Sodium-dependent Action Potentials Research Articles

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections.

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition.

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.

Voltage- and calcium-gated ion channels of neurons in the vertebrate retina.

In this review, we summarize studies investigating the types and distribution of voltage- and calcium-gated ion channels in the different classes of retinal neurons: rods, cones, horizontal cells, bipolar cells, amacrine cells, interplexiform cells, and ganglion cells. We discuss differences among cell subtypes within these major cell classes, as well as differences among species, and consider how different ion channels shape the responses of different neurons. For example, even though second-order bipolar and horizontal cells do not typically generate fast sodium-dependent action potentials, many of these cells nevertheless possess fast sodium currents that can enhance their kinetic response capabilities. Ca2+ channel activity can also shape response kinetics as well as regulating synaptic release. The L-type Ca2+ channel subtype, CaV1.4, expressed in photoreceptor cells exhibits specific properties matching the particular needs of these cells such as limited inactivation which allows sustained channel activity and maintained synaptic release in darkness. The particular properties of K+ and Cl- channels in different retinal neurons shape resting membrane potentials, response kinetics and spiking behavior. A remaining challenge is to characterize the specific distributions of ion channels in the more than 100 individual cell types that have been identified in the retina and to describe how these particular ion channels sculpt neuronal responses to assist in the processing of visual information by the retina.

Generation of Retinal Ganglion Cells With Functional Axons From Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells.

We previously generated self-induced retinal ganglion cells (RGCs) with functional axons from human induced pluripotent stem cells (hiPSCs). We investigated whether self-induced RGCs from mouse embryonic stem cells (mESCs) and induced pluripotent stem cells (miPSCs) are realized by the similar induction protocol. Retinal ganglion cells were induced using a protocol in which floating embryoid bodies (EBs) were differentiated into a retinal cell lineage in three-dimensional culture and subsequently attached to two-dimensional culture dishes with brain-derived neurotrophic factor (BDNF) supplementation. Retinal ganglion cells developed in an attached clump of cells originating from the optic vesicle, and most axons grew from RGC cell bodies at the margins of the clump. The differentiation of RGCs was confirmed by the expression of specific markers, including Brn3a and Math5. The axons contained neurofilament subtypes and tau, and manifested axonal transport and sodium-dependent action potentials. The RGCs derived from mESCs and miPSCs generally showed similar profiles, including RNA and protein expression levels and function. Retinal ganglion cells generated from mESCs and miPSCs, especially the latter, may contribute to research associated with RGCs and to in vitro analyses of genetically modified mice.

Generation of retinal ganglion cells with functional axons from human induced pluripotent stem cells.

We generated self-induced retinal ganglion cells (RGCs) with functional axons from human induced pluripotent stem cells. After development of the optic vesicle from the induced stem cell embryoid body in three-dimensional culture, conversion to two-dimensional culture, achieved by supplementation with BDNF, resulted in differentiation of RGCs at a rate of nearly 90% as indicated by a marginal subregion of an extruded clump of cells, suggesting the formation of an optic vesicle. Axons extended radially from the margin of the clump. Induced RGCs expressed specific markers, such as Brn3b and Math5, as assessed using by quantitative PCR and immunohistochemistry. The long, prominent axons contained neurofilaments and tau and exhibited anterograde axonal transport and sodium-dependent action potentials. The ability to generate RGCs with functional axons uniformly and at a high rate may contribute to both basic and clinical science, including embryology, neurology, pathognomy, and treatment of various optic nerve diseases that threaten vision.

Sequential acquisition of cacophony calcium currents, sodium channels and voltage‐dependent potassium currents affects spike shape and dendrite growth during postembryonic maturation of an identified Drosophila motoneuron

