleting cardiac BDNF/TrkB content, leading to augmented oxidative stress. This comorbidity triggers behavioral deficits and induces hippocampal remodeling, potentially via lower BDNF and TrkB levels. Fund: J.A. was in part supported by Rotary Foundation Global Study Scholarship. G.K. was supported by T32 National Institute of Health (NIH) training grant under award number 1T32AG058527. S.C. was funded by American Heart Association Career Development Award (19CDA34760185). G.A.R.C. was funded by NIH (K01HL133368-01). APB was funded by a Grant from the Friuli Venezia Giulia Region entitled: “Heart failure as the Alzheimer disease of the heart; therapeutic and diagnostic opportunities”. M.C. was supported by PRONAT project (CNR). N.P. was funded by NIH (R01 HL136918) and by the Magic-That-Matters fund (JHU). V.L. was in part supported by institutional funds from Scuola Superiore Sant'Anna (Pisa, Italy), by the TIM-Telecom Italia (WHITE Lab, Pisa, Italy), by a research grant from Pastificio Attilio Mastromauro Granoro s.r.l. (Corato, Italy) and in part by ETHERNA project (Prog. n. 161/16, Fondazione Pisa, Italy). Funding source had no such involvement in study design, in the collection, analysis, interpretation of data, in the writing of the report; and in the decision to submit the paper for publication. Keywords: Brain-heart axis, Brain-derived neurotrophic factor (BDNF), Obesity, Psychosocial stress, Left ventricle, Hippocampus, Tropomyosin receptor kinase B (TrkB), Oxidative stress

Krabbe disease (KD) is a childhood leukodystrophy with no cure currently available. KD is due to a deficiency of a lysosomal enzyme called galactosyl-ceramidase (GALC) and is characterized by the accumulation in the nervous system of the sphingolipid psychosine (PSY), whose cytotoxic molecular mechanism is not fully known yet.Here, we study the expression of some fundamental autophagy markers (LC3, p62, and Beclin-1) in a KD murine model [the twitcher (TWI) mouse] by immunohistochemistry and Western blot. Moreover, the autophagy molecular process is also shown in primary fibroblasts from TWI and WT mice, with and without PSY treatment. Data demonstrate that large p62 cytoplasmic aggregates are present in the brain of both early and late symptomatic TWI mice. p62 expression is also upregulated in TWI sciatic nerves compared to that measured for WT nerves. In vitro data suggest that this effect might not be fully PSY-driven. Finally, we investigate in vitro the capability of autophagy inducers (Rapamycin, RAP and Resveratrol, RESV) to reinstate the WT phenotype in TWI cells. We show that RAP administration can partially restore the autophagy markers levels, while RESV cannot, indicating a line along which new therapeutic approaches can be developed.

Neuronal hyperexcitability often results from an unbalance between excitatory and inhibitory neurotransmission, but the synaptic alterations leading to enhanced seizure propensity are only partly understood. Taking advantage of a mouse model of neocortical epilepsy, we used a combination of photoconversion and electron microscopy to assess changes in synaptic vesicles pools in vivo. Our analyses reveal that epileptic networks show an early onset lengthening of active zones at inhibitory synapses, together with a delayed spatial reorganization of recycled vesicles at excitatory synapses. Proteomics of synaptic content indicate that specific proteins were increased in epileptic mice. Altogether, our data reveal a complex landscape of nanoscale changes affecting the epileptic synaptic release machinery. In particular, our findings show that an altered positioning of release-competent vesicles represent a novel signature of epileptic networks.

