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Astrocytic networks and gap junctional communication mediated by connexins (Cxs) have been repeatedly implicated in seizures, epileptogenesis, and epilepsy. However, the effect of seizures on Cx expression is controversial. The present study focused on the response of Cxs to status epilepticus (SE), which is in turn an epileptogenic insult. The expression of neuronal Cx36 and astrocytic Cx30 and Cx43 mRNAs was investigated in the brain of rats in the first day after pilocarpine-induced SE. In situ hybridization revealed a progressive decrease in Cx43 and Cx30 mRNA levels, significantly marked 24 h after SE onset in neocortical areas and the hippocampus, and in most thalamic domains, whereas Cx36 mRNA did not exhibit obvious changes. Regional evaluation with quantitative real-time-RT-PCR confirmed Cx43 and Cx30 mRNA downregulation 24 h after SE, when ongoing neuronal cell death was found in the same brain regions. Immunolabeling showed at the same time point marked a decrease in Cx43, microglia activation, and interleukin-1β induction in some microglial cells. The data showed a transient downregulation of astroglial Cxs in the cortical and thalamic areas in which SE triggers neurodegenerative events in concomitance with microglia activation and cytokine expression. This could potentially represent a protective response of neuroglial networks to SE-induced acute damage.

Increased exploitation of minerals has led to pollution of confined environments as documented in Nigeria Niger Delta. Information on the effects on brain of such exposure is limited. Due to its exploratory activities, the African giant rat (Cricetomys gambianus) (AGR) provides a unique model for neuroecotoxicological research to determine levels of animal and human exposure to different pollutants. This study aims to unravel neuropathological features of AGR sampled from three agro-ecological zones of Nigeria. Fifteen AGR were sampled according to previously determined data on heavy metal exposure: high vanadium, high lead, and low metals. Eighteen AGR were collected from low metal zone and divided into two groups. Control group received vehicle while SMV exposed group received 3mg/kg sodium metavanadate (SMV) intraperitoneally for 14days. Brain immunohistochemical analyses were conducted, and ultrastructural changes were studied in experimentally exposed group. Results showed significant loss of tyrosin hydroxylase, parvalbumin, orexin-A and melanin concentration hormone containing neuronal populations in brains obtained from high vanadium and high lead zones and in experimentally intoxicated SMV groups. Similarly, significant decrease numbers of dendritic arborations; extracellular matrix density, perineuronal nets; astrocytes and microglia activations are documented in same groups. Ultrastructural studies revealed mass denudation, cilia loss, disintegration of ependymal layer and intense destructions of myelin sheaths in SMV exposed group. These are the first “neuroecotoxicological” findings in distinct neuronal cells. The implications of these findings are highly relevant for human population living in these areas, not only in Nigeria but also in similarly polluted areas elsewhere in the world.

Human African trypanosomiasis (HAT) remains a serious public health problem with diagnostic and treatment challenges in many African countries. The absence of a gold-standard biomarker has been a major difficulty for accurate disease staging and treatment follow-up. We therefore attempted to develop a simple, affordable, and noninvasive biomarker for HAT diagnosis and staging. Simultaneous actigraphy and polysomnography as well as cerebrospinal fluid (CSF) white blood cell (WBC) count, trypanosome presence, and C-X-C motif ligand (CXCL)-10 cytokine levels were performed in 20 HAT patients and nine healthy individuals (controls) using standard procedures. The International HIV Dementia Scale (IHDS) was scored in some patients as a surrogate for clinical assessment. From actigraphic parameters, we developed a novel sleep score and used it to determine correlations with other HAT markers, and compared their performance in differentiating between patients and controls and between HAT stages. The novel actigraphy sleep score (ASS) had the following ranges: 0–25 (healthy controls), 67–103 (HAT stage I), 111–126 (HAT intermediate), and 133–250 (HAT stage II). Compared with controls, stage I patients displayed a 7-fold increase in the ASS (P < 0.01), intermediate stage patients a 10-fold increase (P < 0.001), and HAT stage II patients an almost 20-fold increase (P < 0.001). CXCL-10 showed high interindividual differences. White blood cell counts were only marked in HAT stage II patients with a high interindividual variability. The International HIV Dementia Scale score negatively correlated with the ASS. We report the development and better performance of a new biomarker, ASS, for HAT diagnosis, disease staging, and monitoring that needs to be confirmed in large cohort studies.