During metamorphosis the CNS undergoes profound changes to accommodate the switch from larval to adult behaviors. In Drosophila and other holometabolous insects, adult neurons differentiate either from respecified larval neurons, newly born neurons, or are born embryonically but remain developmentally arrested until differentiation during pupal life. This study addresses the latter in the identified Drosophila flight motoneuron 5. In situ patch-clamp recordings, intracellular dye fills and immunocytochemistry address the interplay between dendritic shape, excitability and ionic current development. During pupal life, changes in excitability and spike shape correspond to a stereotyped, progressive appearance of voltage-gated ion channels. High-voltage-activated calcium current is the first current to appear at pupal stage P4, prior to the onset of dendrite growth. This is followed by voltage-gated sodium as well as transient potassium channel expression, when first dendrites grow, and sodium-dependent action potentials can be evoked by somatic current injection. Sustained potassium current appears later than transient potassium current. During the early stages of rapid dendritic growth, sodium-dependent action potentials are broadened by a calcium component. Narrowing of spike shape coincides with sequential increases in transient and sustained potassium currents during stages when dendritic growth ceases. Targeted RNAi knockdown of pupal calcium current significantly reduces dendritic growth. These data indicate that the stereotyped sequential acquisition of different voltage-gated ion channels affects spike shape and excitability such that activity-dependent calcium influx serves as a partner of genetic programs during critical stages of motoneuron dendrite growth.

Rufinamide: treatment of seizures associated with Lennox-Gastaut syndrome

Rufinamide (RUF) is FDA-approved for adjunctive management of seizures related with Lennox–Gastaut syndrome (LGS). This new anti-epileptic drug (AED) adds to the AEDs previously used for LGS together with valproic acid, lamotrigine, felbamate, and topiramate. Its mechanism of action includes preventive the excessive firing of sodium-dependent action potentials, but RUF also exhibits a broad spectrum of action in animal models. The plasma concentration of other AEDs does not change by the RUF. Dizziness, nausea, diplopia, and ataxia vomiting and somnolence are most common adverse effects taking place with RUF. Status epilepticus has been reported, but were uncommon (0.9%). A recent randomized, double-blinded, placebo-controlled trial of RUF in patients with LGS and generalized seizures, including atypical absence and tonic-atonic seizures, showed a 32.7% median percentage decreased in total seizures and a 42.5% median percentage decreased in tonic-atonic seizures. RUF also considerably decreased seizure severity. RUF has been studied as adjunctive therapy for partial seizures in adults and adolescents. In a study of three healthy volunteers, an oral dose of 600 mg RUF recognized high absorption and monoexponential elimination with a mean half-life (t½) of 9 hrs. Excretion was mainly renal (85%) and complete (98%) within 7 days.

NT2 Derived Neuronal and Astrocytic Network Signalling

A major focus of stem cell research is the generation of neurons that may then be implanted to treat neurodegenerative diseases. However, a picture is emerging where astrocytes are partners to neurons in sustaining and modulating brain function. We therefore investigated the functional properties of NT2 derived astrocytes and neurons using electrophysiological and calcium imaging approaches. NT2 neurons (NT2Ns) expressed sodium dependent action potentials, as well as responses to depolarisation and the neurotransmitter glutamate. NT2Ns exhibited spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling. Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, NT2 astrocytes (NT2As) exhibited morphology and functional properties consistent with this glial cell type. NT2As responded to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. NT2As also generated propagating calcium waves that were gap junction and purinergic signalling dependent. Our results show that NT2 derived astrocytes exhibit appropriate functionality and that NT2N networks interact with NT2A networks in co-culture. These findings underline the utility of such cultures to investigate human brain cell type signalling under controlled conditions. Furthermore, since stem cell derived neuron function and survival is of great importance therapeutically, our findings suggest that the presence of complementary astrocytes may be valuable in supporting stem cell derived neuronal networks. Indeed, this also supports the intriguing possibility of selective therapeutic replacement of astrocytes in diseases where these cells are either lost or lose functionality.

