Search

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Vannini, Restani, Dilillo, McDonnell, Caleo and Marra.)

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).

Currently, high-grade gliomas are the most difficult brain cancers to treat and all the approved experimental treatments do not offer long-term benefits regarding symptom improvement. Epidemiological studies indicate that exercise decreases the risk of brain cancer mortality, but a direct relationship between physical exercise and glioma progression has not been established so far. Here, we exploited a mouse model of high-grade glioma to directly test the impact of voluntary physical exercise on the tumor proliferation and motor capabilities of affected animals. We report that exposing symptomatic, glioma-bearing mice to running wheels (i) reduced the proliferation rate of tumors implanted in the motor cortex and (ii) delayed glioma-induced motor dysfunction. Thus, voluntary physical exercise might represent a supportive intervention that complements existing neuro-oncologic therapies, contributing to the preservation of functional motor ability and counteracting the detrimental effects of glioma on behavioral output.

Foxg1 is an ancient transcription factor gene orchestrating a number of neurodevelopmental processes taking place in the rostral brain. In this study, we investigated its impact on neocortical activity. We found that mice overexpressing Foxg1 in neocortical pyramidal cells displayed an electroencephalography (EEG) with increased spike frequency and were more prone to kainic acid (KA)-induced seizures. Consistently, primary cultures of neocortical neurons gain-of-function for Foxg1 were hyperactive and hypersynchronized. That reflected an unbalanced expression of key genes encoding for ion channels, gamma aminobutyric acid and glutamate receptors, and was likely exacerbated by a pronounced interneuron depletion. We also detected a transient Foxg1 upregulation ignited in turn by neuronal activity and mediated by immediate early genes. Based on this, we propose that even small changes of Foxg1 levels may result in a profound impact on pyramidal cell activity, an issue relevant to neuronal physiology and neurological aberrancies associated to FOXG1 copy number variations., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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., (Copyright © 2020 Allegra Mascaro, Falotico, Petkoski, Pasquini, Vannucci, Tort-Colet, Conti, Resta, Spalletti, Ramalingasetty, von Arnim, Formento, Angelidis, Blixhavn, Leergaard, Caleo, Destexhe, Ijspeert, Micera, Laschi, Jirsa, Gewaltig and Pavone.)

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., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be perceived as a potential conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

Oligophrenin-1 (Ophn1) encodes a Rho GTPase activating protein whose mutations cause X-linked intellectual disability (XLID) in humans. Loss of function of Ophn1 leads to impairments in the maturation and function of excitatory and inhibitory synapses, causing deficits in synaptic structure, function and plasticity. Epilepsy is a frequent comorbidity in patients with Ophn1-dependent XLID, but the cellular bases of hyperexcitability are poorly understood. Here we report that male mice knock-out (KO) for Ophn1 display hippocampal epileptiform alterations, which are associated with changes in parvalbumin-, somatostatin- and neuropeptide Y-positive interneurons. Because loss of function of Ophn1 is related to enhanced activity of Rho-associated protein kinase (ROCK) and protein kinase A (PKA), we attempted to rescue Ophn1-dependent pathological phenotypes by treatment with the ROCK/PKA inhibitor fasudil. While acute administration of fasudil had no impact on seizure activity, seven weeks of treatment in adulthood were able to correct electrographic, neuroanatomical and synaptic alterations of Ophn1 deficient mice. These data demonstrate that hyperexcitability and the associated changes in GABAergic markers can be rescued at the adult stage in Ophn1-dependent XLID through ROCK/PKA inhibition. SIGNIFICANCE STATEMENT In this study we demonstrate enhanced seizure propensity and impairments in hippocampal GABAergic circuitry in Ophn1 mouse model of X-linked intellectual disability (XLID). Importantly, the enhanced susceptibility to seizures, accompanied by an alteration of GABAergic markers were rescued by Rho-associated protein kinase (ROCK)/protein kinase A (PKA) inhibitor fasudil, a drug already tested on humans. Because seizures can significantly impact the quality of life of XLID patients, the present data suggest a potential therapeutic pathway to correct alterations in GABAergic networks and dampen pathological hyperexcitability in adults with XLID., (Copyright © 2020 the authors.)

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., Competing Interests: The authors have no competing interests to declare., (© 2020 The Authors.)

