Anticonvulsant effect of equilibrative nucleoside transporters 1 inhibitor in a mouse model of Dravet syndrome.

There are limited therapeutic options for patients with Dravet syndrome (DS). The equilibrative nucleoside transporters 1 (ENT1) mediate both the influx and efflux of adenosine across the cell membrane exerted beneficial effects in the treatment of epilepsy. This study aimed to evaluate the anticonvulsant effect of the ENT1 inhibitor in an animal model of DS (Scn1aE1099X/+ mice). J7 (5 mg/kg) treatment was efficacious in elevating seizure threshold in Scn1aE1099X/+ mice after hyperthermia exposure. Moreover, the J7 treatment significantly reduced the frequency of spontaneous excitatory post-synaptic currents (sEPSCs, ~35% reduction) without affecting the amplitude in dentate gyrus (DG) granule cells. Pretreatment with the adenosine A1 receptor (A1R) antagonist, DPCPX, abolished the J7 effects on sEPSCs. These observations suggest that the J7 shows an anticonvulsant effect in hyperthermia-induced seizures in Scn1aE1099X/+ mice. This effect possibly acts on presynaptic A1R-mediated signaling modulation in granule cells.

Perampanel Reduces Hyperthermia-Induced Seizures in Dravet Syndrome Mouse Model.

Treatment options for Dravet syndrome are limited. The aim of this study was to evaluate the antiepileptic effect of the AMPA receptor antagonist perampanel (PER) on a mouse model of Dravet syndrome (Scn1a E1099X/+ ). We report here that the PER (2 mg/kg) treatment inhibited the spontaneous recurrent seizures and attenuated epileptic activity in Scn1a E1099X/+ mice. In the hyperthermia-induced seizure experiment, PER clearly increased temperature tolerance and significantly ameliorated seizure frequency and discharge duration. PER also demonstrated antiepileptic effects in a cross-over study and a synergistic effect for attenuating heat-induced seizure when given in combination with stiripentol or valproic acid. The results showed that PER effectively decreased the occurrence of spontaneous recurrent seizures and showed significant therapeutic potential for hyperthermia-induced seizures with regard to both susceptibility and severity in a Dravet syndrome mouse model. Potential therapeutic effects of PER for treatment of Dravet syndrome were demonstrated.

Equilibrative Nucleoside Transporters-1 Inhibitors Act as Anti-epileptic Agents by Inhibiting Glutamatergic Transmission.

Background and Purpose: Adenosine dysregulation is associated with the occurrence of the epilepsy and equilibrative nucleoside transporters-1 (ENT-1) functions as an important regulator of extracellular adenosine in the brain. This study was aimed to prove the anti-epileptic effect of BBB permeable ENT-1 inhibitors, JMF1907 and J4, on animal models of various epilepsy, and the mechanisms that are involved. Experimental Approach: Maximal electroshock seizure (MES), pentylenetetrazol (PTZ)-induced seizure and kindling models were used as mouse models of generalized tonic-clonic epilepsy, generalized myoclonic epilepsy, and partial epilepsy, respectively. The epilepsy frequency, duration, and Racine score were evaluated. Whole-cell recordings were made from the hippocampal dentate granule cells by using a patch-clamp technique in the brain slice of the mice. Key Results: In MES, JMF1907 at a dose of 5 mg kg-1 was efficacious in decreasing hindlimb extension, while J4 did not decrease hindlimb extension until a higher dose (10 mg kg-1). Both JMF1907 and J4 were more potent in lengthening onset latency than ethosuximide (ETH) in PTZ-induced myoclonic epilepsy model, whereas ETH had better effects on the Racine score. In kindling model, JMF1907 and J4 at a dose of 1 mg kg-1 had effects on seizure frequency and duration, and the effects of JMF1907 were comparable with those of carbamazepine. Both JMF1907 and J4 can reduce the glutamatergic spontaneous excitatory post-synaptic currents (sEPSCs) frequency. The maximal inhibition was about 50% for JMF1907 at a concentration of 1 µg L-1, whereas J4 only inhibited 40% of sEPSCs frequency at a dose of 10 µg L-1. Conclusion and Implications: ENT-1 inhibitors, JMF1907 and J4, showed anti-epileptic effects in different epilepsy models and the effects involved pre-synaptic neuronal modulation.

