Neuroinflammation: A Signature or a Cause of Epilepsy?

Epilepsy can be both a primary pathology and a secondary effect of many neurological conditions. Many papers show that neuroinflammation is a product of epilepsy, and that in pathological conditions characterized by neuroinflammation, there is a higher probability to develop epilepsy. However, the bidirectional mechanism of the reciprocal interaction between epilepsy and neuroinflammation remains to be fully understood. Here, we attempt to explore and discuss the relationship between epilepsy and inflammation in some paradigmatic neurological and systemic disorders associated with epilepsy. In particular, we have chosen one representative form of epilepsy for each one of its actual known etiologies. A better understanding of the mechanistic link between neuroinflammation and epilepsy would be important to improve subject-based therapies, both for prophylaxis and for the treatment of epilepsy.

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo.

Intracellular chloride ([Cl-]i) and pH (pHi) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-]i and pHi, but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl- and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-]i and pHi at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-]i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-]i in response to hypercapnia and seizure activity.

The bacterial protein toxin, cytotoxic necrotizing factor 1 (CNF1) provides long-term survival in a murine glioma model.

BACKGROUND: Glioblastomas are largely unresponsive to all available treatments and there is therefore an urgent need for novel therapeutics. Here we have probed the antineoplastic effects of a bacterial protein toxin, the cytotoxic necrotizing factor 1 (CNF1), in the syngenic GL261 glioma cell model. CNF1 produces a long-lasting activation of Rho GTPases, with consequent blockade of cytodieresis in proliferating cells and promotion of neuron health and plasticity. METHODS: We have tested the antiproliferative effects of CNF1 on GL261 cells and human glioma cells obtained from surgical specimens. For the in vivo experiments, we injected GL261 cells into the adult mouse visual cortex, and five days later we administered either a single intracerebral dose of CNF1 or vehicle. To compare CNF1 with a canonical antitumoral drug, we infused temozolomide (TMZ) via minipumps for 1 week in an additional animal group. RESULTS: In culture, CNF1 was very effective in blocking proliferation of GL261 cells, leading them to multinucleation, senescence and death within 15 days. CNF1 had a similar cytotoxic effect in primary human glioma cells. CNF1 also inhibited motility of GL261 cells in a scratch-wound migration assay. Low dose (2 nM) CNF1 and continuous TMZ infusion significantly prolonged animal survival (median survival 35 days vs. 28 days in vehicle controls). Remarkably, increasing CNF1 concentration to 80 nM resulted in a dramatic enhancement of survival with no obvious toxicity. Indeed, 57% of the CNF1-treated animals survived up to 60 days following GL261 glioma cell transplant. CONCLUSIONS: The activation of Rho GTPases by CNF1 represents a novel potential therapeutic strategy for the treatment of central nervous system tumors.

Shank3 deletion in PV neurons is associated with abnormal behaviors and neuronal functions that are rescued by increasing GABAergic signaling.

BACKGROUND: Phelan-McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by developmental delay, intellectual disability, and autistic-like behaviors and is primarily caused by haploinsufficiency of SHANK3 gene. Currently, there is no specific treatment for PMS, highlighting the need for a better understanding of SHANK3 functions and the underlying pathophysiological mechanisms in the brain. We hypothesize that SHANK3 haploinsufficiency may lead to alterations in the inhibitory system, which could be linked to the excitatory/inhibitory imbalance observed in models of autism spectrum disorder (ASD). Investigation of these neuropathological features may shed light on the pathogenesis of PMS and potential therapeutic interventions. METHODS: We recorded local field potentials and visual evoked responses in the visual cortex of Shank3∆11-/- mice. Then, to understand the impact of Shank3 in inhibitory neurons, we generated Pv-cre+/- Shank3Fl/Wt conditional mice, in which Shank3 was deleted in parvalbumin-positive neurons. We characterized the phenotype of this murine model and we compared this phenotype before and after ganaxolone administration. RESULTS: We found, in the primary visual cortex, an alteration of the gain control of Shank3 KO compared with Wt mice, indicating a deficit of inhibition on pyramidal neurons. This alteration was rescued after the potentiation of GABAA receptor activity by Midazolam. Behavioral analysis showed an impairment in grooming, memory, and motor coordination of Pv-cre+/- Shank3Fl/Wt compared with Pv-cre+/- Shank3Wt/Wt mice. These deficits were rescued with ganaxolone, a positive modulator of GABAA receptors. Furthermore, we demonstrated that treatment with ganaxolone also ameliorated evocative memory deficits and repetitive behavior of Shank3 KO mice. LIMITATIONS: Despite the significant findings of our study, some limitations remain. Firstly, the neurobiological mechanisms underlying the link between Shank3 deletion in PV neurons and behavioral alterations need further investigation. Additionally, the impact of Shank3 on other classes of inhibitory neurons requires further exploration. Finally, the pharmacological activity of ganaxolone needs further characterization to improve our understanding of its potential therapeutic effects. CONCLUSIONS: Our study provides evidence that Shank3 deletion leads to an alteration in inhibitory feedback on cortical pyramidal neurons, resulting in cortical hyperexcitability and ASD-like behavioral problems. Specifically, cell type-specific deletion of Shank3 in PV neurons was associated with these behavioral deficits. Our findings suggest that ganaxolone may be a potential pharmacological approach for treating PMS, as it was able to rescue the behavioral deficits in Shank3 KO mice. Overall, our study highlights the importance of investigating the role of inhibitory neurons and potential therapeutic interventions in neurodevelopmental disorders such as PMS.