Stroke is one of the leading causes of long-term disability. Advanced technological solutions ("neurotechnologies") exploiting robotic systems and electrodes that stimulate the nervous system can increase the efficacy of stroke rehabilitation. Recent studies on these approaches have shown promising results. However, a paradigm shift in the development of new approaches must be made to significantly improve the clinical outcomes of neurotechnologies compared with those of traditional therapies. An "evolutionary" change can occur only by understanding in great detail the basic mechanisms of natural stroke recovery and technology-assisted neurorehabilitation. In this review, we first describe the results achieved by existing neurotechnologies and highlight their current limitations. In parallel, we summarize the data available on the mechanisms of recovery from electrophysiological, behavioral, and anatomical studies in humans and rodent models. Finally, we propose new approaches for the effective use of neurotechnologies in stroke survivors, as well as in people with other neurological disorders., Competing Interests: Declaration of Interests S.M. holds shares of the companies IUVO, GTX, Sensars Neurotechnologies, and AGO Neurotechnologies, which are all developing neurotechnologies to restore the sensory-motor functions of disabled people. All the other authors have no conflicts of interest to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The correct morphofunctional shaping of the cerebral cortex requires a continuous interaction between intrinsic (genes/molecules expressed within the tissue) and extrinsic (e.g., neural activity) factors at all developmental stages. Forkhead Box G1 (FOXG1) is an evolutionarily conserved transcription factor, essential for the cerebral cortex patterning and layering. FOXG1-related disorders, including the congenital form of Rett syndrome, can be caused by deletions, intragenic mutations or duplications. These genetic alterations are associated with a complex phenotypic spectrum, spanning from intellectual disability, microcephaly, to autistic features, and epilepsy. We investigated the functional correlates of dysregulated gene expression by performing electrophysiological assays on FoxG1 +/- mice. Local Field Potential (LFP) recordings on freely moving animals detected cortical hyperexcitability. On the other hand, patch-clamp recordings showed a downregulation of spontaneous glutamatergic transmission. These findings were accompanied by overactivation of Akt/S6 signaling. Furthermore, the expression of vesicular glutamate transporter 2 (vGluT2) was increased, whereas the level of the potassium/chloride cotransporter KCC2 was reduced, thus indicating a higher excitation/inhibition ratio. Our findings provide evidence that altered expression of a key gene for cortical development can result in specific alterations in neural circuit function at the macro- and micro-scale, along with dysregulated intracellular signaling and expression of proteins controlling circuit excitability.

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.

Krabbe disease is a rare, childhood lysosomal storage disorder caused by a deficiency of galactosylceramide beta-galactosidase (GALC). The major effect of GALC deficiency is the accumulation of psychosine in the nervous system and widespread degeneration of oligodendrocytes and Schwann cells, causing rapid demyelination. The molecular mechanisms of Krabbe disease are not yet fully elucidated and a definite cure is still missing. Here we report the first in-depth characterization of the proteome of the Twitcher mouse, a spontaneous mouse model of Krabbe disease, to investigate the proteome changes in the Central and Peripheral Nervous System. We applied a TMT-based workflow to compare the proteomes of the corpus callosum, motor cortex and sciatic nerves of littermate homozygous Twitcher and wild-type mice. More than 400 protein groups exhibited differences in expression and included proteins involved in pathways that can be linked to Krabbe disease, such as inflammatory and defense response, lysosomal proteins accumulation, demyelination, reduced nervous system development and cell adhesion. These findings provide new insights on the molecular mechanisms of Krabbe disease, representing a starting point for future functional experiments to study the molecular pathogenesis of Krabbe disease. Data are available via ProteomeXchange with identifier PXD010594., (© 2019 Pellegrini et al.)

The Forkhead Box G1 (FOXG1) gene encodes a transcription factor with an essential role in mammalian telencephalon development. FOXG1-related disorders, caused by deletions, intragenic mutations or duplications, are usually associated with severe intellectual disability, autistic features, and, in 87% of subjects, epileptiform manifestations. In a subset of patients with FoxG1 mutations, seizures remain intractable, prompting the need for novel therapeutic options. To address this issue, we took advantage of a haploinsufficient animal model, the FoxG1 +/- mouse. In vivo electrophysiological analyses of FoxG1 +/- mice detected hippocampal hyperexcitability, which turned into overt seizures upon delivery of the proconvulsant kainic acid, as confirmed by behavioral observations. These alterations were associated with decreased expression of the chloride transporter KCC2. Next, we tested whether a triheptanoin-based anaplerotic diet could have an impact on the pathological phenotype of FoxG1 +/- mice. This manipulation abated altered neural activity and normalized enhanced susceptibility to proconvulsant-induced seizures, in addition to rescuing altered expression of KCC2 and increasing the levels of the GABA transporter vGAT. In conclusion, our data show that FoxG1 haploinsufficiency causes dysfunction of hippocampal circuits and increases the susceptibility to a proconvulsant insult, and that these alterations are rescued by triheptanoin dietary treatment., (Copyright © 2019. Published by Elsevier Ltd.)

