Valerio Rizzo

Hippocampal Hyperexcitability is Modulated by Microtubule-Active Agent: Evidence from In Vivo and In Vitro Epilepsy Models in the Rat

The involvement of microtubule dynamics on bioelectric activity of neurons and neurotransmission represents a fascinating target of research in the context of neural excitability. It has been reported that alteration of microtubule cytoskeleton can lead to profound modifications of neural functioning, with a putative impact on hyperexcitability phenomena. Altogether, in the present study we pointed at exploring the outcomes of modulating the degree of microtubule polymerization in two electrophysiological models of epileptiform activity in the rat hippocampus. To this aim, we used in vivo maximal dentate activation (MDA) and in vitro hippocampal epileptiform bursting activity (HEBA) paradigms to assess the effects of nocodazole (NOC) and paclitaxel (PAC), that respectively destabilize and stabilize microtubule structures. In particular, in the MDA paroxysmal discharge is electrically induced, whereas the HEBA is obtained by altering extracellular ionic concentrations. Our results provided evidence that NOC 10 μM was able to reduce the severity of MDA seizures, without inducing neurotoxicity as verified by the immunohistochemical assay. In some cases, paroxysmal discharge was completely blocked during the maximal effect of the drug. These data were also in agreement with the outcomes of in vitro HEBA, since NOC markedly decreased burst activity that was even silenced occasionally. In contrast, PAC at 10 μM did not exert a clear action in both paradigms. The present study, targeting cellular mechanisms not much considered so far, suggests the possibility that microtubule-active drugs could modulate brain hyperexcitability. This contributes to the hypothesis that cytoskeleton function may affect synaptic processes, relapsing on bioelectric aspects of epileptic activity.

Evidences of cannabinoids-induced modulation of paroxysmal events in an experimental model of partial epilepsy in the rat

The anticonvulsant effect of cannabinoids (CB) has been shown to be mediated by the activation of the CB(1) receptor. This study evaluates the anticonvulsant activity of (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-Yl]-1-naphthalenylmethanone (WIN55,212-2, CB agonist) alone or preceded by the administration of N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, selective CB(1) antagonist) in an experimental in vivo model of complex partial seizures (maximal dentate gyrus activation - MDA) in the rat. WIN55,212-2 (21mgkg(-1)) exerted an anticonvulsant effect, significantly reduced by the pre-treatment with AM251 (1mgkg(-1), 30 min interval). Surprisingly, AM251, administered alone at the same dose, failed to induce any modification in MDA responses. Our data suggest the involvement of the CB system in the inhibitory control of hyperexcitability phenomena in a model of acute partial epilepsy. Although the MDA model per se does not induce a basal activation of CB(1) receptors, as suggested by the lack of efficacy of AM251 when administered alone, the partial suppression of WIN55,212-2-induced effects in rats pre-treated with AM251 allows to hypothesise that the WIN55,212-2-induced antiepileptic effect is strictly linked to an increased CB(1) receptor activation or to the involvement of further receptor subtypes.

Role of CB2 receptors and cGMP pathway on the cannabinoid-dependent antiepileptic effects in an in vivo model of partial epilepsy

This study aimed at providing an insight on the possible role of cannabinoid (CB) type 2 receptors (CB2R) and cGMP pathway in the antiepileptic activity of WIN 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-Yl]-1-naphthalenylmethanone, a non-selective CB agonist, in the maximal dentate activation (MDA) model of partial epilepsy in adult male rats. We evaluated the activity of a CB2 antagonist/inverse agonist AM630, [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone or 6-iodopravadoline, alone or in co-administration with WIN 55,212-2. Also, in the MDA model it was investigated the co-treatment of WIN 55,212-2 and 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a specific inhibitor of the nitric oxide (NO)-activated soluble guanylyl cyclase (sGC), the cGMP producing enzyme. The WIN 55,212-2-dependent (21mg/kg) antiepileptic effects were significantly increased by the co-administration with AM630 and by the co-treatment with ODQ (10mg/kg). Whereas, the administration of AM630 (2mg/kg), alone exerts no effects on hippocampal hyperexcitability. Our data show that pharmacological blockade of CB2 receptors and of sGC seems to cooperate with WIN in its antiepileptic action. These findings shed light on CB signaling mechanisms, hinting that the modulation of the effects of CB agonist in the hyperexcitability phenomena may be exerted both by targeting CB receptors and their possible downstream effectors, such as nitrergic-dependent cGMP pathway.