Highlights • MIMO2 had myelin-potentiating effects at 10 mg/kg. • MIMO2 showed no side-effects. • MIMO2 reduced neuropathologies., Vanadium, a transition series metal released during some industrial activities, induces oxidative stress and lipid peroxidation. Ameliorative effect of a pure compound from the methanolic extract of Moringa oleifera leaves, code-named MIMO2, in 14-day old mice administered with vanadium (as sodium metavanadate 3 mg/kg) for 2 weeks was assessed. Results from body weight monitoring, muscular strength, and open field showed slight reduction in body weight and locomotion deficit in vanadium-exposed mice, ameliorated with MIMO2 co-administration. Degeneration of the Purkinje cell layer and neuronal death in the hippocampal CA1 region were observed in vanadium-exposed mice and both appeared significantly reduced with MIMO2 co-administration. Demyelination involving the midline of the corpus callosum, somatosensory and retrosplenial cortices was also reduced with MIMO2. Microglia activation and astrogliosis observed through immunohistochemistry were also alleviated. Immunohistochemistry for myelin, axons and oligodendrocyte lineage cells were also carried out and showed that in vanadium-treated mice brains, oligodendrocyte progenitor cells increased NG2 immunolabelling with hypertrophy and bushy, ramified appearance of their processes. MIMO2 displayed ameliorative and antioxidative effects in vanadium-induced neurotoxicity in experimental murine species. This is likely the first time MIMO2 is being used in vivo in an animal model.

Background: The European PharmaCog study (http://www.pharmacog.org) has reported a reduction in delta (1–6 Hz) electroencephalographic (EEG) power (density) during cage exploration (active condition) compared with quiet wakefulness (passive condition) in PDAPP mice (hAPP Indiana V717F mutation) modeling Alzheimer’s disease (AD) amyloidosis and cognitive deficits. Objective: Here, we tested the reproducibility of that evidence in TASTPM mice (double mutation in APP KM670/671NL and PSEN1 M146V), which develop brain amyloidosis and cognitive deficits over aging. The reliability of that evidence was examined in four research centers of the PharmaCog study. Methods: Ongoing EEG rhythms were recorded from a frontoparietal bipolar channel in 29 TASTPM and 58 matched “wild type” C57 mice (range of age: 12–24 months). Normalized EEG power was calculated. Frequency and amplitude of individual delta and theta frequency (IDF and ITF) peaks were considered during the passive and active conditions. Results: Compared with the “wild type” group, the TASTPM group showed a significantly lower reduction in IDF power during the active over the passive condition (p

Carbon‐based nanomaterials (CNMs) are being explored for neurological applications. However, systematic in vivo studies investigating the effects of CNM nanocarriers in the brain and how brain cells respond to such nanomaterials are scarce. To address this, functionalized multiwalled carbon nanotubes and graphene oxide (GO) sheets are injected in mice brain and compared with charged liposomes. The induction of acute neuroinflammatory and neurotoxic effects locally and in brain structures distant from the injection site are assessed up to 1 week postadministration. While significant neuronal cell loss and sustained microglial cell activation are observed after injection of cationic liposomes, none of the tested CNMs induces either neurodegeneration or microglial activation. Among the candidate nanocarriers tested, GO sheets appear to elicit the least deleterious neuroinflammatory profile. At molecular level, GO induces moderate activation of proinflammatory markers compared to vehicle control. At histological level, brain response to GO is lower than after vehicle control injection, suggesting some capacity for GO to reduce the impact of stereotactic injection on brain. While these findings are encouraging and valuable in the selection and design of nanomaterial‐based brain delivery systems, they warrant further investigations to better understand the mechanisms underlying GO immunomodulatory properties in brain., This work was partly supported by the EU H2020 RTD Framework Program: FET Graphene Flagship project (GrapheneCore3, Grant agreement ID: 881603), the Centre National de la Recherche Scientifique (CNRS), the International Center for Frontier Research in Chemistry (icFRC), and the Agence Nationale de la Recherche (ANR) through the LabEx project Chemistry of Complex Systems (ANR‐10‐LABX‐0026_CSC). M.P., as the recipient of the AXA Carbon Bionanotechnology Chair, is grateful to the AXA Research Fund for financial support. MP was also supported by the Spanish Ministry of Economy and Competitiveness MINECO (project CTQ2016‐76721‐R), the University of Trieste, and the Spanish State Research Agency (Maria de Maeztu Units of Excellence Program Grant No. MDM‐2017‐0720). Financial support to this project was partially provided by the Fondazione Cariverona (“Verona Nanomedicine Initiative”) and funding from an intramural (University of Verona) international cooperation program (“CooperInt”) was obtained. K.K. would like to acknowledge the Severo Ochoa Centre of Excellence Award to ICN2.

Hydrocephalus is especially prevalent in countries with limited resources, where its treatment is still a challenge. However, long-term neuropathological changes in untreated hydrocephalus remain largely unexplored. The present study looks at cortical parenchyma and neuroinflammation in acquired, chronic hydrocephalus. Intracisternal kaolin injections were performed in 3-week-old rats, followed by -1, 4- and 8-week survival; matched control rats received saline injections. Ventriculomegaly has been previously reported to stabilize by the third week in this model. Single and triple immunocytochemical approaches were used to highlight neurones, astrocytes, microglia, and the pro-inflammatory cytokine interleukin (IL)-1β in the parietal cortex, utilizing cell counts and densitometry. Microglial protein ionized calcium binding adaptor molecule 1 (Iba1) and IL-1β expressions were monitored with Western blotting in the parietal cortex and hippocampus. In the parietal cortex, which showed progressive disruption of cytoarchitecture, neuronal density was significantly increased at 8 weeks post-induction but not at earlier time points, indicating on-going cortical damage in chronic hydrocephalus. Astrocyte and microglia hypertrophy, and Iba1 expression indicated glial cell activation which peaked at 4 weeks. IL-1β expression also peaked at 4 weeks and was then down-regulated. Overall the findings indicate that neuroinflammatory features build up in the first month after hydrocephalus induction implicating marked IL-1β upregulation. The data also show that astrocytes are the main source of IL-1β in this disorder.