Botulinum neurotoxin Type A (BoNT/A) is an effective treatment for several movement disorders, including spasticity and dystonia. BoNT/A acts by cleaving synaptosomal-associated protein of 25 kDa (SNAP-25) at the neuromuscular junction, thus blocking synaptic transmission and weakening overactive muscles. However, not all the therapeutic benefits of the neurotoxin are explained by peripheral neuroparalysis, suggesting an action of BoNT/A on central circuits. Currently, the specific targets of BoNT/A central activity remain unclear. Here, we show that catalytically active BoNT/A is transported to the facial nucleus (FN) after injection into the nasolabial musculature of rats and mice. BoNT/A-mediated cleavage of SNAP-25 in the FN is prevented by intracerebroventricular delivery of antitoxin antibodies, demonstrating that BoNT/A physically leaves the motoneurons to enter second-order neurons. Analysis of intoxicated terminals within the FN shows that BoNT/A is transcytosed preferentially into cholinergic synapses. The cholinergic boutons containing cleaved SNAP-25 are associated with a larger size, suggesting impaired neuroexocytosis. Together, the present findings indicate a previously unrecognized source of reduced motoneuron drive after BoNT/A via blockade of central, excitatory cholinergic inputs. These data highlight the ability of BoNT/A to selectively target and modulate specific central circuits, with consequent impact on its therapeutic effectiveness in movement disorders. SIGNIFICANCE STATEMENT Botulinum neurotoxins are among the most potent toxins known. Despite this, their specific and reversible action prompted their use in clinical practice to treat several neuromuscular pathologies (dystonia, spasticity, muscle spasms) characterized by hyperexcitability of peripheral nerve terminals or even in nonpathological applications (i.e., cosmetic use). Substantial experimental and clinical evidence indicates that not all botulinum neurotoxin Type A (BoNT/A) effects can be explained solely by the local action (i.e., silencing of the neuromuscular junction). In particular, there are cases in which the clinical benefit exceeds the duration of peripheral neurotransmission blockade. In this study, we demonstrate that BoNT/A is transported to facial motoneurons, released, and internalized preferentially into cholinergic terminals impinging onto the motoneurons. Our data demonstrate a direct central action of BoNT/A., (Copyright © 2018 the authors 0270-6474/18/3810329-09$15.00/0.)

Rodent models are decisive for translational research in healthy and pathological conditions of motor function thanks to specific similarities with humans. Here, we present an upgraded version of the M-Platform, a robotic device previously designed to train mice during forelimb retraction tasks. This new version significantly extends its possibilities for murine experiments during motor tasks: 1) an actuation system for friction adjustment allows to automatically adapt pulling difficulty; 2) the device can be used both for training, with a retraction task, and for assessment, with an isometric task; and 3) the platform can be integrated with a neurophysiology systems to record simultaneous cortical neural activity. Results of the validation experiments with healthy mice confirmed that the M-Platform permits precise adjustments of friction during the task, thus allowing to change its difficulty and that these variations induce a different improvement in motor performance, after specific training sessions. Moreover, simultaneous and high quality (high signal-to-noise ratio) neural signals can be recorded from the rostral forelimb area (RFA) during task execution. With the novel features presented herein, the M-Platform may allow to investigate the outcome of a customized motor rehabilitation protocol after neural injury, to analyze task-related signals from brain regions interested by neuroplastic events and to perform optogenetic silencing or stimulation during experiments in transgenic mice.