Transcriptome analyses of adult mouse brain reveal enrichment of lncRNAs in specific brain regions and neuronal populations

Despite the importance of the long non-coding RNAs (lncRNAs) in regulating biological functions, the expression profiles of lncRNAs in the sub-regions of the mammalian brain and neuronal populations remain largely uncharacterized. By analyzing RNASeq datasets, we demonstrate region specific enrichment of populations of lncRNAs and mRNAs in the mouse hippocampus and pre-frontal cortex (PFC), the two major regions of the brain involved in memory storage and neuropsychiatric disorders. We identified 2759 lncRNAs and 17,859 mRNAs in the hippocampus and 2561 lncRNAs and 17,464 mRNAs expressed in the PFC. The lncRNAs identified correspond to ~14% of the transcriptome of the hippocampus and PFC and ~70% of the lncRNAs annotated in the mouse genome (NCBIM37) and are localized along the chromosomes as varying numbers of clusters. Importantly, we also found that a few of the tested lncRNA-mRNA pairs that share a genomic locus display specific co-expression in a region-specific manner. Furthermore, we find that sub-regions of the brain and specific neuronal populations have characteristic lncRNA expression signatures. These results reveal an unexpected complexity of the lncRNA expression in the mouse brain.

Effects of nitric oxide-active drugs on the discharge of subthalamic neurons : microiontophoretic evidence in the rat

The presence of nitric oxide (NO) synthase and of soluble guanylyl cyclase, the main NO‐activated metabolic pathway, has been demonstrated in many cells of the subthalamic nucleus. In this study, the effects induced on the firing of 96 subthalamic neurons by microiontophoretically administering drugs modifying NO neurotransmission were explored in anaesthetized rats. Recorded neurons were classified into regularly and irregularly discharging on the basis of their firing pattern. Nω‐nitro‐l‐arginine methyl ester (L‐NAME; a NO synthase inhibitor), 3‐morpholino‐sydnonimin‐hydrocloride (SIN‐1; a NO donor), S‐nitroso‐glutathione (SNOG; another NO donor) and 8‐Br‐cGMP (a cell‐permeable analogue of cGMP, the main second‐messenger of NO neurotransmission) were iontophoretically applied while performing single‐unit extracellular recordings. The activity of most neurons was influenced in a statistically significant way: in particular, both current‐related inhibitory L‐NAME‐induced effects (20/39 tested cells) and excitatory effects of SIN‐1 (25/41 tested neurons), SNOG (19/32 tested cells) and 8‐Br‐cGMP (13/19 tested neurons) were demonstrated. Neither statistically significant differences between the responses of regularly and irregularly discharging cells, nor specific topographical clustering of responding neurons, were demonstrated. Neurons administered drugs oppositely modulating the NO neurotransmission often displayed responses to only one treatment. We hypothesize that NO neurotransmission could exert a modulatory influence upon subthalamic neurons, with a prevalent excitatory effect. However, in the light of the presence of some responses of opposite sign to the same drug displayed by different subthalamic neurons, more complex effects of NO neurotransmission could be suggested, probably due to interactions with other classical neurotransmitter systems.

Pathologies of axonal transport in neurodegenerative diseases

Gene products such as organelles, proteins and RNAs are actively transported to synaptic terminals for the remodeling of pre-existing neuronal connections and formation of new ones. Proteins described as molecular motors mediate this transport and utilize specialized cytoskeletal proteins that function as molecular tracks for the motor based transport of cargos. Molecular motors such as kinesins and dynein’s move along microtubule tracks formed by tubulins whereas myosin motors utilize tracks formed by actin. Deficits in active transport of gene products have been implicated in a number of neurological disorders. We describe such disorders collectively as “transportopathies”. Here we review current knowledge of critical components of active transport and their relevance to neurodegenerative diseases.