Static Magnetic Field Induced Neural Stem/Progenitor Cell Early Differentiation and Promotes Maturation.

The potential impacts of magnetic field exposures on brain development have raised public concern. In the present study, we aimed to investigate the biophysical effects of moderate-intensity (0.5 T, Tesla) static magnetic field (SMF) on mice neural progenitor cells (mNPCs). Our results showed that the SMF exposure increased the number of neurosphere formation and enhanced proliferative activity in mNPCs. In addition, our flow cytometry data demonstrated that the proportions of S phase and G2/M phase mNPCs were remarkably increased following 5 days of SMF exposure. Moreover, the level of a mitotic regulatory protein, cyclin B, was upregulated after SMF exposure. Furthermore, the mNPCs exposed to SMF exhibited a significant increase in Sox2 expression. When mNPCs were induced to differentiation, our immunofluorescence assay revealed that the percentage of neurons (Tuj-1-positive cells) but not astrocyte (s100ß-positive cells) was significantly higher and displayed morphological complexity in the SMF group. Finally, our electrophysiological results demonstrated the mNPC-derived neurons from the SMF group showing a significantly increased in input resistance, which indicated more functional maturation. Based on these findings, it appears reasonable to suggest that SMF exposure could affect normal neurogenesis and promote neural lineage differentiation as well as neuronal maturation.

Electrical Abnormalities in Dopaminergic Neurons of the Substantia Nigra in Mice With an Aromatic L-Amino Acid Decarboxylase Deficiency.

Aromatic L-acid decarboxylase (AADC) deficiency causes severe motor disturbances in affected children. A putamen-targeted gene therapy improves the motor function of patients. The present study investigated the electrical properties of dopaminergic (DA) neurons in the substantia nigra compacta (SNc) of mice with an AADC deficiency (DdcKI). The basal firing of DA neurons, which determines DA release in the putamen, was abnormal in the DdcKI mice, including a low frequency and irregular firing pattern, because of a decrease in the after-hyperpolarization (AHP) amplitude of action potentials (APs). The frequency of spontaneous excitatory postsynaptic currents (sEPSCs) increased and that of spontaneous inhibitory PSCs (sIPSCs) decreased in the SNc DA neurons from the DdcKI mice, suggesting an elevation in glutamatergic excitatory stimuli and a reduction in GABAergic inhibitory stimuli, respectively. Altered expression patterns of genes encoding receptors and channels were also observed in the DdcKI mice. Administration of a widespread neuron-specific gene therapy to the brains of the DdcKI mice partially corrected these electric abnormalities. The overexcitability of SNc DA neurons in the presence of generalized dopamine deficiency likely underlies the occurrence of motor disturbances.

SDF-1/CXCR4 Signaling Maintains Stemness Signature in Mouse Neural Stem/Progenitor Cells.

SDF-1 and its primary receptor, CXCR4, are highly expressed in the embryonic central nervous system (CNS) and play a crucial role in brain architecture. Loss of SDF-1/CXCR4 signaling causes abnormal development of neural stem/progenitor cells (NSCs/NPCs) in the cerebellum, hippocampus, and cortex. However, the mechanism of SDF-1/CXCR4 axis in NSCs/NPCs regulation remains unknown. In this study, we found that elimination of SDF-1/CXCR4 transduction caused NSCs/NPCs to lose their stemness characteristics and to encounter neurogenic differentiation. Moreover, Notch and RE1 silencing transcription factor (REST) both play an essential role in NSCs/NPCs maintenance and neuronal differentiation and were dramatically downregulated following SDF-1/CXCR4 cascade inhibition. Finally, we demonstrated that the expression of achaete-scute homolog 1 (Ascl1), a proneural gene, and p27, an antiproliferative gene, were significantly increased after genetic elimination of SDF-1 alleles. Our results support that the loss of functional SDF-1/CXCR4 signaling pathway in NSCs/NPCs induces exit of cell cycle and promotes premature neural differentiation.

Autism-associated gene Dlgap2 mutant mice demonstrate exacerbated aggressive behaviors and orbitofrontal cortex deficits.