Local intramuscular injections of botulinum neurotoxin type A (BoNT/A) are effective in the treatment of focal dystonias, muscle spasms, and spasticity. However, not all clinical effects of BoNT/A may be explained by its action at peripheral nerve terminals. For example, the therapeutic benefit may exceed the duration of the peripheral neuroparalysis induced by the neurotoxin. In cellular and animal models, evidence demonstrates retrograde transport of catalytically active BoNT/A in projection neurons. This process of long-range trafficking is followed by transcytosis and action at second-order synapses. In humans, several physiological changes have been described following intramuscular delivery of BoNT/A. In particular, clinical studies have documented a decrease in Renshaw cell-mediated inhibition (i.e., recurrent inhibition), which may be important therapeutically for normalizing uncoordinated movements and overflow of muscle activity. In this review, we present data obtained in animal and experimental models that support direct central actions of BoNT/A mediated via retrograde axonal trafficking. We also discuss the reorganization of central circuitry induced by BoNT/A in patients, and the potential contribution of these effects to the therapeutic efficacy of the neurotoxin., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pathogenic bacteria produce toxins to promote host invasion and, therefore, their survival. The extreme potency and specificity of these toxins confer to this category of proteins an exceptionally strong potential for therapeutic exploitation. In this review, we deal with cytotoxic necrotizing factor (CNF1), a cytotoxin produced by Escherichia coli affecting fundamental cellular processes, including cytoskeletal dynamics, cell cycle progression, transcriptional regulation, cell survival and migration. First, we provide an overview of the mechanisms of action of CNF1 in target cells. Next, we focus on the potential use of CNF1 as a pharmacological treatment in central nervous system's diseases. CNF1 appears to impact neuronal morphology, physiology, and plasticity and displays an antineoplastic activity on brain tumors. The ability to preserve neural functionality and, at the same time, to trigger senescence and death of proliferating glioma cells, makes CNF1 an encouraging new strategy for the treatment of brain tumors.

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 (T 0 ), immediately after (T 1 ) 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 T 1 , 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 T 2 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.

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.

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Increasing evidence points to the importance of dendritic spines in the formation and allocation of memories, and alterations of spine number and physiology are associated to memory and cognitive disorders. Modifications of the activity of subsets of synapses are believed to be crucial for memory establishment. However, the development of a method to directly test this hypothesis, by selectively controlling the activity of potentiated spines, is currently lagging. Here we introduce a hybrid RNA/protein approach to regulate the expression of a light-sensitive membrane channel at activated synapses, enabling selective tagging of potentiated spines following the encoding of a novel context in the hippocampus. This approach can be used to map potentiated synapses in the brain and will make it possible to re-activate the neuron only at previously activated synapses, extending current neuron-tagging technologies in the investigation of memory processes.

Local field potential (LFP) recordings were performed from the visual cortex (V1) of a focal epilepsy mouse model. Epilepsy was induced by a unilateral injection of the synaptic blocker tetanus neurotoxin (TeNT). LFP signals were simultaneously recorded from V1 of both hemispheres of each animal under acute and chronic conditions (i.e. during and after the period of TeNT action). All data were analysed by using nonlinear time series methods. Suitable values of the lag time and embedding dimension for phase space reconstruction were estimated by employing well-known methods. The results showed that lag times are sensitive to the presence of TeNT. Interestingly, TeNT promoted an increase in the level of linear and nonlinear correlation of LFP signals. The values of the embedding dimension failed to show any dependence on the presence of the neurotoxin. However, a local nonlinear prediction method showed that the presence of TeNT increases the predictability, quantified by the normalized prediction error, of the neural recordings. From a neurophysiological point of view, the above results suggest that TeNT injected in one hemisphere strongly impacts the local electrical activity of the neural populations in the opposite hemisphere. We hypothesize that this could arise from a qualitative and quantitative alteration of the transmission properties of the callosal fibers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A large series of clinical and experimental studies supports a link between inflammation and epilepsy, indicating that inflammatory processes within the brain are important contributors to seizure recurrence and precipitation. Systemic inflammation can precipitate seizures in children suffering from epileptic encephalopathies, and hallmarks of a chronic inflammatory state have been found in patients with temporal lobe epilepsy. Research performed on animal models of epilepsy further corroborates the idea that seizures upregulate inflammatory mediators, which in turn may enhance brain excitability and neuronal degeneration. Several inflammatory molecules and their signaling pathways have been implicated in epilepsy. Among these, the chemokine pathway has increasingly gained attention. Chemokines are small cytokines secreted by blood cells, which act as chemoattractants for leukocyte migration. Recent studies indicate that chemokines and their receptors are also produced by brain cells, and are involved in various neurological disorders including epilepsy. In this review, we will focus on a subset of pro-inflammatory chemokines (namely CCL2, CCL3, CCL5, CX3CL1) and their receptors, and their increasingly recognized role in seizure control., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mass spectrometry imaging (MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue. Rapid direct tissue analysis is essential for MSI in order to maintain spatial localization and acceptable measurement times. The absence of an explicit analyte separation/purification step means MSI lacks the depth of coverage of LC-MS/MS. In this work, we demonstrate how atmospheric pressure MALDI-MSI enables the same tissue section to be first analyzed by MSI, to identify regions of interest that exhibit distinct molecular signatures, followed by localized proteomics analysis using laser capture microdissection isolation and LC-MS/MS.