In the rat maximal dentate activation model of partial complex epilepsy, the anticonvulsant activity of levetiracetam is modulated by nitric oxide-active drugs

The effects of nitric oxide-active drugs on the anticonvulsant action of the antiepileptic drug levetiracetam in an experimental model of partial complex seizures named maximal dentate gyrus activation were studied in rats. Levetiracetam was given alone or in combination with 7-nitroindazole, a preferential inhibitor of neuronal nitric oxide synthase, or with l-arginine, the precursor of nitric oxide synthesis. The maximal dentate activation parameters were the time of latency and the durations of maximal dentate activation and afterdischarge responses. The administration of levetiracetam showed an anticonvulsant effect that was increased when given in combination with 7-nitroindazole. The co-administration of levetiracetam and l-arginine, which is pro-convulsant, did not significantly modify all the parameters. The present results indicate that the acute administration of levetiracetam, at the lower effective dose, exerts an efficacious inhibitory effect on the severity of maximal dentate activation seizures. Levetiracetam-induced antiepileptic effect is significantly increased by the simultaneous inhibition of neuronal nitric oxide synthase.

Antiepileptic effect of dimethyl sulfoxide in a rat model of temporal lobe epilepsy

Dimethyl sulfoxide (DMSO) is an amphipathic molecule widely used to solubilize water-insoluble compounds. In many studies it was reported that DMSO is capable of affecting several biological processes, thus resulting in a potential cause for the misinterpretation of experimental data. Recent papers showed that DMSO modified the brain bioelectric activity in animal models of epilepsy. In an in vivo model of temporal lobe epilepsy in the rat, we examined the effects of different doses (10%, 50% and 100%) of DMSO on the maximal dentate activation (MDA). The results show that DMSO induced a dose-dependent significant reduction of the electrically induced paroxysmal activity.

Involvement of nitric oxide-soluble guanylyl cyclase pathway in the control of maximal dentate gyrus activation in the rat.

Summary.Nitric oxide/soluble Guanylyl cyclase (NO/sGC) pathway on the maximal dentate gyrus activation (MDA) was studied in rats. The cerebral NO levels were modified by administrating 7-Nitroindazole (7-NI), a selective inhibitor of neuronal NOS, and L-arginine, a precursor of the synthesis of NO. 1H-[1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ), a specific inhibitor of the NO-sGC pathway, was administered to study the involvement of cGMP pathway. The epileptic activity of the dentate gyrus was obtained through the repetitive stimulation of the angular bundle; MDA parameters studied were: onset time, MDA duration and post-stimulus afterdischarge (AD) duration. 7-NI caused an increase of MDA onset time and a decrease of MDA and AD duration. L-arginine, induced an aggravation of the epileptiform phenomena. ODQ induced modifications of MDA parameters as those caused by 7-NI. Our results indicate that the nitrergic neurotransmission exerts a modulatory role in the proneness to the epileptogenic phenomena through the activation of sGC metabolic pathway.

Cannabinoid and nitric oxide signaling interplay in the modulation of hippocampal hyperexcitability: Study on electrophysiological and behavioral models of temporal lobe epilepsy in the rat

A growing bulk of evidence suggests that cannabinoid system plays a pivotal role in the control of hyperexcitability phenomena. Notwithstanding, the anticonvulsant action of cannabinoids has not been fully addressed, in particular the involvement of potential cellular neuromodulators, for instance nitric oxide. In the current study, we focused on two distinct rat models of temporal lobe epilepsy, the Maximal Dentate Activation and the pilocarpine-induced acute seizures, providing both electrophysiological and behavioral data on cannabinoid and nitrergic system interplay. We evaluated the antiepileptic effects of WIN 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-Yl]-1-naphthalenylmethanone (WIN), a CB agonist, and of 7-Nitroindazole (7NI), a preferential neuronal nitric oxide synthase (nNOS) inhibitor, at different doses, alone and in combination. MDA study showed that these drugs protected animals in a dose-dependent manner from electrically induced epileptiform discharges. In pilocarpine model, a dose-related activity of 7NI and WIN: a) decreased the behavioral scoring, used to describe the severity of chemically induced acute seizures; b) affected latency of the onset of acute convulsions; c) dampened mortality rate. Interestingly, the combination of the treatments brought to light that individually ineffective doses of WIN turn into effective when nNOS activity is pharmacologically inhibited in both experimental conditions. This effect is mediated by CB1 receptor since the co-administration of N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), a CB1 receptor specific antagonist, thwarted the 7NI-WIN convergent action. In the light of this, our findings suggest a putative antagonism between CBr-activated pathway and NO signaling in the context of neuronal hyperexcitability and contribute to elucidate possible synaptic processes underlying neuroprotective properties of cannabinoids, with a view to better integrate antiepileptic therapy.