The goal of this paper is to examine existing methods to study the "Human Brain Connectome" with a specific focus on the neurophysiological ones. In recent years, a new approach has been developed to evaluate the anatomical and functional organization of the human brain: the aim of this promising multimodality effort is to identify and classify neuronal networks with a number of neurobiologically meaningful and easily computable measures to create its connectome. By defining anatomical and functional connections of brain regions on the same map through an integrated approach, comprising both modern neurophysiological and neuroimaging (i.e. flow/metabolic) brain-mapping techniques, network analysis becomes a powerful tool for exploring structural-functional connectivity mechanisms and for revealing etiological relationships that link connectivity abnormalities to neuropsychiatric disorders. Following a recent IFCN-endorsed meeting, a panel of international experts was selected to produce this current state-of-art document, which covers the available knowledge on anatomical and functional connectivity, including the most commonly used structural and functional MRI, EEG, MEG and non-invasive brain stimulation techniques and measures of local and global brain connectivity.

For targeted brain delivery, nanoparticles (NPs) should bypass the blood-brain barrier (BBB). Novel functionalization strategies, based on low-density lipoprotein receptor (LDLR) binding domain, have been here tested to increase the brain targeting efficacy of poly d,l-lactic-co-glycolic acid (PLGA) NPs, biodegradable and suited for biomedical applications. Custom-made PLGA NPs were functionalized with an apolipoprotein E modified peptide (pep-apoE) responsible for LDLR binding, or with lipocalin-type prostaglandin-d-synthase (L-PGDS), highly expressed in the brain. At the comparison of pep-apoE and L-PGDS sequences, a highly homologs region was here identified, indicating that also L-PGDS could bind LDLR. Non-functionalized and functionalized NPs did not affect the viability of cultured human dendritic cells, protagonists of the immune response, and did not activate them to a proinflammatory profile. At 2 h after intravenous injection in mice, functionalized, but not the non-functionalized ones, fluorescent-tagged NPs were observed in the cerebral cortex parenchyma. The NPs were mostly internalized by neurons and microglia; glial cells showed a weak activation. The findings indicate that the tested functionalization strategies do not elicit adverse immune responses and that the peptidic moieties enable BBB traversal of the NPs, thus providing potential brain drug carriers. These could be especially effective for brain diseases in which LDLR is involved. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 847-858, 2017.

Sleep is frequently altered in systemic infections as a component of sickness behavior in response to inflammation. Sleepiness in sickness behavior has been extensively investigated. Much less attention has instead been devoted to sleep and wake alterations in brain infections. Most of these, as other neuroinfections, are prevalent in sub-Saharan Africa. The present overview highlights the importance of this topic from both the clinical and pathogenetic points of view. Vigilance states and their regulation are first summarized, emphasizing that key nodes in this distributed brain system can be targeted by neuroinflammatory signaling. Sleep-wake changes in the parasitic disease human African trypanosomiasis (HAT) and its animal models are then reviewed and discussed. Experimental data have revealed that the suprachiasmatic nucleus, the master circadian pacemaker, and peptidergic cell populations of the lateral hypothalamus (the wake-promoting orexin neurons and the sleep-promoting melanin-concentrating hormone neurons) are targeted by African trypanosome infection. It is then discussed how prominent and disturbing are sleep changes in HIV/AIDS, also when the infection is cured with antiretroviral therapy. This recalls attention on the bidirectional interactions between sleep and immune system, including the specialized brain immune response of which microglial cells are protagonists. Sleep changes in an ancient viral disease, rabies, and in the emerging infection due to Zika virus which causes a congenital syndrome, are also dealt with. Altogether the findings indicate that sleep-wake regulation is targeted by brain infections caused by different pathogens and, although the relevant pathogenetic mechanisms largely remain to be clarified, these alterations differ from hypersomnia occurring in sickness behavior. Thus, brain infections point to the vulnerability of the neural network of sleep-wake regulation as a highly relevant clinical and basic science challenge.