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.

A large body of evidence that has accumulated over the past decade strongly supports the role of inflammation in the pathophysiology of human epilepsy. Specific inflammatory molecules and pathways have been identified that influence various pathologic outcomes in different experimental models of epilepsy. Most importantly, the same inflammatory pathways have also been found in surgically resected brain tissue from patients with treatment-resistant epilepsy. New antiseizure therapies may be derived from these novel potential targets. An essential and crucial question is whether targeting these molecules and pathways may result in anti-ictogenesis, antiepileptogenesis, and/or disease-modification effects. Therefore, preclinical testing in models mimicking relevant aspects of epileptogenesis is needed to guide integrated experimental and clinical trial designs. We discuss the most recent preclinical proof-of-concept studies validating a number of therapeutic approaches against inflammatory mechanisms in animal models that could represent novel avenues for drug development in epilepsy. Finally, we suggest future directions to accelerate preclinical to clinical translation of these recent discoveries., (Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.)

Glioblastoma (GBM) is the most aggressive type of brain tumor. In this context, animal models represent excellent tools for the early detection and longitudinal mapping of neuronal dysfunction, that are critical in the preclinical validation of new therapeutic strategies. In a mouse glioma model, we developed sensitive behavioral readouts that allow early recognizing and following neurological symptoms. We injected GL261 cells into the primary motor cortex of syngenic mice and we used a battery of behavioral tests to longitudinally monitor the dysfunction induced by tumor growth. Grip strength test revealed an early onset of functional deficit associated to the glioma growth, with a significant forelimb weakness appearing 9 days after tumor inoculation. A later deficit was observed in the rotarod and in the grid walk tasks. Using this model, we found reduced tumor growth and maintenance of behavioral functions following treatment with Cytotoxic Necrotizing Factor 1 (CNF1) at a symptomatic stage. Our data provide a detailed and precise examination of how tumor growth reverberates on the behavioral functions of glioma-bearing mice, providing normative data for the study of therapeutic strategies for glioma treatment. The reduced tumor volume and robust functional sparing observed in CNF1-treated, glioma-bearing mice strengthen the notion that CNF1 delivery is a promising strategy for glioma therapy.

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., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Besides its classical function of bone metabolism regulation, 1alpha, 25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ), acts on a variety of tissues including the nervous system, where the hormone plays an important role as neuroprotective, antiproliferating and differentiating agent. Sphingolipids are bioactive lipids that play critical and complex roles in regulating cell fate. In the present paper we have investigated whether sphingolipids are involved in the protective action of 1,25(OH) 2 D 3. We have found that 1,25(OH) 2 D 3 prevents amyloid-β peptide (Aβ(1-42)) cytotoxicity both in differentiated SH-SY5Y human neuroblastoma cells and in vivo. In differentiated SH-SY5Y cells, Aβ(1-42) strongly reduces the sphingosine-1-phosphate (S1P)/ceramide (Cer) ratio while 1,25(OH) 2 D 3 partially reverts this effect. 1,25(OH) 2 D 3 reverts also the Aβ(1-42)-induced reduction of sphingosine kinase activity. We have also studied the crosstalk between 1,25(OH) 2 D 3 and S1P signaling pathways downstream to the activation of S1P receptor subtype S1P1. Notably, we found that 1,25(OH) 2 D 3 prevents the reduction of S1P1 expression promoted by Aβ(1-42) and thereby it modulates the downstream signaling leading to ER stress damage (p38MAPK/ATF4). Similar effects were observed by using ZK191784. In addition, chronic treatment with 1,25(OH) 2 D 3 protects from aggregated Aβ(1-42)-induced damage in the CA1 region of the rat hippocampus and promotes cell proliferation in the hippocampal dentate gyrus of adult mice. In conclusion, these results represent the first evidence of the role of 1,25(OH) 2 D 3 and its structural analogue ZK191784 in counteracting the Aβ(1-42) peptide-induced toxicity through the modulation of S1P/S1P1/p38MAPK/ATF4 pathway in differentiated SH-SY5Y cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ischemic damage to the brain triggers substantial reorganization of spared areas and pathways, which is associated with limited, spontaneous restoration of function. A better understanding of this plastic remodeling is crucial to develop more effective strategies for stroke rehabilitation. In this review article, we discuss advances in the comprehension of post-stroke network reorganization in patients and animal models. We first focus on rodent studies that have shed light on the mechanisms underlying neuronal remodeling in the perilesional area and contralesional hemisphere after motor cortex infarcts. Analysis of electrophysiological data has demonstrated brain-wide alterations in functional connectivity in both hemispheres, well beyond the infarcted area. We then illustrate the potential use of non-invasive brain stimulation (NIBS) techniques to boost recovery. We finally discuss rehabilitative protocols based on robotic devices as a tool to promote endogenous plasticity and functional restoration.