Recent studies have demonstrated an active role for neurons in glioma progression. Specifically, peritumoral neurons establish functional excitatory synapses with glioma cells, and optogenetic stimulation of cortical pyramidal neurons drives tumor progression. However, the specific role of different subsets of cortical neurons, such as GABAergic interneurons, remains unexplored. Here, we directly compared the effects of optogenetic stimulation of pyramidal cells vs. fast-spiking, GABAergic neurons. In mice inoculated with GL261 cells into the motor cortex, we show that optogenetic stimulation of pyramidal neurons enhances glioma cell proliferation. In contrast, optogenetic stimulation of fast-spiking, parvalbumin-positive interneurons reduces proliferation as measured by BrdU incorporation and Ki67 immunolabelling. Since both principal cells and fast-spiking interneurons are directly activated by sensory afferent input, we next placed tumors in the occipital cortex to test the impact of visual stimulation/deprivation. We report that total lack of visual input via dark rearing enhances the density of proliferating glioma cells, while daily visual stimulation by gratings of different spatial frequencies and contrast reduces tumor growth. The effects of sensory input are region-specific, as visual deprivation has no significant effect on tumor proliferation in mice with gliomas in the motor cortex. We also report that sensory stimulation combined with temozolomide administration delays the loss of visual responses in peritumoral neurons. Altogether, these data demonstrate complex effects of different neuronal subtypes in the control of glioma proliferation.

Being able to replicate real experiments with computational simulations is a unique opportunity to refine and validate models with experimental data and redesign the experiments based on simulations. However, since it is technically demanding to model all components of an experiment, traditional approaches to modeling reduce the experimental setups as much as possible. In this study, our goal is to replicate all the relevant features of an experiment on motor control and motor rehabilitation after stroke. To this aim, we propose an approach that allows continuous integration of new experimental data into a computational modeling framework. First, results show that we could reproduce experimental object displacement with high accuracy via the simulated embodiment in the virtual world by feeding a spinal cord model with experimental registration of the cortical activity. Second, by using computational models of multiple granularities, our preliminary results show the possibility of simulating several features of the brain after stroke, from the local alteration in neuronal activity to long-range connectivity remodeling. Finally, strategies are proposed to merge the two pipelines. We further suggest that additional models could be integrated into the framework thanks to the versatility of the proposed approach, thus allowing many researchers to achieve continuously improved experimental design.

Neurological diseases constitute a quarter of global disease burden and are expected to rise worldwide with the ageing of human populations. There is an increasing need to develop new molecular systems which can deliver drugs specifically into neurons, non-dividing cells meant to last a human lifetime. Neuronal drug delivery must rely on agents which can recognise neurons with high specificity and affinity. Here we used a recently introduced ‘stapling’ system to prepare macromolecules carrying duplicated binding domains from the clostridial family of neurotoxins. We engineered individual parts of clostridial neurotoxins separately and combined them using a strong alpha-helical bundle. We show that combining two identical binding domains of tetanus and botulinum type D neurotoxins, in a sterically defined way by protein stapling, allows enhanced intracellular delivery of molecules into neurons. We also engineered a botulinum neurotoxin type C variant with a duplicated binding domain which increased enzymatic delivery compared to the native type C toxin. We conclude that duplication of the binding parts of tetanus or botulinum neurotoxins will allow production of high avidity agents which could deliver imaging reagents and large therapeutic enzymes into neurons with superior efficiency. Keywords: Botulinum, Tetanus, Duplicated, Double, Multivalent, Neuronal delivery

Summary: The capability of generating neural precursor cells with distinct types of regional identity in vitro has recently opened new opportunities for cell replacement in animal models of neurodegenerative diseases. By manipulating Wnt and BMP signaling, we steered the differentiation of mouse embryonic stem cells (ESCs) toward isocortical or hippocampal molecular identity. These two types of cells showed different degrees of axonal outgrowth and targeted different regions when co-transplanted in healthy or lesioned isocortex or in hippocampus. In hippocampus, only precursor cells with hippocampal molecular identity were able to extend projections, contacting CA3. Conversely, isocortical-like cells were capable of extending long-range axonal projections only when transplanted in motor cortex, sending fibers toward both intra- and extra-cortical targets. Ischemic damage induced by photothrombosis greatly enhanced the capability of isocortical-like cells to extend far-reaching projections. Our results indicate that neural precursors generated by ESCs carry intrinsic signals specifying axonal extension in different environments. : In this article, Terrigno and colleagues show that Wnt and BMB signaling control the differentiation of mouse ESCs toward isocortical or hippocampal identity in vitro. The two types of cells contact different regions when transplanted in adult brain. Photothrombotic lesion favors neurite elongation of cortical transplanted cells, which can improve the motor performance after ischemic damage of motor cortex. Keywords: mouse embryonic stem cells, WNT signaling, neuronal identity, transplantation, stroke, cell replacement, isocortex, hippocampus, axonal extension, axonal projection