Sleeping sickness is a fatal disease caused by Trypanosoma brucei, a unicellular parasite that lives in the bloodstream and interstitial spaces of peripheral tissues and the brain. Patients have altered sleep/wake cycles, body temperature, and endocrine profiles, but the underlying causes are unknown. Here, we show that the robust circadian rhythms of mice become phase advanced upon infection, with abnormal activity occurring during the rest phase. This advanced phase is caused by shortening of the circadian period both at the behavioral level as well as at the tissue and cell level. Period shortening is T. brucei specific and independent of the host immune response, as co-culturing parasites with explants or fibroblasts also shortens the clock period, whereas malaria infection does not. We propose that T. brucei causes an advanced circadian rhythm disorder, previously associated only with mutations in clock genes, which leads to changes in the timing of sleep., African sleeping sickness is well known for the alterations of sleeping patterns, but it is not known how circadian biology is altered by the causative pathogen Trypanosoma brucei. Here the authors show T. brucei causes a disorder of the cellular circadian clock that is unrelated to the immune response to the parasite.

Trypanosoma brucei (T. b.) gambiense is the parasite subspecies responsible for most reported cases of human African trypanosomiasis (HAT) or sleeping sickness. This severe infection leads to characteristic disruption of the sleep-wake cycle, recalling attention on the circadian timing system. Most animal models of the disease have been hitherto based on infection of laboratory rodents with the T. b. brucei subspecies, which is not infectious to humans. In these animal models, functional, rather than structural, alterations of the master circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), have been reported. Information on the SCN after infection with the human pathogenic T. b. gambiense is instead lacking. The present study was aimed at the examination of the SCN after T. b. gambiense infection of a susceptible rodent, the multimammate mouse, Mastomys natalensis, compared with T. b. brucei infection of the same host species. The animals were examined at 4 and 8 weeks post-infection, when parasites (T. b. gambiense or T. b. brucei) were detected in the brain parenchyma, indicating that the disease was in the encephalitic stage. Neuron and astrocyte changes were examined with Nissl staining, immunophenotyping and quantitative analyses. Interestingly, significant neuronal loss (about 30% reduction) was documented in the SCN during the progression of T. b. gambiense infection. No significant neuronal density changes were found in the SCN of T. b. brucei-infected animals. Neuronal cell counts in the hippocampal dentate gyrus of T. b. gambiense-infected M. natalensis did not point out significant changes, indicating that no widespread neuron loss had occurred in the brain. Marked activation of astrocytes was detected in the SCN after both T. b. gambiense and T. b. brucei infections. Altogether the findings reveal that neurons of the biological clock are highly susceptible to the infection caused by human pathogenic African trypanosomes, which have the capacity to cause permanent partial damage of this structure.

Background: It has been shown that theta (6-10 Hz) and delta (1-6 Hz) ongoing electroencephalographic (EEG) rhythms revealed variations in the cortical arousal in C57 Wild Type (WT) mice during cage exploration (active condition) compared to awake quiet behavior (passive condition; IMI PharmaCog project, www.pharmacog.eu). Objective: The objective was to test if these EEG rhythms might be abnormal in old PDAPP mice modeling Alzheimer's disease (AD) with a hAPP Indiana V717F mutation (They show abnormal neural transmission, cognitive deficits, and brain accumulation of Aβ1-42). Methods: Ongoing EEG rhythms were recorded by a frontoparietal bipolar channel in 15 PDAPP and 23 WT C57 male mice (mean age of 22.8 months ±0.4 and 0.3 standard error, respectively). EEG absolute power (density) was calculated. Frequency and amplitude of individual delta and theta frequency (IDF and ITF) peaks were considered during passive and active states in the wakefulness. Results: Compared with the WT group, the PDAPP group showed higher frequency of the IDF during the passive condition and lower frequency of the ITF during the active state. Furthermore, the WT but not PDAPP group showed significant changes in the frontoparietal EEG power (IDF, ITF) during active over passive state. Conclusion: PDAPP mice were characterized by less changes in the brain arousal during an active state as revealed by frontoparietal EEG rhythms. Future studies will have to cross-validate the present results on large animal groups, clarify the neurophysiological underpinning of the effect, and test if the disease modifying drugs against AD amyloidosis normalize those candiate EEG biomarkers in PDAPP mice

Orexin (OX)/hypocretin-containing neurons are main regulators of wakefulness stability, arousal, and energy homeostasis. Their activity varies in relation to the animal's behavioral state. We here tested whether such variation is subserved by synaptic plasticity phenomena in basal conditions. Mice were sacrificed during day or night, at times when sleep or wake, respectively, predominates, as assessed by electroencephalography in matched mice. Triple immunofluorescence was used to visualize OX-A perikarya and varicosities containing the vesicular glutamate transporter (VGluT)2 or the vesicular GABA transporter (VGAT) combined with synaptophysin (Syn) as a presynaptic marker. Appositions on OX-A+ somata were quantitatively analyzed in pairs of sections in epifluorescence and confocal microscopy. The combined total number of glutamatergic (Syn+/VGluT2+) and GABAergic (Syn+/VGAT+) varicosities apposed to OX-A somata was similar during day and night. However, glutamatergic varicosities were significantly more numerous at night, whereas GABAergic varicosities prevailed in the day. Triple immunofluorescence in confocal microscopy was employed to visualize synapse scaffold proteins as postsynaptic markers and confirmed the nighttime prevalence of VGluT2+ together with postsynaptic density protein 95+ excitatory contacts, and daytime prevalence of VGAT+ together with gephyrin+ inhibitory contacts, while also showing that they formed synapses on OX-A+ cell bodies. The findings reveal a daily reorganization of axosomatic synapses in orexinergic neurons, with a switch from a prevalence of excitatory innervation at a time corresponding to wakefulness to a prevalence of inhibitory innervations in the antiphase, at a time corresponding to sleep. This reorganization could represent a key mechanism of plasticity of the orexinergic network in basal conditions.