Background: Glioblastomas are the most aggressive type of brain tumor. A successful treatment should aim at halting tumor growth and protecting neuronal cells to prevent functional deficits and cognitive deterioration. Here, we exploited a Rho GTPase-activating bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1), to interfere with glioma cell growth in vitro and vivo. We also investigated whether this toxin spares neuron structure and function in peritumoral areas., Methods: We performed a microarray transcriptomic and in-depth proteomic analysis to characterize the molecular changes triggered by CNF1 in glioma cells. We also examined tumor cell senescence and growth in vehicle- and CNF1-treated glioma-bearing mice. Electrophysiological and morphological techniques were used to investigate neuronal alterations in peritumoral cortical areas., Results: Administration of CNF1 triggered molecular and morphological hallmarks of senescence in mouse and human glioma cells in vitro. CNF1 treatment in vivo induced glioma cell senescence and potently reduced tumor volumes. In peritumoral areas of glioma-bearing mice, neurons showed a shrunken dendritic arbor and severe functional alterations such as increased spontaneous activity and reduced visual responsiveness. CNF1 treatment enhanced dendritic length and improved several physiological properties of pyramidal neurons, demonstrating functional preservation of the cortical network., Conclusions: Our findings demonstrate that CNF1 reduces glioma volume while at the same time maintaining the physiological and structural properties of peritumoral neurons. These data indicate a promising strategy for the development of more effective antiglioma therapies., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A deeper understanding of post-stroke plasticity is critical to devise more effective pharmacological and rehabilitative treatments. The GABAergic system is one of the key modulators of neuronal plasticity, and plays an important role in the control of "critical periods" during brain development. Here, we report a key role for GABAergic inhibition in functional restoration following ischemia in the adult mouse forelimb motor cortex. After stroke, the majority of cortical sites in peri-infarct areas evoked simultaneous movements of forelimb, hindlimb and tail, consistent with a loss of inhibitory signalling. Accordingly, we found a delayed decrease in several GABAergic markers that accompanied cortical reorganization. To test whether reductions in GABAergic signalling were causally involved in motor improvements, we treated animals during an early post-stroke period with a benzodiazepine inverse agonist, which impairs GABA A receptor function. We found that hampering GABA A signalling led to significant restoration of function in general motor tests (i.e., gridwalk and pellet reaching tasks), with no significant impact on the kinematics of reaching movements. Improvements were persistent as they remained detectable about three weeks after treatment. These data demonstrate a key role for GABAergic inhibition in limiting motor improvements after cortical stroke.

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 the 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 (MD). 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 the 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 and recovery in various disorders of the visual pathway.

Retinitis pigmentosa (RP) comprises a group of inherited pathologies characterized by progressive photoreceptor degeneration. In rodent models of RP, expression of defective genes and retinal degeneration usually manifest during the first weeks of postnatal life, making it difficult to distinguish consequences of primary genetic defects from abnormalities in retinal development. Moreover, mouse eyes are small and not always adequate to test pharmacological and surgical treatments. An inducible paradigm of retinal degeneration potentially extensible to large animals is therefore desirable. Starting from the serendipitous observation that intraocular injections of a Rho GTPase activator, the bacterial toxin Cytotoxic Necrotizing Factor 1 (CNF1), lead to retinal degeneration, we implemented an inducible model recapitulating most of the key features of Retinitis Pigmentosa. The model also unmasks an intrinsic vulnerability of photoreceptors to the mechanism of CNF1 action, indicating still unexplored molecular pathways potentially leading to the death of these cells in inherited forms of retinal degeneration.