Ischemic damage in brain tissue triggers a cascade of molecular and structural plastic changes, thus influencing a wide range of cell-to-cell interactions. Understanding and manipulating this scenario of intercellular connections is the Holy Grail for post-stroke neurorehabilitation. Here, we discuss the main findings in the literature related to post-stroke alterations in cell-to-cell interactions, which may be either detrimental or supportive for functional recovery. We consider both neural and non-neural cells, starting from astrocytes and reactive astrogliosis and moving to the roles of the oligodendrocytes in the support of vulnerable neurons and sprouting inhibition. We discuss the controversial role of microglia in neural inflammation after injury and we conclude with the description of post-stroke alterations in pyramidal and GABAergic cells interactions. For all of these sections, we review not only the spontaneous evolution in cellular interactions after ischemic injury, but also the experimental strategies which have targeted these interactions and that are inspiring novel therapeutic strategies for clinical application.

Glioblastoma Multiforme (GBM) is a brain tumor with a poor prognosis and low survival rates. GBM is diagnosed at an advanced stage, so little information is available on the early stage of the disease and few improvements have been made for earlier diagnosis. Longitudinal murine models are a promising platform for biomarker discovery as they allow access to the early stages of the disease. Nevertheless, their use in proteomics has been limited owing to the low sample amount that can be collected at each longitudinal time point. Here we used optimized microproteomics workflows to investigate longitudinal changes in the protein profile of serum, serum small extracellular vesicles (sEVs), and cerebrospinal fluid (CSF) in a GBM murine model. Baseline, pre-symptomatic, and symptomatic tumor stages were determined using non-invasive motor tests. Forty-four proteins displayed significant differences in signal intensities during GBM progression. Dysregulated proteins are involved in cell motility, cell growth, and angiogenesis. Most of the dysregulated proteins already exhibited a difference from baseline at the pre-symptomatic stage of the disease, suggesting that early effects of GBM might be detectable before symptom onset.

Changes to subsets of dendritic spines are thought to be important for memory formation. Here, the authors develop a hybrid RNA/protein tool that allows for optogenetic stimulation of single synapses that have been tagged in an activity-dependent manner

Abstract Transplantation of human bone marrow mesenchymal stromal cells (hBM-MSC) promotes functional recovery after stroke in animal models, but the mechanisms underlying these effects remain incompletely understood. We tested the efficacy of Good Manufacturing Practices (GMP) compliant hBM-MSC, injected intravenously 3.5 hours after injury in mice subjected to transient middle cerebral artery occlusion (tMCAo). We addressed whether hBM-MSC are efficacious and if this efficacy is associated with cortical circuit reorganization using neuroanatomical analysis of GABAergic neurons (parvalbumin; PV-positive cells) and perineuronal nets (PNN), a specialized extracellular matrix structure which acts as an inhibitor of neural plasticity. tMCAo mice receiving hBM-MSC, showed early and lasting improvement of sensorimotor and cognitive functions compared to control tMCAo mice. Furthermore, 5 weeks post-tMCAo, hBM-MSC induced a significant rescue of ipsilateral cortical neurons; an increased proportion of PV-positive neurons in the perilesional cortex, suggesting GABAergic interneurons preservation; and a lower percentage of PV-positive cells surrounded by PNN, indicating an enhanced plastic potential of the perilesional cortex. These results show that hBM-MSC improve functional recovery and stimulate neuroprotection after stroke. Moreover, the downregulation of “plasticity brakes” such as PNN suggests that hBM-MSC treatment stimulates plasticity and formation of new connections in the perilesional cortex.