BACKGROUND: As elegant structures designed for neural communication, synapses are the building bricks of our mental functions. Recently, many studies have pointed out that synaptic protein-associated mutations may lead to dysfunctions of social cognition. Dlgap2, which encodes one of the main components of scaffold proteins in postsynaptic density (PSD), has been addressed as a candidate gene in autism spectrum disorders. To elucidate the disturbance of synaptic balance arising from Dlgap2 loss-of-function in vivo, we thus generated Dlgap2 (-/-) mice to investigate their phenotypes of synaptic function and social behaviors. METHODS: The creation of Dlgap2 (-/-) mice was facilitated by the recombineering-based method, Cre-loxP system and serial backcross. Reversal learning in a water T-maze was used to determine repetitive behaviors. The three-chamber approach task, resident-intruder test and tube task were performed to characterize the social behaviors of mutant mice. Cortical synaptosomal fraction, Golgi-Cox staining, whole-cell patch electrophysiology and transmission electron microscopy were all applied to investigate the function and structure of synapses in the orbitofrontal cortex (OFC) of Dlgap2 (-/-) mice. RESULTS: Dlgap2 (-/-) mice displayed exacerbated aggressive behaviors in the resident-intruder task, and elevated social dominance in the tube test. In addition, Dlgap2 (-/-) mice exhibited a clear reduction of receptors and scaffold proteins in cortical synapses. Dlgap2 (-/-) mice also demonstrated lower spine density, decreased peak amplitude of miniature excitatory postsynaptic current and ultra-structural deficits of PSD in the OFC. CONCLUSIONS: Our findings clearly demonstrate that Dlgap2 plays a vital role in social behaviors and proper synaptic functions of the OFC. Moreover, these results may provide valuable insights into the neuropathology of autism.

Protein kinase mζ is necessary for cocaine-induced synaptic potentiation in the ventral tegmental area.

BACKGROUND: Much progress has been made in our understanding of brain regions and specific receptors that are involved in the action of cocaine addiction. Although long-term modifications of mesolimbic reward circuit following cocaine exposure are responsible for cocaine-addicted behaviors, the underlying molecular mechanism at the cellular level is still obscure. Here, we investigated the possible participation of protein kinase Mζ (PKMζ) in synaptic potentiation following cocaine exposure. METHODS: Spontaneous and evoked synaptic activity of glutamate synapse in saline- and cocaine-treated rats were examined by preparing acute brain slices and performing whole-cell voltage-clamp recordings from individual dopamine neurons in the ventral tegmental area (VTA). We also assessed the role of PKMζ on the behavioral responses by cocaine conditioned place preference. RESULTS: Chelerythrine, an inhibitor of PKMζ, reversed the cocaine-induced facilitation of spontaneous synaptic transmission in the VTA. PKMζ inhibition by chelerythrine or myristoylated ζ inhibitory peptide significantly attenuated the cocaine exposure-induced enhancement of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor/N-methyl-D-aspartate receptor ratio. Myristoylated ζ inhibitory peptide had no effect on spike timing-dependent long-term potentiation in rats previously injected with saline but remarkably restored spike timing-dependent long-term potentiation in VTA dopamine neurons in slices prepared from rats that received single or multiple cocaine exposure. Western blot analyses showed that both single and five consecutive cocaine injections induced a significant increase in PKMζ level. Furthermore, intracranial infusion of myristoylated ζ inhibitory peptide in the VTA disrupted cocaine conditioned place preference. CONCLUSIONS: Our results suggest that persistent activity of PKMζ is a requisite for cocaine-induced enhancement of synaptic plasticity in the VTA and cocaine conditioned place preference.

Mechanism of beta-bungarotoxin in facilitating spontaneous transmitter release at neuromuscular synapse.

The mechanism of the action of beta-bungarotoxin (beta-BuTx) in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture using whole-cell patch clamp recording. Exposure of the culture to beta-BuTx dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by beta-BuTx. The beta-BuTx-enhanced SSC frequency was reduced when the pharmacological Ca2+ -ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ store. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by beta-BuTx. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished the beta-BuTx-induced facilitation of synaptic transmission. The beta-BuTx-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDPbetaS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that beta-BuTx elicits Ca2+ release from the IP3 sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PI3K/PLC signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.