Objective: To investigate functional changes underlying photosensitivity, we studied the response of the visual cortex to low-frequency, inhibitory repetitive transcranial magnetic stimulation (rTMS) in drug-free patients with photosensitive seizures and healthy volunteers., Methods: Visual evoked potentials (VEPs) triggered by grating stimuli of different contrasts were recorded in both hemispheres before and after transient functional inactivation of the occipital cortex of one side via low-frequency rTMS (0.5Hz for 20'). VEPs were recorded before (T0), immediately after (T1) and 45' following the completion of rTMS (T2)., Results: Baseline amplitudes of the early VEP components (N1 and P1) were enhanced in photosensitive patients. At T1, rTMS produced an inhibitory effect on VEPs amplitudes at all contrasts in the targeted side and a concurrent facilitation of responses in the contralateral hemisphere. Compared with PSE subjects, VEP amplitudes remained persistently dampened in the stimulated hemisphere of controls (Holm-Sidak post-hoc method, p<0.05). In the contralateral hemisphere, we found a clear enhancement of VEP amplitude in photosensitive subjects but not controls at T2 (Holm-Sidak test, p<0.001)., Conclusions: Visual responses recovered more quickly in the stimulated hemisphere, and disinhibition persisted in the contralateral side of photosensitive subjects., Significance: The rapid recovery of excitability and the persistent transcallosal disinhibition following perturbation of cortical activity may play a role in the pathophysiology of photosensitive epilepsy., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Epilepsy is characterized by impaired circuit function and a propensity for spontaneous seizures, but how plastic rearrangements within the epileptic focus trigger cortical dysfunction and hyperexcitability is only partly understood. Here we have examined alterations in sensory processing and the underlying biochemical and neuroanatomical changes in tetanus neurotoxin (TeNT)-induced focal epilepsy in mouse visual cortex. We documented persistent epileptiform electrographic discharges and upregulation of GABAergic markers at the completion of TeNT effects. We also found a significant remodeling of the dendritic arbors of pyramidal neurons, with increased dendritic length and branching, and overall reduction in spine density but significant preservation of mushroom, mature spines. Functionally, spontaneous neuronal discharge was increased, visual responses were less reliable, and electrophysiological and behavioural visual acuity was consistently impaired in TeNT-injected mice. These data demonstrate robust, long-term remodeling of both inhibitory and excitatory circuitry associated with specific disturbances of network function in neocortical epilepsy.

Epilepsy is a chronic disorder characterized by spontaneous recurrent seizures. Several lines of evidence demonstrate that inflammatory processes within the brain parenchyma contribute to recurrence and precipitation of seizures. In both epileptic patients and animal models, seizures upregulate inflammatory mediators, which in turn may enhance brain excitability. We recently showed that the C-C motif ligand 2 (CCL2) chemokine (also known as monocyte chemoattractant protein-1 [MCP-1]) mediates the seizure-promoting effects of inflammation. Systemic inflammatory challenge in chronically epileptic mice markedly enhanced seizure frequency and upregulated CCL2 expression in the brain. Selective pharmacological blockade of CCL2 synthesis or C-C chemokine receptor type 2 (CCR2) significantly suppressed inflammation-induced seizures. These results have important implications for the development of novel anticonvulsant therapies: drugs interfering with CCL2 signaling are used clinically for several human disorders and might be redirected for use in pharmacoresistant epilepsy. Here we review the role of CCL2/CCR2 signaling in linking systemic inflammation with seizure susceptibility and discuss some open questions that arise from our recent studies.

Epilepsy is a chronic disorder characterized by spontaneous recurrent seizures. Brain inflammation is increasingly recognized as a critical factor for seizure precipitation, but the molecular mediators of such proconvulsant effects are only partly understood. The chemokine CCL2 is one of the most elevated inflammatory mediators in patients with pharmacoresistent epilepsy, but its contribution to seizure generation remains unexplored. Here, we show, for the first time, a crucial role for CCL2 and its receptor CCR2 in seizure control. We imposed a systemic inflammatory challenge via lipopolysaccharide (LPS) administration in mice with mesial temporal lobe epilepsy. We found that LPS dramatically increased seizure frequency and upregulated the expression of many inflammatory proteins, including CCL2. To test the proconvulsant role of CCL2, we administered systemically either a CCL2 transcription inhibitor (bindarit) or a selective antagonist of the CCR2 receptor (RS102895). We found that interference with CCL2 signaling potently suppressed LPS-induced seizures. Intracerebral administration of anti-CCL2 antibodies also abrogated LPS-mediated seizure enhancement in chronically epileptic animals. Our results reveal that CCL2 is a key mediator in the molecular pathways that link peripheral inflammation with neuronal hyperexcitability., Significance Statement: Substantial evidence points to a role for inflammation in epilepsy, but currently there is little insight as to how inflammatory pathways impact on seizure generation. Here, we examine the molecular mediators linking peripheral inflammation with seizure susceptibility in mice with mesial temporal lobe epilepsy. We show that a systemic inflammatory challenge via lipopolysaccharide administration potently enhances seizure frequency and upregulates the expression of the chemokine CCL2. Remarkably, selective pharmacological interference with CCL2 or its receptor CCR2 suppresses lipopolysaccharide-induced seizure enhancement. Thus, CCL2/CCR2 signaling plays a key role in linking systemic inflammation with seizure susceptibility., (Copyright © 2016 the authors 0270-6474/16/363777-12$15.00/0.)