Oligophrenin-1 (OPHN1) is a Rho GTPase activating protein whose mutations cause X-linked intellectual disability (XLID). How loss of function of Ophn1 affects neuronal development is only partly understood. Here we have exploited adult hippocampal neurogenesis to dissect the steps of neuronal differentiation that are affected by Ophn1 deletion. We found that mice lacking Ophn1 display a reduction in the number of newborn neurons in the dentate gyrus. A significant fraction of the Ophn1-deficient newly generated neurons failed to extend an axon towards CA3, and showed an altered density of dendritic protrusions. Since Ophn1-deficient mice display overactivation of Rho-associated protein kinase (ROCK) and protein kinase A (PKA) signaling, we administered a clinically approved ROCK/PKA inhibitor (fasudil) to correct the neurogenesis defects. While administration of fasudil was not effective in rescuing axon formation, the same treatment completely restored spine density to control levels, and enhanced the long-term survival of adult-born neurons in mice lacking Ophn1. These results identify specific neurodevelopmental steps that are impacted by Ophn1 deletion, and indicate that they may be at least partially corrected by pharmacological treatment.

Summary: Rehabilitation is considered the most effective treatment for promoting the recovery of motor deficits after stroke. One of the most challenging experimental goals is to unambiguously link brain rewiring to motor improvement prompted by rehabilitative therapy. Previous work showed that robotic training combined with transient inactivation of the contralesional cortex promotes a generalized recovery in a mouse model of stroke. Here, we use advanced optical imaging and manipulation tools to study cortical remodeling induced by this rehabilitation paradigm. We show that the stabilization of peri-infarct synaptic contacts accompanies increased vascular density induced by angiogenesis. Furthermore, temporal and spatial features of cortical activation recover toward pre-stroke conditions through the progressive formation of a new motor representation in the peri-infarct area. In the same animals, we observe reinforcement of inter-hemispheric connectivity. Our results provide evidence that combined rehabilitation promotes the restoration of structural and functional features distinctive of healthy neuronal networks. : Robotic training combined with transient contralesional inactivation was recently shown to promote a generalized recovery after stroke. Here, Allegra Mascaro et al. investigate how this rehabilitation paradigm affects neuronal and vascular plasticity of the mouse cortex. Synaptic stabilization is associated with angiogenesis and recovery of a segregated motor representation. Keywords: robotic rehabilitation, pharmacological therapy, two-photon microscopy, in vivo, cerebral cortex, spine plasticity, photothrombosis, optogenetics, calcium imaging, angiogenesis

Brain injuries causing chronic sensory or motor deficit, such as stroke, are among the leading causes of disability worldwide, according to the World Health Organization; furthermore, they carry heavy social and economic burdens due to decreased quality of life and need of assistance. Given the limited effectiveness of rehabilitation, novel therapeutic strategies are required to enhance functional recovery. Since cell-based approaches have emerged as an intriguing and promising strategy to promote brain repair, many efforts have been made to study the functional integration of neurons derived from pluripotent stem cells (PSCs), or fetal neurons, after grafting into the damaged host tissue. PSCs hold great promises for their clinical applications, such as cellular replacement of damaged neural tissues with autologous neurons. They also offer the possibility to create in vitro models to assess the efficacy of drugs and therapies. Notwithstanding these potential applications, PSC-derived transplanted neurons have to match the precise sub-type, positional and functional identity of the lesioned neural tissue. Thus, the requirement of highly specific and efficient differentiation protocols of PSCs in neurons with appropriate neural identity constitutes the main challenge limiting the clinical use of stem cells in the near future. In this Review, we discuss the recent advances in the derivation of telencephalic (cortical and hippocampal) neurons from PSCs, assessing specificity and efficiency of the differentiation protocols, with particular emphasis on the genetic and molecular characterization of PSC-derived neurons. Second, we address the remaining challenges for cellular replacement therapies in cortical brain injuries, focusing on electrophysiological properties, functional integration and therapeutic effects of the transplanted neurons.