Resting state electroencephalographic (EEG) rhythms reflect the fluctuation of cortical arousal and vigilance in a typical clinical setting, namely the EEG recording for few minutes with eyes closed (i.e., passive condition) and eyes open (i.e., active condition). Can this procedure be back-translated to C57 (wild type) mice for aging studies? On-going EEG rhythms were recorded from a frontoparietal bipolar channel in 85 (19 females) C57 mice. Male mice were subdivided into 3 groups: 25 young (4.5–6 months), 18 middle-aged (12–15 months), and 23 old (20–24 months) mice to test the effect of aging. EEG power density was compared between short periods (about 5 minutes) of awake quiet behavior (passive) and dynamic exploration of the cage (active). Compared with the passive condition, the active condition induced decreased EEG power at 1–2 Hz and increased EEG power at 6–10 Hz in the group of 85 mice. Concerning the aging effects, the passive condition showed higher EEG power at 1–2 Hz in the old group than that in the others. Furthermore, the active condition exhibited a maximum EEG power at 6–8 Hz in the former group and 8–10 Hz in the latter. In the present conditions, delta and theta EEG rhythms reflected changes in cortical arousal and vigilance in freely behaving C57 mice across aging. These changes resemble the so-called slowing of resting state EEG rhythms observed in humans across physiological and pathological aging. The present EEG procedures may be used to enhance preclinical phases of drug discovery in mice for understanding the neurophysiological effects of new compounds against brain aging.

Studies on the molecular clockwork during aging have been hitherto addressed to core clock genes. These previous investigations indicate that circadian profiles of core clock gene expression at an advanced age are relatively preserved in the master circadian pacemaker and the hypothalamic suprachiasmatic nucleus (SCN), and relatively impaired in peripheral tissues. It remains to be clarified whether the effects of aging are confined to the primary loop of core clock genes, or also involve secondary clock loop components, including Rev-erbα and the clock-controlled genes Dbp and Dec1. Using quantitative real-time RT-PCR, we here report a comparative analysis of the circadian expression of canonical core clock genes (Per1, Per2, Cry1, Cry2, Clock and Bmal1) and non-core clock genes (Rev-erbα, Dbp and Dec1) in the SCN, liver, and heart of 3month-old vs 22month-old mice. The results indicate that circadian clock gene expression is significantly modified in the SCN and peripheral oscillators of aged mice. These changes are not only highly tissue-specific, but also involve different clock gene loops. In particular, we here report changes of secondary clock loop components in the SCN, changes of the primary clock loop in the liver, and minor changes of clock gene expression in the heart of aged mice. The present findings outline a track to further understanding of the role of primary and secondary clock loop components and their crosstalk in the impairment of circadian output which characterizes aging.

Different kinds of challenge can alter cognitive process and electroencephalographic (EEG) rhythms in humans. This can provide an alternative paradigms to evaluate treatment effects in drug discovery. Here, we report recent findings on the effects of challenges represented by sleep deprivation (SD), transient hypoxia, and transcranial magnetic stimulation (TMS) in healthy volunteers on cognitive processes and EEG rhythms to build a knowledge platform for novel research for drug discovery in AD Alzheimer's disease (AD). Sleep pressure enhanced frontal delta rhythms (< 4 Hz) during the night, while SD increased slow rhythms in the theta range (4-7 Hz), and reduced resting state alpha rhythms (8-12 Hz) after the following day. Furthermore, SD transiently affected cognitive performance. In contrast, transient experimental hypoxia induced abnormal posterior resting state delta and alpha rhythms in healthy volunteers that resemble the abnormal EEG rhythms typically recorded in AD patients. However, the relationship between the cognitive and EEG effects of such challenges is poorly understood. TMS reversibly interfered with higher brain functions during EEG recordings, but few studies have investigated the relationship between the cognitive and EEG effects of TMS. In conclusion, SD is the most mature challenge model for testing new drugs for AD. Future investigation is needed to better understand the opportunities offered by TMS and hypoxia challenges.