Histamine Elevates Free Intracellular Calcium in Mouse Retinal Dopaminergic Cells via H1-Receptors

Previously, retinopetal axons containing histamine and dopaminergic neurons expressing histamine H(1)-receptor had been localized in mouse retinas using anatomic techniques. The goal of these experiments was to demonstrate that these receptors are functional. Dopaminergic cells were acutely isolated from retinas of transgenic mice expressing red fluorescent protein under control of the tyrosine hydroxylase promoter and loaded with the calcium indicator Fura-2. Under control conditions, there were spontaneous oscillations in the levels of free intracellular calcium in dopaminergic cells. These oscillations were abolished in nominally calcium-free extracellular medium and in 1 μM tetrodotoxin, findings suggesting that the oscillations were mediated by calcium entry across the plasma membrane in response to sodium-dependent action potentials. Histamine increased the mean free intracellular calcium in the dopaminergic cells by increasing the frequency and/or amplitude of the calcium oscillations. The effects of histamine were dose-dependent and reached maximum at 5 μM. With this dose, there was a 65% increase in the mean free intracellular calcium concentration. The histamine H(1)-receptor antagonist, pyrilamine, blocked the effects of 5 μM histamine when applied at 50 μM. The selective histamine H(1)-receptor agonists, 2-(3-trifluoromethylphenyl) histamine and methylhistaprodifen significantly increased mean free intracellular calcium when applied at 5 μM. Histamine released from retinopetal axons in the mouse retina can elevate intracellular calcium levels in the perikarya of dopaminergic cells via the activation of histamine H(1)-receptors.

Rufinamide for Pediatric Patients with Lennox-Gastaut Syndrome

Rufinamide is a triazole derivative with broad-spectrum antiepileptic effects that is unrelated to any antiepileptic drug currently on the market. The European Commission and the US FDA approved rufinamide in 2007 and 2008, respectively, for adjunctive treatment of seizures associated with Lennox-Gastaut syndrome in children 4 years of age or older and adults. The mechanism of action of rufinamide is not completely understood but it is believed to prolong the inactive state of sodium channels, therefore limiting excessive firing of sodium-dependent action potentials. Rufinamide is well absorbed when taken with food, with an absolute bioavailability between 70% and 85%. The elimination half-life of the drug is around 6-10 hours, with a time to maximum plasma concentration (C(max)) of approximately 4-6 hours. The C(max) at a dosage of 10 mg/kg/day and 30 mg/kg/day is 4.01 μg/mL and 8.68 μg/mL, respectively, and the area under the plasma concentration-time curve from time 0 to 12 hours was 37.8 ± 47 μg · h/mL and 89.3 ± 58 μg · h/mL, respectively. Rufinamide exerts non-linear pharmacokinetics with increasing doses. The volume of distribution in children is similar to that in adults (0.8-1.2 L/kg) and the drug binds rather poorly to plasma protein (26.2-34.8%). Rufinamide is mainly metabolized by carboxylesterases to an inactive metabolite (CGP 47292), and the majority of the metabolites are excreted in the urine (91%). No dosage adjustment is required in patients with renal dysfunction. Rufinamide does not affect the plasma concentration of other antiepileptics, but phenytoin, phenobarbital, valproate, and primidone affect the clearance of rufinamide. In a clinical study of 138 patients averaging 12 years of age, rufinamide used as an adjunctive therapy (with an initial dosage of 10 mg/kg/day up to a target dosage of 45 mg/kg/day) in patients with Lennox-Gastaut syndrome reduced the median total seizure frequency by 32.7% versus 11.7% in the placebo group (p = 0.0015). Similar reduction in total seizure frequency was maintained in the extension phase of this study. In other studies, rufinamide also seemed to provide improvement in both partial seizures and refractory epilepsy, but further studies need to validate this observation and to identify its clinical significance. Rufinamide is usually started orally at 10 mg/kg/day, titrating up by 10 mg/kg/day every 2 days to a target dosage of 45 mg/kg/day divided twice daily (maximum dosage of 3200 mg/day). Dosing of rufinamide has not been established in patients <4 years of age. Rufinamide is available as 100, 200, and 400 mg tablets in Europe, and 200 and 400 mg tablets in the US; a suspension of 40 mg/mL can be prepared extemporaneously. Rufinamide is well tolerated, with the most common adverse effects being dizziness, fatigue, nausea, vomiting, diplopia, and somnolence. From the current data, rufinamide serves as an adjunctive therapy in the management of Lennox-Gastaut syndrome. Further studies need to evaluate its efficacy as a first-line agent in the management of this neurologic disorder.