Current strategies for glioma treatment are only partly effective because of the poor selectivity for tumoral cells. Hence, the necessity to identify novel approaches is urgent. Recent studies highlighted the effectiveness of the bacterial protein cytotoxic necrotizing factor 1 (CNF1) in reducing tumoral mass, increasing survival of glioma-bearing mice and protecting peritumoral neural tissue from dysfunction. However, native CNF1 needs to be delivered into the brain, because of its incapacity to cross the blood–brain barrier (BBB) per se, thus hampering its clinical translation. To allow a non-invasive administration of CNF1, we here developed a chimeric protein (CTX-CNF1) conjugating CNF1 with chlorotoxin (CTX), a peptide already employed in clinics due to its ability of passing the BBB and selectively binding glioma cells. After systemic administration, we found that CTX-CNF1 is able to target glioma cells and significantly prolong survival of glioma-bearing mice. Our data point out the potentiality of CTX-CNF1 as a novel effective tool to treat gliomas.

Krabbe disease (KD, or globoid cell leukodystrophy; OMIM #245200) is an inherited neurodegenerative condition belonging to the class of the lysosomal storage disorders. It is caused by genetic alterations in the gene encoding for the enzyme galactosylceramidase, which is responsible for cleaving the glycosydic linkage of galatosylsphingosine (psychosine or PSY), a highly cytotoxic molecule. Here, we describe morphological and functional alterations in the visual system of the Twitcher (TWI) mouse, the most used animal model of Krabbe disease. We report in vivo electrophysiological recordings showing defective basic functional properties of the TWI primary visual cortex. In particular, we demonstrate a reduced visual acuity and contrast sensitivity, and a delayed visual response. Specific neuropathological alterations are present in the TWI visual cortex, with reduced myelination, increased astrogliosis and microglia activation, and around the whole brain. Finally, we quantify PSY content in the brain and optic nerves by high-pressure liquid chromatography-mass spectrometry methods. An increasing PSY accumulation with time, the characteristic hallmark of KD, is found in both districts. These results represent the first complete characterization of the TWI visual system. Our data set a baseline for an easy testing of potential therapies for this district, which is also dramatically affected in KD patients.

Objective: Amblyopia is a neurodevelopmental disorder characterized by visual acuity and contrast sensitivity loss, refractory to pharmacological and optical treatments in adulthood. In animals, the corpus callosum (CC) contributes to suppression of visual responses of the amblyopic eye. To investigate the role of interhemispheric pathways in amblyopic patients, we studied the response of the visual cortex to transcranial Direct Current Stimulation (tDCS) applied over the primary visual area (V1) contralateral to the “lazy eye.”Methods: Visual acuity (logMAR) was assessed before (T0), immediately after (T1) and 60’ following the application of cathodal tDCS (2.0 mA, 20’) in 12 amblyopic patients. At each time point, Visual Evoked Potentials (VEPs) triggered by grating stimuli of different contrasts (K90%, K20%) were recorded in both hemispheres and compared to those obtained in healthy volunteers.Results: Cathodal tDCS improved visual acuity respect to baseline (p < 0.0001), whereas sham polarization had no significant effect. At T1, tDCS induced an inhibitory effect on VEPs amplitudes at all contrasts in the targeted side and a facilitation of responses in the hemisphere ipsilateral to the amblyopic eye; compared with controls, the facilitation persisted at T2 for high contrasts (K90%; Holm–Sidak post hoc method, p < 0.001), while the stimulated hemisphere recovered more quickly from inhibition (Holm–Sidak post hoc method, p < 0.001).Conclusions: tDCS is a promising treatment for amblyopia in adults. The rapid recovery of excitability and the concurrent transcallosal disinhibition following perturbation of cortical activity may support a critical role of interhemispheric balance in the pathophysiology of amblyopia.