Daily rhythm in cortical chloride homeostasis underpins functional changes in visual cortex excitability.

Cortical activity patterns are strongly modulated by fast synaptic inhibition mediated through ionotropic, chloride-conducting receptors. Consequently, chloride homeostasis is ideally placed to regulate activity. We therefore investigated the stability of baseline [Cl-]i in adult mouse neocortex, using in vivo two-photon imaging. We found a two-fold increase in baseline [Cl-]i in layer 2/3 pyramidal neurons, from day to night, with marked effects upon both physiological cortical processing and seizure susceptibility. Importantly, the night-time activity can be converted to the day-time pattern by local inhibition of NKCC1, while inhibition of KCC2 converts day-time [Cl-]i towards night-time levels. Changes in the surface expression and phosphorylation of the cation-chloride cotransporters, NKCC1 and KCC2, matched these pharmacological effects. When we extended the dark period by 4 h, mice remained active, but [Cl-]i was modulated as for animals in normal light cycles. Our data thus demonstrate a daily [Cl-]i modulation with complex effects on cortical excitability.

In vivo detection of murine glioblastoma through Raman and reflectance fiber-probe spectroscopies.

Significance: Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults. With a worldwide incidence rate of 2 to 3 per 100,000 people, it accounts for more than 60% of all brain cancers; currently, its 5-year survival rate is < 5 % . GBM treatment relies mainly on surgical resection. In this framework, multimodal optical spectroscopy could provide a fast and label-free tool for improving tumor detection and guiding the removal of diseased tissues. Aim: Discriminating healthy brain from GBM tissues in an animal model through the combination of Raman and reflectance spectroscopies. Approach: EGFP-GL261 cells were injected into the brains of eight laboratory mice for inducing murine GBM in these animals. A multimodal optical fiber probe combining fluorescence, Raman, and reflectance spectroscopy was used to localize in vivo healthy and tumor brain areas and to collect their spectral information. Results: Tumor areas were localized through the detection of EGFP fluorescence emission. Then, Raman and reflectance spectra were collected from healthy and tumor tissues, and later analyzed through principal component analysis and linear discriminant analysis in order to develop a classification algorithm. Raman and reflectance spectra resulted in 92% and 93% classification accuracy, respectively. Combining together these techniques allowed improving the discrimination between healthy and tumor tissues up to 97%. Conclusions: These preliminary results demonstrate the potential of multimodal fiber-probe spectroscopy for in vivo label-free detection and delineation of brain tumors, and thus represent an additional, encouraging step toward clinical translation and deployment of fiber-probe spectroscopy.

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter.

Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.

Understanding Spreading Depression from Headache to Sudden Unexpected Death.

Spreading depression (SD) is a neurophysiological phenomenon characterized by abrupt changes in intracellular ion gradients and sustained depolarization of neurons. It leads to loss of electrical activity, changes in the synaptic architecture, and an altered vascular response. Although SD is often described as a unique phenomenon with homogeneous characteristics, it may be strongly affected by the particular triggering event and by genetic background. Furthermore, SD may contribute differently to the pathogenesis of widely heterogeneous clinical conditions. Indeed, clinical disorders related to SD vary in their presentation and severity, ranging from benign headache conditions (migraine syndromes) to severely disabling events, such as cerebral ischemia, or even death in people with epilepsy. Although the characteristics and mechanisms of SD have been dissected using a variety of approaches, ranging from cells to human models, this phenomenon remains only partially understood because of its complexity and the difficulty of obtaining direct experimental data. Currently, clinical monitoring of SD is limited to patients who require neurosurgical interventions and the placement of subdural electrode strips. Significantly, SD events recorded in humans display electrophysiological features that are essentially the same as those observed in animal models. Further research using existing and new experimental models of SD may allow a better understanding of its core mechanisms, and of their differences in different clinical conditions, fostering opportunities to identify and develop targeted therapies for SD-related disorders and their worst consequences.