Epilepsy

Approximately 30% of patients with epilepsy are medically refractory despite multiple antiepileptic drug (AED) trials due to continued seizures or intolerable side effects. Novel AEDs and other therapies are needed that are more effective with improved tolerability. New treatments and new insights into management of comorbidities are discussed, with a focus on adult patients with epilepsy. The Food and Drug Administration (FDA) recently approved 3 new AEDs, and a fourth AED may be near approval. Lacosamide (LCS) is approved by the FDA for adjunctive treatment for partial-onset seizures. It has novel mechanisms of action, enhancing the slow inactivation of voltage-gated sodium channels and binding collapsing response mediator protein 2. LCS has a low potential for drug–drug interactions, is renally excreted, and is dosed twice daily. It does not affect plasma concentrations of concomitant AEDs. The most common adverse effects were dizziness, headache, diplopia, nausea, and vomiting, which were mild or moderate. Small increases in the PR interval were observed, but were not of clinical consequence. In a recent randomized, double-blinded, placebo-controlled trial of adjunctive treatment with LCS for medically refractory partial-onset seizures at doses of 400 to 600 mg daily, median reductions in seizure frequency were 37.3% for the 400 mg/day and 37.8% for the 600 mg/day groups. For secondarily generalized tonic-clonic seizures, median reductions in seizure frequency were more robust at 59.4% for the 400 mg/day group and 93.0% for the 600 mg/day groups. In addition, 8% of the patients in the 600 mg/day group became seizure-free.1 Rufinamide (RUF) is FDA-approved for adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS). This new AED adds to the AEDs already used for LGS including valproic acid, lamotrigine, felbamate, and topiramate. Its mechanism of action includes limiting the excessive firing of sodium-dependent action potentials, but RUF also exhibits a broad …

Lacosamide

The mechanism of action of several antiepileptic drugs (AEDs) rests on their ability to modulate the activity of voltage-gated sodium currents that are responsible for fast action potential generation. Recent data indicate that lacosamide (a compound with analgesic and anticonvulsant effects in animal models) shares a similar mechanism. When compared with other AEDs, lacosamide has the unique ability to interact with sodium channel slow inactivation without affecting fast inactivation. This article reviews these findings and discusses their relevance within the context of neuronal activity seen during epileptiform discharges generated by limbic neuronal networks in the presence of chemical convulsants. These seizure-like events are characterized by sustained discharges of sodium-dependent action potentials supported by robust depolarizations, thus providing synchronization within neuronal networks. Generally, AEDs such as phenytoin, carbamazepine and lamotrigine block sodium channels when activated. In contrast, lacosamide facilitates slow inactivation of sodium channels both in terms of kinetics and voltage dependency. This effect may be relatively selective for repeatedly depolarized neurons, such as those participating in seizure activity in which the persistence of sodium currents is more pronounced and promotes neuronal excitation. The clinical effectiveness of lacosamide has been demonstrated in randomized, double-blind, parallel-group, placebo-controlled, adjunctive-therapy trials in patients with refractory partial seizures. Further studies should determine whether the effects of lacosamide in animal models and in clinical settings are fully explained by its selective action on sodium current slow inactivation or whether other effects (e.g. interactions with the collapsin-response mediator protein-2) play a contributory role.

An Autonomous Circadian Clock in the Inner Mouse Retina Regulated by Dopamine and GABA