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.

Background: Transcutaneous spinal Direct Current Stimulation (tsDCS) is a noninvasive technique based on the application of weak electrical currents over spinal cord., New Method: We studied the effects of tsDCS on interhemispheric motor connectivity and visual processing by evaluating changes in ipsilateral Silent Period (iSP), Transcallosal Conduction Time (TCT) and hemifield Visual Evoked Potentials (hVEPs), before (T0) and at a different intervals following sham, anodal and cathodal tsDCS (T9-T11 level, 2.0 mA, 20'). Motor Evoked Potentials (MEPs) were recorded from abductor pollicis brevis (APB), abductor hallucis (AH) and deltoid muscles. hVEPs were recorded bilaterally by reversal of a horizontal square wave grating with the display positioned in the right hemifield., Results: Anodal tsDCS increased TCT (p < 0.001) and the interhemispheric delay for both the main VEP components (N1: p = 0.0003; P1: p < 0.0001), dampening at the same time iSP duration (APB: p < 0.0001; AH: p = 0.0005; deltoid: p < 0.0001), while cathodal stimulation elicited opposite effects (p < 0.0001)., Discussion: tsDCS modulates interhemispheric processing in a polarity-specific manner, with anodal stimulation leading to a functional disconnection between hemispheres. tsDCS would be a new promising therapeutic tool in managing a number of human diseases characterized by an impaired interhemispheric balance, or an early rehabilitation strategy in patients with acute brain lesions, when other non-invasive brain stimulation techniques (NIBS) are not indicated due to safety concerns., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background and Objective: Kinematic analysis of reaching movements is increasingly used to evaluate upper extremity function after cerebrovascular insults in humans and has also been applied to rodent models. Such analyses can require time-consuming frame-by-frame inspections and are affected by the experimenter's bias. In this study, we introduce a semi-automated algorithm for tracking forepaw movements in mice. This methodology allows us to calculate several kinematic measures for the quantitative assessment of performance in a skilled reaching task before and after a focal cortical stroke., Methods: Mice were trained to reach for food pellets with their preferred paw until asymptotic performance was achieved. Photothrombosis was then applied to induce a focal ischemic injury in the motor cortex, contralateral to the trained limb. Mice were tested again once a week for 30 days. A high frame rate camera was used to record the movements of the paw, which was painted with a nontoxic dye. An algorithm was then applied off-line to track the trajectories and to compute kinematic measures for motor performance evaluation., Results: The tracking algorithm proved to be fast, accurate, and robust. A number of kinematic measures were identified as sensitive indicators of poststroke modifications. Based on end-point measures, ischemic mice appeared to improve their motor performance after 2 weeks. However, kinematic analysis revealed the persistence of specific trajectory adjustments up to 30 days poststroke, indicating the use of compensatory strategies., Conclusions: These results support the use of kinematic analysis in mice as a tool for both detection of poststroke functional impairments and tracking of motor improvements following rehabilitation. Similar studies could be performed in parallel with human studies to exploit the translational value of this skilled reaching analysis., (© The Author(s) 2014.)

Botulinum neurotoxin type A (BoNT/A) is a metalloprotease that produces a sustained yet transitory blockade of transmitter release from peripheral nerve terminals. Local delivery of this neurotoxin is successfully employed in clinical practice to reduce muscle hyperactivity such as in spasticity and dystonia, and to relieve pain with long-lasting therapeutic effects. However, not all BoNT/A effects can be explained by an action at peripheral nerve terminals. Indeed, it appears that BoNT/A is endowed with trafficking properties similar to the parental tetanus neurotoxin and thus be able to directly affect the CNS. In this review, we present and discuss novel compelling evidence for a direct central effect of BoNT/A in both dorsal and ventral horns of the animal and human spinal cord after peripheral injection of the neurotoxin, with important consequences on pain and motor control. This new knowledge is expected to radically change the approach to the use of BoNT/A in the future. As BoNT/A central action appears to also contribute to functional improvement, for instance in human spastic gait, the challenge will be to develop new subtypes or BoNT derivatives with deliberate, cell-specific central effects in order to fully exploit the spectrum of BoNT/A therapeutic activity., (© The Author(s) 2014.)