A rapid, nongenomic pathway facilitates the synaptic transmission induced by retinoic acid at the developing synapse.

We have previously shown that retinoic acid (RA), a factor highly expressed in spinal cord, rapidly and specifically enhances the spontaneous acetylcholine release at developing neuromuscular synapses in Xenopus cell culture, using whole-cell patch-clamp recording. We have now further investigated the underlying mechanisms that are involved in RA-induced facilitation on the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by RA. The prompt RA-enhanced SSC frequency was not abolished when Ca2+ was eliminated from the culture medium or there was bath application of the pharmacological Ca2+ channel inhibitor Cd2+, indicating that Ca2+ influx through voltage-activated Ca2+ channels are not required. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or ryanodine receptors effectively blocked the increase of SSC frequency elicited by RA. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) and with the phospholipase Cgamma (PLCgamma) inhibitor U73122, abolished RA-induced facilitation of synaptic transmission. Preincubation of the cultures with pharmacological inhibitors, either genistein, a broad-spectrum tyrosine kinase inhibitor, or PP2, which predominantly inhibits the Src family of nonreceptor tyrosine kinase, completely abolished RA-induced synaptic facilitation. Taken collectively, these results suggest that RA elicits Ca2+ release from Ins1,4,5P3 and/or ryanodine-sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PLCgamma/PI 3-kinase signaling cascades and Src tyrosine kinase activation, leading to an enhancement of spontaneous transmitter release.

Non-genomic regulation of transmitter release by retinoic acid at developing motoneurons in Xenopus cell culture.

Although the long-term effects of all-trans retinoic acid (RA) on neuronal growth and differentiation have been intensively studied, nothing is known about its effect on synaptic transmission. Here we show that RA rapidly and specifically enhances the spontaneous acetylcholine release at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch-clamp recording. Acute addition of RA dose-dependently and reversibly enhances the frequency of spontaneous synaptic currents (SSCs). Application of the lipophilic RA analogue all-trans retinol or RA metabolites produced by light-induced decomposition failed to provoke similar changes in SSC frequency, indicating the specificity of RA-induced facilitation of spontaneous transmitter release. Protein synthesis inhibitors anisomycin or cycloheximide had no effect on RA-induced SSC frequency facilitation. Treating cells with pan RA receptor (RAR) selective agonist or RARbeta-selective agonist, but not RARalpha-, RARgamma- or retinoid X receptor (RXR)-selective agonists, mimicked the action of RA. These results suggest that RA acts through the activation of RARbeta, to induce a rapid, non-genomic increase in the frequency of spontaneous transmitter release at developing neuromuscular synapses.

Potentiation of quantal secretion by insulin-like growth factor-1 at developing motoneurons in Xenopus cell culture.

Although evidence suggests that insulin-like growth factor (IGF) plays an important role in the development and growth of the nervous system, the effect of IGF-1 in the regulation of neurotransmitter release in the peripheral nervous system remains unknown. Here we show that acute application of IGF-1, a factor widely expressed in developing myocytes, dose-dependently enhances the spontaneous acetylcholine (ACh) secretion at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch clamp recording. We studied the role of endogenously released IGF-1 by examining the effect of IGF-1 antibody on the frequency of spontaneous synaptic currents (SSCs) at high-activity synapses, and found SSC frequency was markedly reduced at these high-activity synapses. The IGF-1-induced synaptic potentiation was not abolished when Ca2+ was eliminated from the culture medium or there was bath-application of the pharmacological Ca2+ channel inhibitor Cd2+, indicating that Ca2+ influxes through voltage-activated Ca2+ channels are not required. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) or ryanodine receptors effectively occluded the increase of SSC frequency elicited by IGF-I. Treating cells with the phosphoinositide-3 kinase (PI3-K) inhibitors wortmannin or LY294002, and with phospholipase Cgamma (PLCgamma) inhibitor U73122, but not the inhibitor of mitogen-activated protein (MAP) kinase PD98059, abolished IGF-1-induced synaptic potentiation. Taken collectively, these results suggest that endogenously released IGF-1 from myocytes elicits Ca2+ release from IP3- and/or ryanodine-sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PI3-K and PLCgamma signalling cascades, leading to an enhancement of spontaneous transmitter release.