Characterization of a mice model of human epilepsy with Multi-Electrode Arrays

We applied microelectrode array (MEA) recordings to study the generation and propagation of epileptform activity in various connected regions of cortico-hippocampal slices obtained from SynapsinI/II/III knockout (TKO) mice and the effects of the synaptic vesicle-targeted anti epileptic drug levetiracetam (LEV). Synapsins (SynI, SynII and SynIII) are synaptic vesicle phosphoproteins playing a role in synaptic transmission and plasticity. TKO mice display an epileptic phenotype and mutation of the SYN1 gene is associated with epilepsy in man. We found that both interictal (IIC) and ictal (IC) discharges induced by 4AP were more pronounced and widespread in TKO mice, revealing a state of hyperexcitability of TKO networks. To get insight into the frequencies characterizing the IC seizures, we analyzed the average IC power spectral density (PSD) in the 10-50 Hz range in different cortical regions. TKO slices exhibited an increase of power for frequencies above 20Hz with respect to Wild- Type (TWT). To determine whether the hyperexcitability of TKO slices is also reflected by an increased spread of IC discharges and taking advantage of the spatial resolution of the MEA device, we measured the percentage of electrodes recording IC discharges over the total number of cortical electrodes. The spread of excitation was significantly higher in TKO slices than in TWT ones and treatment with LEV decreased the spread of IC discharges in the entorhinal of TKO slices. In order to better characterize the propagation of the IIC events in the hippocampus, we recently coupled MEA recordings with optical imaging using voltage-sensitive dyes by exploiting the possibility of simultaneous recordings with a high spatial and temporal resolution to reveal more detailed patterns of propagation

Cell adhesion molecule L1 contributes to neuronal excitability regulating the function of voltage-gated Na+ channels

L1 (also known as L1CAM) is a trans-membrane glycoprotein mediating neuron-neuron adhesion through homophilic and heterophilic interactions. Although experimental evidence has implicated L1 in axonal outgrowth, fasciculation and pathfinding, its contribution to voltage-gated Na+ channel function and membrane excitability has remained unknown. Here, we show that firing rate, single cell spiking frequency and Na+ current density are all reduced in hippocampal excitatory neurons from L1-deficient mice both in culture and in slices owing to an overall reduced membrane expression of Na+ channels. Remarkably, normal firing activity was restored when L1 was reintroduced into L1-deficient excitatory neurons, indicating that abnormal firing patterns are not related to developmental abnormalities, but are a direct consequence of L1 deletion. Moreover, L1 deficiency leads to impairment of action potential initiation, most likely due to the loss of the interaction of L1 with ankyrin G that produces the delocalization of Na+ channels at the axonal initial segment. We conclude that L1 contributes to functional expression and localization of Na+ channels to the neuronal plasma membrane, ensuring correct initiation of action potential and normal firing activity

Validation of a high-density microelectrode array for acute brain slice recordings

Microelectrode arrays (MEAs) are employed to study extracellular electrical activity in neuronal tissues. Nevertheless, commercially available MEAs provide a limited number of recording sites and do not allow a precise identification of the spatio-temporal characterization of the recorded signal. To overcome this limitation, high density MEAs (HDMEA), based on CMOS technology, were recently developed and validated on dissociated preparations. The platform enables extracellular electrophysiological recordings from 4096 electrodes arranged in a squared area of 2.7 mm x 2.7 mm with inter-electrode distance of 21 μm at a sampling rate of 7.7 kHz/electrode. Here, we demonstrate the performances of this HDMEA platform for the acquisition of electrophysiological activity from acute brain slices. The unique recording performances and the large recording area of the chip permit the observation of fast propagating activities involving multiple areas. In our experimental paradigm, epileptic-like discharges were induced by treating hippocampal slices with 4-aminopyridine and/ or bicuculline. The HDMEA allowed us to clearly identify epileptic foci and to describe the involvement of cortical and hippocampal circuitries in the generation of the epileptiform activity. Furthermore, the HDMEA can be coupled with conventional extracellular electrodes for both stimulation and recording, giving the opportunity to perform standard short- and long-term plasticity protocols. We also show that HDMEA can be used in combination with fluorescence live imaging techniques such as Voltage Sensitive Dye recordings. The combination of complementary methodologies supports the HDMEA platform validation and paves the way to detailed electrophysiological studies