Focal cortical stroke often leads to persistent motor deficits, prompting the need for more effective interventions. The efficacy of rehabilitation can be increased by ‘plasticity-stimulating’ treatments that enhance experience-dependent modifications in spared areas. Transcallosal pathways represent a promising therapeutic target, but their role in post-stroke recovery remains controversial. Here, we demonstrate that the contralesional cortex exerts an enhanced interhemispheric inhibition over the perilesional tissue after focal cortical stroke in mouse forelimb motor cortex. Accordingly, we designed a rehabilitation protocol combining intensive, repeatable exercises on a robotic platform with reversible inactivation of the contralesional cortex. This treatment promoted recovery in general motor tests and in manual dexterity with remarkable restoration of pre-lesion movement patterns, evaluated by kinematic analysis. Recovery was accompanied by a reduction of transcallosal inhibition and ‘plasticity brakes’ over the perilesional tissue. Our data support the use of combinatorial clinical therapies exploiting robotic devices and modulation of interhemispheric connectivity.

Vision is a very important sensory modality in humans. Visual disorders are numerous and arising from diverse and complex causes. Deficits in visual function are highly disabling from a social point of view and in addition cause a considerable economic burden. For all these reasons there is an intense effort by the scientific community to gather knowledge on visual deficit mechanisms and to find possible new strategies for recovery and treatment. In this review we focus on an important and sometimes neglected player of the visual function, the corpus callosum (CC). The CC is the major white matter structure in the brain and is involved in information processing between the two hemispheres. In particular, visual callosal connections interconnect homologous areas of visual cortices, binding together the two halves of the visual field. This interhemispheric communication plays a significant role in visual cortical output. Here, we will first review essential literature on the physiology of the callosal connections in normal vision. The available data support the view that the callosum contributes to both excitation and inhibition to the target hemisphere, with a dynamic adaptation to the strength of the incoming visual input. Next, we will focus on data showing how callosal connections may sense visual alterations and respond to the classical paradigm for the study of visual plasticity, i.e. monocular deprivation. This is a prototypical example of a model for the study of callosal plasticity in pathological conditions (e.g. strabismus and amblyopia) characterized by unbalanced input from the two eyes. We will also discuss findings of callosal alterations in blind subjects. Noteworthy, we will discuss data showing that inter-hemispheric transfer mediates recovery of visual responsiveness following cortical damage. Finally, we will provide an overview of how callosal projections dysfunction could contribute to pathologies such as neglect and occipital epilepsy. A particular focus will be on reviewing noninvasive brain stimulation techniques and optogenetic approaches that allow to selectively manipulate callosal function and to probe its involvement in cortical processing and plasticity. Overall, the data indicate that experience can potently impact on transcallosal connectivity, and that the callosum itself is crucial for plasticity

The therapeutic potential of botulinum neurotoxin type A (BoNT/A) has recently been widely recognized. BoNT/A acts to silence synaptic transmission via specific proteolytic cleavage of an essential neuronal protein, SNAP25. The advantages of BoNT/A-mediated synaptic silencing include very long duration, high potency and localized action. However, there is a fear of possible side-effects of BoNT/A due to its diffusible nature which may lead to neuromuscular blockade away from the injection site. We recently developed a “protein-stapling” technology which allows re-assembly of BoNT/A from two separate fragments. This technology allowed, for the first time, safe production of this popular neuronal silencing agent. Here we evaluated the re-assembled toxin in several CNS assays and assessed its systemic effects in an animal model. Our results show that the re-assembled toxin is potent in inhibiting CNS function at 1 nM concentration but surprisingly does not exhibit systemic toxicity after intraperitoneal injection even at 200 ng/kg dose. This shows that the re-assembled toxin represents a uniquely safe tool for neuroscience research and future medical applications.