Summary Objective SCN1A encodes the alpha subunit of the voltage-gated sodium channel and plays a crucial role in several epilepsy syndromes. The common SCN1A splice-site polymorphism rs3812718 (IVS5N+5 G>A) might contribute to the pathophysiology underlying genetic generalized epilepsies and is associated with electrophysiologic properties of the channel and the effect of sodium-channel blocking antiepileptic drugs. We assessed the effects of the rs3812718 genotype on cortical excitability at baseline and after administration of carbamazepine in order to investigate the mechanism of this association. Methods Paired-pulse transcranial magnetic stimulation (TMS) was applied in 92 healthy volunteers with the homozygous genotypes AA or GG of rs3812718 at baseline and after application of 400 mg of carbamazepine or placebo in a double-blind, randomized, crossover design. Resting motor threshold (RMT), short interval intracortical inhibition (SICI), intracortical facilitation (ICF), and cortical silent period (CSP) were determined. Results At baseline there was no significant difference in any TMS parameter. Genotype GG was associated with a higher carbamazepine-induced increase in CSP duration as compared to AA (multivariate analysis of covariance [MANCOVA], p = 0.013). An expected significant increase in RMT was genotype independent. Significance We found that the rs3812718 genotype modifies the effect of carbamazepine on CSP duration (mainly reflecting modulation of γ-aminobutyric acid (GABA)ergic inhibition), but not on RMT (mainly reflecting modulation of voltage-gated sodium channels). This provides evidence that rs3812718 affects the pharmacoresponse to carbamazepine via an effect on GABAergic cortical interneurons. Our results also confirm that TMS is useful to investigate the effect of genetic variants on cortical excitability and pharmacoresponse.

Motoneuron degeneration is the hallmark of amyotrophic lateral sclerosis (ALS). The cause and predisposing factors for sporadic ALS are still unknown. Exposure to a specific environmental risk factors in subjects with a susceptibility genotype may increase the risk of the disease. The role of physical activity and the use of anabolic steroids are still debated in epidemiological studies on patients and murine models of ALS. To assess at the cellular level the role (beneficial or detrimental) of physical exercise and the use of anabolic steroid, we here investigated, in SOD1(G93A) (mSOD1) mice and wild-type littermates, changes in the ventral horn after regular exercise, treatment with the anabolic androgenic steroid 19-nortestosterone (nandrolone), and their combination, compared with matched control sedentary mice. The experiments were pursued for several weeks until symptom onset in mSOD1 mice. Lumbar motoneurons, astrocytes and microglia were analyzed. In wild-type mice, cytological alterations of motoneurons were observed especially after nandrolone treatment. The following main findings were observed in treated mSOD1 mice versus untreated ones: i) nandrolone treatment markedly enhanced motoneuron loss; this detrimental effect was reverted by the combination with exercise, resulting in increased motoneuron survival; ii) astrocytic activation was most marked after nandrolone treatment when motoneuron damage was most severe; iii) microglia activation was most marked after physical exercise when motoneuron damage was less severe. The results indicate a vulnerability of mSOD1 motoneurons to nandrolone treatment, a potential neuroprotective effect of physical exercise, and a modulation by glial cells in the ALS murine model in the examined paradigms.

Aim: To unravel key aspects of the use of lanthanide-doped nanoparticles (NPs) in biomedicine, the interaction with immune and brain cells. Materials & methods: Effects of citrate-stabilized CaF2 and SrF2: Yb, Er NPs (13–15 nm) on human dendritic cells and neurons were assessed in vitro. In vivo distribution was analyzed in mice at tissue and ultrastructural levels, and with glia immunophenotyping. Results: The NPs did not elicit dendritic cell activation and were internalized by cultured neurons, without viability changes. After intravenous injection, NPs were found in the brain parenchyma, without features of glial neuroinflammatory response. Conclusion: Lanthanide-doped NPs do not activate cells protagonists of systemic and brain immune responses, are endocytosed by neurons and can cross an intact blood–brain barrier.

Graphical abstract, Anabolic/androgenic steroids (AAS) are drugs that enhance muscle mass, and are often illegally utilized in athletes to improve their performances. Recent data suggest that the increased risk for amyotrophic lateral sclerosis (ALS) in male soccer and football players could be linked to AAS abuse. ALS is a motor neuron disease mainly occurring in sporadic (sALS) forms, but some familial forms (fALS) exist and have been linked to mutations in different genes. Some of these, in their wild type (wt) form, have been proposed as risk factors for sALS, i.e. superoxide dismutase 1 (SOD1) gene, whose mutations are causative of about 20% of fALS. Notably, SOD1 toxicity might occur both in motor neurons and in muscle cells. Using gastrocnemius muscles of mice overexpressing human mutant SOD1 (mutSOD1) at different disease stages, we found that the expression of a selected set of genes associated to muscle atrophy, MyoD, myogenin, atrogin-1, and transforming growth factor (TGF)β1, is up-regulated already at the presymptomatic stage. Atrogin-1 gene expression was increased also in mice overexpressing human wtSOD1. Similar alterations were found in axotomized mouse muscles and in cultured ALS myoblast models. In these ALS models, we then evaluated the pharmacological effects of the synthetic AAS nandrolone on the expression of the genes modified in ALS muscle. Nandrolone administration had no effects on MyoD, myogenin, and atrogin-1 expression, but it significantly increased TGFβ1 expression at disease onset. Altogether, these data suggest that, in fALS, muscle gene expression is altered at early stages, and AAS may exacerbate some of the alterations induced by SOD1 possibly acting as a contributing factor also in sALS.