Electrophysiological imaging of epileptic brain slices reveals pharmacologically confined functional changes

Microelectrode arrays (MEAs) are employed to study extracellular electrical activity in neuronal tissues. Neverthe- less, commercially available MEAs provide a limited number of recording sites and do not allow a precise identifi- cation of the spatio-temporal characterization of the recorded signal. To overcome this limitation, high density MEAs, based on CMOS technology, were recently developed and validated on dissociated preparations (Ber- dondini et al. 2009). We show the platform capability to record extracellular electrophysiological signal from 4096 electrodes arranged in a squared area of 2.7 mm x 2.7 mm with inter-electrode distance of 21 μm at a sampling rate of 7.7 kHz/electrode. Here, we demonstrate the performances of these platforms for the acquisition chemi- cally evoked epileptiform activity from brain slices. Moreover the high spatial resolutions allow us to estimate the effect of drugs in spatially modulating Inter-Ictal ((I-IC) activity

The PRRT2 knockout mouse recapitulates the neurological diseases associated with PRRT2 mutations.

Heterozygous and rare homozygous mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia episodic ataxia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function. Recently, an important role for PRTT2 in the neurotransmitter release machinery, brain development and synapse formation has been uncovered. In this work, we have characterized the phenotype of a mouse in which the PRRT2 gene has been constitutively inactivated (PRRT2 KO)beta-galactosidase staining allowed to map the regional expression of PRRT2 that was more intense in the cerebellum, hindbrain and spinal cord, while it was localized to restricted areas in the fore-brain. PRRT2 KO mice are normal at birth, but display paroxysmal movements at the onset of locomotion that persist in the adulthood. In addition, adult PRRT2 KO mice present abnormal motor behaviors characterized by wild running and jumping in response to audiogenic stimuli that are ineffective in wild type mice and an increased sensitivity to the convulsive effects of pentylentetrazol. Patch-clamp electrophysiology in hippocampal and cerebellar slices revealed specific effects in the cerebellum, where PRRT2 is highly expressed, consisting in a higher excitatory strength at parallel fiber-Purkinje cell synapses during high frequency stimulation. The results show that the PRRT2 KO mouse reproduces the motor paroxysms present in the human PRRT2-linked pathology and can be proposed as an experimental model for the study of the pathogenesis of the disease as well as for testing personalized therapeutic approaches

Large-scale, high-resolution electrophysiological imaging of field potentials in brain slices with microelectronic multielectrode arrays

Multielectrode arrays (MEAs) are extensively used for electrophysiological studies on brain slices, but the spatial resolution and field of recording of conventional arrays are limited by the low number of electrodes available. Here, we present a large-scale array recording simultaneously from 4096 electrodes used to study propagating spontaneous and evoked network activity in acute murine cortico-hippocampal brain slices at unprecedented spatial and temporal resolution. We demonstrate that multiple chemically induced epileptiform episodes in the mouse cortex and hippocampus can be classified according to their spatio-temporal dynamics. Additionally, the large-scale and high-density features of our recording system enable the topological localization and quantification of the effects of antiepileptic drugs in local neuronal microcircuits, based on the distinct field potential propagation patterns. This novel high-resolution approach paves the way to detailed electrophysiological studies in brain circuits spanning spatial scales from single neurons up to the entire slice network

Late and Severe Myopathy in a Patient With Glycogenosis VII Worsened by Cyclosporine and Amiodarone

Glycogenosis VII (GSD VII) is a rare autosomal recessive glycogen storage disorder caused by mutations in the PFKM gene encoding the phosphofructokinase (PFK) enzyme. A classical form with exercise intolerance, contractures, and myoglobinuria, a severe multisystem infantile form, an hemolytic variant and a late-onset form usually presenting with muscle pain and mild fixed proximal weakness have been reported. We describe a 65-year-old man affected by muscle PFK deficiency who, since the age of 33, presented with exercise intolerance and myoglobinuria. Muscle biopsy showed a vacuolar myopathy with glycogen storage. The biochemical assay of PFK-M showed very low residual activity (6%). Genetic analysis of PFKM gene evidenced the presence of the heterozygote c.1817A>C (p.Asp543Ala) and c.488 G>A (p.Arg100Gln) pathogenic mutations. In his fifth decade, he started cyclosporine after liver transplantation for hepatocellular carcinoma and, then, amiodarone because of atrial fibrillation. In the following years, he developed a progressive and severe muscle weakness, mainly involving lower limbs, up to a loss of independent walking. Muscle MRI showed adipose substitution of both anterior and posterior thigh muscles with selective sparing of the medial compartment. Marked signs of adipose substitution were also documented in the legs with a selective replacement of gemelli and peroneus muscles. The temporal relationship between the patient's clinical worsening and chronic treatment with cyclosporine and amiodarone suggests an additive toxic damage by these two potentially myotoxic drugs determining such an unusually severe phenotype, also confirmed by muscle MRI findings