Botulinum neurotoxins are metalloproteases that specifically cleave N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in synaptic terminals, resulting in a potent inhibition of vesicle fusion and transmitter release. The family comprises different serotypes (BoNT/A to BoNT/G). The natural target of these toxins is represented by the neuromuscular junction, where BoNTs block acetylcholine release. In this review, we describe the actions of botulinum toxins after direct delivery to the central nervous system (CNS), where BoNTs block exocytosis of several transmitters, with near-complete silencing of neural networks. The use of clostridial neurotoxins in the CNS has allowed us to investigate specifically the role of synaptic activity in different physiological and pathological processes. The silencing properties of BoNTs can be exploited for therapeutic purposes, for example to counteract pathological hyperactivity and seizures in epileptogenic brain foci, or to investigate the role of activity in degenerative diseases like prion disease. Altogether, clostridial neurotoxins and their derivatives hold promise as powerful tools for both the basic understanding of brain function and the dissection and treatment of activity-dependent pathogenic pathways.

PURPOSE:Limited restoration of function is known to occur spontaneously after an ischemic injury to the primary motor cortex. Evidence suggests that Pre-Motor Areas (PMAs) may "take over" control of the disrupted functions. However, little is known about functional reorganizations in PMAs. Forelimb movements in mice can be driven by two cortical regions, Caudal and Rostral Forelimb Areas (CFA and RFA), generally accepted as primary motor and pre-motor cortex, respectively. Here, we examined longitudinal changes in functional coupling between the two RFAs following unilateral photothrombotic stroke in CFA (mm from Bregma: +0.5 anterior, +1.25 lateral). METHODS:Local field potentials (LFPs) were recorded from the RFAs of both hemispheres in freely moving injured and naïve mice. Neural signals were acquired at 9, 16 and 23 days after surgery (sub-acute period in stroke animals) through one bipolar electrode per hemisphere placed in the center of RFA, with a ground screw over the occipital bone. LFPs were pre-processed through an efficient method of artifact removal and analysed through: spectral,cross-correlation, mutual information and Granger causality analysis. RESULTS:Spectral analysis demonstrated an early decrease (day 9) in the alpha band power in both the RFAs. In the late sub-acute period (days 16 and 23), inter-hemispheric functional coupling was reduced in ischemic animals, as shown by a decrease in the cross-correlation and mutual information measures. Within the gamma and delta bands, correlation measures were already reduced at day 9. Granger analysis, used as a measure of the symmetry of the inter-hemispheric causal connectivity, showed a less balanced activity in the two RFAs after stroke, with more frequent oscillations of hemispheric dominance. CONCLUSIONS:These results indicate robust electrophysiological changes in PMAs after stroke. Specifically, we found alterations in transcallosal connectivity, with reduced inter-hemispheric functional coupling and a fluctuating dominance pattern. These reorganizations may underlie vicariation of lost functions following stroke.

The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT) is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs) block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer of diverse cargoes from nerve terminals to the soma, and represents a general gateway for the delivery of virulence factors and pathogens to the central nervous system.

In chronic neurodegenerative diseases associated with aggregates of misfolded proteins (such as Alzheimer's, Parkinson's and prion disease), there is an early degeneration of presynaptic terminals prior to the loss of the neuronal somata. Identifying the mechanisms that govern synapse degeneration is of paramount importance, as cognitive decline is strongly correlated with loss of presynaptic terminals in these disorders. However, very little is known about the processes that link the presence of a misfolded protein to the degeneration of synapses. It has been suggested that the process follows a simple linear sequence in which terminals that become dysfunctional are targeted for death, but there is also evidence that high levels of activity can speed up degeneration. To dissect the role of activity in synapse degeneration, we infused the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of mice with prion disease and assessed synapse loss at the electron microscopy level. We found that injection of BoNT/A in naïve mice caused a significant enlargement of excitatory presynaptic terminals in the hippocampus, indicating transmission impairment. Long-lasting blockade of activity by BoNT/A caused only minimal synaptic pathology and no significant activation of microglia. In mice with prion disease infused with BoNT/A, rates of synaptic degeneration were indistinguishable from those observed in control diseased mice. We conclude that silencing synaptic activity neither prevents nor enhances the degree of synapse degeneration in prion disease. These results challenge the idea that dysfunction of synaptic terminals dictates their elimination during prion-induced neurodegeneration.