Background: Gap junctions are specialized channels composed of several connexins, membrane proteins that mediate electrical and metabolic coupling between cells. Previous data have suggested that changes in the expression of Cx43, the main astrocytic Cx isoform, may be involved in seizure activity in human epileptic tissue. However, Cx43 has never been examined in focal cortical dysplasia (FCD) and in other human refractory epilepsies. Methods: We analyzed Cx43 protein localization and Cx43 mRNA levels in surgical specimens of cortex from a cohort of patients with intractable epilepsy vs control nonepileptic tissue. Samples had neuropathologically defined diagnosis of cryptogenic epilepsy or epilepsy secondary to FCD. Results: Cx43 immunoreactivity, which labeled punctate elements, did not reveal distinctive features in cryptogenic epilepsy and FCD type IA and IIA. A peculiar pattern of immunolabeling was instead observed in FCD type IIB, in which large aggregates of Cx43-immunopositive puncta were clustered around subsets of balloon cells and astrocytes. Further characterization revealed that these balloon cells do not express markers of precursor cells, such as CD34. Quantitative real-time reverse transcriptase PCR showed elevated levels of Cx43 transcript in a subgroup (25%) of cryptogenic epilepsy specimens compared to control and FCD ones. Conclusions: Our study points out that a rearrangement of Cx43-positive elements is part of abnormal tissue organization in FCD type IIB, and that cryptogenic epilepsies include forms with increased Cx43 mRNA expression. The data implicate functional consequences of altered Cx43 expression, and therefore of altered gap junctional coupling, in abnormal network properties of subtypes of human refractory epilepsies.

Author(s): Bentivoglio, Marina; Jones, Edward G; Mazzarello, Paolo; Ribak, Charles E; Shepherd, Gordon M; Swanson, Larry W

An overview of current concepts on neuroinflammation and on the dialogue between neurons and non-neuronal cells in three important infections of the central nervous systems (rabies, cerebral malaria, and human African trypanosomiasis or sleeping sickness) is here presented. Large numbers of cases affected by these diseases are currently reported. In the context of an issue dedicated to Camillo Golgi, historical notes on seminal discoveries on these diseases are also presented. Neuroinflammation is currently closely associated with pathogenetic mechanisms of chronic neurodegenerative diseases. Neuroinflammatory signaling in brain infections is instead relatively neglected in the neuroscience community, despite the fact that the above infections provide paradigmatic examples of alterations of the intercellular crosstalk between neurons and non-neuronal cells. In rabies, strategies of immune evasion of the host lead to silencing neuroinflammatory signaling. In the intravascular pathology which characterizes cerebral malaria, leukocytes and Plasmodium do not enter the brain parenchyma. In sleeping sickness, leukocytes and African trypanosomes invade the brain parenchyma at an advanced stage of infection. Both the latter pathologies leave open many questions on the targeting of neuronal functions and on the pathogenetic role of non-neuronal cells, and in particular astrocytes and microglia, in these diseases. All three infections are hallmarked by very severe clinical pictures and relative sparing of neuronal structure. Multidisciplinary approaches and a concerted action of the neuroscience community are needed to shed light on intercellular crosstalk in these dreadful brain diseases. Such effort could also lead to new knowledge on non-neuronal mechanisms which determine neuronal death or survival.

The extracellular parasite Trypanosoma brucei causes human African trypanosomiasis (HAT), also known as sleeping sickness. Trypanosomes are transmitted by tsetse flies and HAT occurs in foci in sub-Saharan Africa. The disease, which is invariably lethal if untreated, evolves in a first hemo-lymphatic stage, progressing to a second meningo-encephalitic stage when the parasites cross the blood-brain barrier. At first, trypanosomes are restricted to circumventricular organs and choroid plexus in the brain outside the blood-brain barrier, and to dorsal root ganglia. Later, parasites cross the blood-brain barrier at post-capillary venules, through a multi-step process similar to that of lymphocytes. Accumulation of parasites in the brain is regulated by cytokines and chemokines. Trypanosomes can alter neuronal function and the most prominent manifestation is represented by sleep alterations. These are characterized, in HAT and experimental rodent infections, by disruption of the sleep-wake 24h cycle and internal sleep structure. Trypanosome infections alter also some, but not all, other endogenous biological rhythms. A number of neural pathways and molecules may be involved in such effects. Trypanosomes secrete prostaglandins including the somnogenic PGD2, and they interact with the host's immune system to cause release of pro-inflammatory cytokines. From the sites of early localization of parasites in the brain and meninges, such molecules could affect adjacent brain areas implicated in sleep-wakefulness regulation, including the suprachiasmatic nucleus and its downstream targets, to cause the changes characteristic of the disease. This raises challenging issues on the effects of cytokines on synaptic functions potentially involved in sleep-wakefulness alterations.