Impact of social determinants on antiretroviral therapy access and outcomes entering the era of universal treatment for people living with HIV in Italy

Background: Social determinants are known to be a driving force of health inequalities, even in high income countries. Aim of our study was to determine if these factors can limit antiretroviral therapy (ART) access, outcome and retention in care of people living with HIV (PLHIV) in Italy. Methods: All ART naïve HIV+ patients (pts) of Italian nationality enrolled in the ICONA Cohort from 2002 to 2016 were included. The association of socio-demographic characteristics (age, sex, risk factor for HIV infection, educational level, occupational status and residency area) with time to: ART initiation (from the first positive anti-HIV test), ART regimen discontinuation, and first HIV-RNA < 50 cp/mL, were evaluated by Cox regression analysis, Kaplan Meier method and log-rank test. Results: A total of 8023 HIV+ pts (82% males, median age at first pos anti-HIV test 36 years, IQR: 29-44) were included: 6214 (77.5%) started ART during the study period. Women, people who inject drugs (PWID) and residents in Southern Italy presented the lowest levels of education and the highest rate of unemployment compared to other groups. Females, pts aged > 50 yrs., unemployed vs employed, and people with lower educational levels presented the lowest CD4 count at ART initiation compared to other groups. The overall median time to ART initiation was 0.6 years (yrs) (IQR 0.1-3.7), with a significant decrease over time [2002-2006 = 3.3 yrs. (0.2-9.4); 2007-2011 = 1.0 yrs. (0.1-3.9); 2012-2016 = 0.2 yrs. (0.1-2.1), p < 0.001]. By multivariate analysis, females (p < 0.01) and PWID (p < 0.001), presented a longer time to ART initiation, while older people (p < 0.001), people with higher educational levels (p < 0.001), unemployed (p = 0.02) and students (p < 0.001) were more likely to initiate ART. Moreover, PWID, unemployed vs stable employed, and pts. with lower educational levels showed a lower 1-year probability of achieving HIV-RNA suppression, while females, older patients, men who have sex with men (MSM), unemployed had higher 1-year risk of first-line ART discontinuation. Conclusions: Despite median time to ART start decreased from 2002 to 2016, socio-demographic factors still contribute to disparities in ART initiation, outcome and durability

Economic consequences of investing in anti-HCV antiviral treatment from the Italian NHS perspective : a real-world-based analysis of PITER data

OBJECTIVE: We estimated the cost consequence of Italian National Health System (NHS) investment in direct-acting antiviral (DAA) therapy according to hepatitis C virus (HCV) treatment access policies in Italy. METHODS: A multistate, 20-year time horizon Markov model of HCV liver disease progression was developed. Fibrosis stage, age and genotype distributions were derived from the Italian Platform for the Study of Viral Hepatitis Therapies (PITER) cohort. The treatment efficacy, disease progression probabilities and direct costs in each health state were obtained from the literature. The break-even point in time (BPT) was defined as the period of time required for the cumulative costs saved to recover the Italian NHS investment in DAA treatment. Three different PITER enrolment periods, which covered the full DAA access evolution in Italy, were considered. RESULTS: The disease stages of 2657 patients who consecutively underwent DAA therapy from January 2015 to December 2017 at 30 PITER clinical centres were standardized for 1000 patients. The investment in DAAs was considered to equal €25 million, €15 million, and €9 million in 2015, 2016, and 2017, respectively. For patients treated in 2015, the BPT was not achieved, because of the disease severity of the treated patients and high DAA prices. For 2016 and 2017, the estimated BPTs were 6.6 and 6.2 years, respectively. The total cost savings after 20 years were €50.13 and €55.50 million for 1000 patients treated in 2016 and 2017, respectively. CONCLUSIONS: This study may be a useful tool for public decision makers to understand how HCV clinical and epidemiological profiles influence the economic burden of HCV