Biological rhythms are frequently disturbed with advancing age, and aging-related changes of glia in the hypothalamic suprachiasmatic nucleus (SCN), the master circadian pacemaker, require special attention. In particular, astrocytes contribute to SCN function, and aging is associated with increased inflammatory activity in the brain, in which microglia could be especially implicated. On this basis, we investigated in the SCN of young and old mice glial transcripts and cell features, and the glial cell response to a central inflammatory challenge. Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyze the expression of mRNAs encoding the astrocytic glial fibrillary acidic protein and the microglial antigen CD11b. Both these transcripts, here investigated in the SCN for the first time, were significantly increased in the old SCN. Glial cell phenotyping with immunohistochemistry revealed hypertrophic and intensely stained astrocytes and microglia in the aged SCN. In both age groups, microglia were scattered throughout the SCN and astrocytes were prominent in the ventral portion, where retinal fibers are densest; in the aged SCN, astrocytes were also numerous in the dorsal portion. After intracerebroventricular injections of a mixture of interferon-gamma and tumor necrosis factor-alpha, or phosphate-buffered saline as control, immunolabeling was evaluated with stereological cell counts and confocal microscopy. Phenotypic features of astrocyte and microglia activation in response to cytokine injections were markedly enhanced in the aged SCN. Subregional variations in glial cell density were also documented in the aged compared to the young SCN. Altogether, the findings show increases in the expression of glial transcripts and hypertrophy of astrocytes and microglia in the aged SCN, as well as age-dependent variation in the responses of immune-challenged SCN glia. The data thus point out an involvement of glia in aging-related changes of the biological clock.

We present here findings obtained on a large number of human tissues over a period of more than ten years, by our modification of the Osmium maceration method for high resolution scanning electron microscopy (HRSEM). Data are documented by original pictures which illustrate both some 3-D intracellular features not previously shown in human tissues, and results obtained in our current studies on mitochondrial morphology and on the secretory process of salivary glands. We have demonstrated that mitochondria of cells of practically all human tissues and organs have usually tubular cristae, and that even the cristae that look lamellar are joined to the inner mitochondrial membrane by tubular connexions similar to the crista junctions later seen by electron tomography. Concerning salivary glands an important result is the development of a morphometric method that allows the quantitative evaluation of the secretory events.

Summary Purpose: To analyze cellular mechanisms of limbic-seizure suppression, the response to pilocarpine-induced seizures was investigated in cortex and thalamus, comparing epilepsy-resistant rats Proechimys guyannensis with Wistar rats. Methods: Fos immunoreactivity revealing neuronal activation, and degenerating neurons labeled by Fluoro-Jade B (FJB) histochemistry were analyzed on the first day after onset of seizures lasting 3 h. Subpopulations of γ-aminobutyric acid (GABA)ergic cells were characterized with double Fos-parvalbumin immunohistochemistry. Results: In both cortex and thalamus, degenerating neurons were much fewer in Proechimys than Wistar rats. Fos persisted at high levels at 24 h only in the Proechimys thalamus and cortex, especially in layer VI where corticothalamic neurons reside. In the parietal cortex, about 50% of parvalbumin-containing interneurons at 8 h, and 10–20% at 24 h, were Fos-positive in Wistar rats, but in Proechimys, Fos was expressed in almost all parvalbumin-containing interneurons at 8 h and dropped at 24 h. Fos positivity in cingulate cortex interneurons was similar in both species. In the Wistar rat thalamus, Fos was induced in medial and midline nuclei up to 8 h, when

Lower motoneuron abnormalities have been extensively documented in the murine model of familial amyotrophic lateral sclerosis, whereas information on corticospinal neurons in these mice is very limited. We investigated 1) mRNA levels of inflammation-related molecules in the deep layers in which corticospinal neurons reside, 2) corticospinal neurons labeled from tracer injections in the corticospinal tract at the cervical level, 3) axonal damage revealed by beta-amyloid precursor protein accumulation, and 4) glial cell activation in the sensorimotor cortex of presymptomatic and end-stage superoxide dismutase (SOD)-1 (G93A) mice. We demonstrated induction of inflammatory gene transcripts in the deep layers, early and progressive shrinkage of corticospinal cell bodies and activation of surrounding astrocytes and microglia with upregulation of major histocompatibility complex class I antigen. Accumulation of beta-amyloid precursor protein in proximal axonal swellings indicating axonal injury was also evident at the terminal stage in the motor cortex and internal capsule. Glial and axon changes were not observed elsewhere in the cortex. These data reveal that the entire motor circuit is affected in this murine amyotrophic lateral sclerosis model as it is in human amyotrophic lateral sclerosis. Sensorimotor cortical inflammation and progressive corticospinal cell body and fiber damage may reflect transsynaptic signaling of damage from lower motoneurons.