The influence of the mammalian retinal circadian clock on retinal physiology and function is widely recognized, yet the cellular elements and neural regulation of retinal circadian pacemaking remain unclear due to the challenge of long-term culture of adult mammalian retina and the lack of an ideal experimental measure of the retinal circadian clock. In the current study, we developed a protocol for long-term culture of intact mouse retinas, which allows retinal circadian rhythms to be monitored in real time as luminescence rhythms from a PERIOD2::LUCIFERASE (PER2::LUC) clock gene reporter. With this in vitro assay, we studied the characteristics and location within the retina of circadian PER2::LUC rhythms, the influence of major retinal neurotransmitters, and the resetting of the retinal circadian clock by light. Retinal PER2::LUC rhythms were routinely measured from whole-mount retinal explants for 10 d and for up to 30 d. Imaging of vertical retinal slices demonstrated that the rhythmic luminescence signals were concentrated in the inner nuclear layer. Interruption of cell communication via the major neurotransmitter systems of photoreceptors and ganglion cells (melatonin and glutamate) and the inner nuclear layer (dopamine, acetylcholine, GABA, glycine, and glutamate) did not disrupt generation of retinal circadian PER2::LUC rhythms, nor did interruption of intercellular communication through sodium-dependent action potentials or connexin 36 (cx36)-containing gap junctions, indicating that PER2::LUC rhythms generation in the inner nuclear layer is likely cell autonomous. However, dopamine, acting through D1 receptors, and GABA, acting through membrane hyperpolarization and casein kinase, set the phase and amplitude of retinal PER2::LUC rhythms, respectively. Light pulses reset the phase of the in vitro retinal oscillator and dopamine D1 receptor antagonists attenuated these phase shifts. Thus, dopamine and GABA act at the molecular level of PER proteins to play key roles in the organization of the retinal circadian clock.

Sodium-dependent action potentials induced by brevetoxin-3 trigger both IP 3 increase and intracellular Ca 2+ release in rat skeletal myotubes

Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca2+ levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca2+ signals evoked by PbTx-3 were inhibited by blocking either IP3 receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP3 mass levels, dependent on extra-cellular Na+. A similar increase in IP3 mass was induced by high K+ depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP3 mass induced by either PbTx-3 or K+ was also detected in Ca2+-free medium. These results establish that the effect of the toxin on both intracellular Ca2+ and IP3 levels occurs via a membrane potential sensor instead of directly by Na+ flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP3-dependent calcium signal involved in regulation of gene expression.

Circadian- and Light-Dependent Regulation of Resting Membrane Potential and Spontaneous Action Potential Firing of Drosophila Circadian Pacemaker Neurons

The ventral lateral neurons (LNvs) of adult Drosophila brain express oscillating clock proteins and regulate circadian behavior. Whole cell current-clamp recordings of large LNvs in freshly dissected Drosophila whole brain preparations reveal two spontaneous activity patterns that correlate with two underlying patterns of oscillating membrane potential: tonic and burst firing of sodium-dependent action potentials. Resting membrane potential and spontaneous action potential firing are rapidly and reversibly regulated by acute changes in light intensity. The LNv electrophysiological light response is attenuated, but not abolished, in cry(b) mutant flies hypomorphic for the cell-autonomous light-sensing protein CRYPTOCHROME. The electrical activity of the large LNv is circadian regulated, as shown by significantly higher resting membrane potential and frequency of spontaneous action potential firing rate and burst firing pattern during circadian subjective day relative to subjective night. The circadian regulation of membrane potential, spontaneous action potential firing frequency, and pattern of Drosophila large LNvs closely resemble mammalian circadian neuron electrical characteristics, suggesting a general evolutionary conservation of both physiological and molecular oscillator mechanisms in pacemaker neurons.

Rufinamide: a new anti-epileptic medication

Rufinamide (1-[2,6-difluorobenzyl]-1H-1,2,3-triazole-4-carboxamide) is a new anti-epileptic drug with a novel triazole derivative structure. The suspected mechanism of action is limitation of sodium-dependent action potentials, thought to result in a membrane stabilizing effect. Rufinamide is extensively metabolized in the liver by non-CYP450 enzymes with an elimination half-life of 8 – 12 h. Three randomized, placebo-controlled trials have shown that rufinamide is effective against partial seizures in adults. Efficacy in the Lennox-Gastaut syndrome, a severe, disabling childhood onset epilepsy syndrome, was shown in a single, randomized, placebo-controlled trial. It has recently been approved for treatment of Lennox-Gastaut syndrome in Europe. In the US it is under regulatory review. Most common adverse effects are somnolence, fatigue, dizziness, dipolopia, nausea and ataxia. Rufinamide has shown promise as adjunctive treatment for Lennox-Gastaut syndrome and may have some role in localization related epilepsies as well.

Rufinamide: pharmacology, clinical trials, and role in clinical practice

Rufinamide is a structurally novel compound with anticonvulsant activity that is undergoing evaluation through the European Medicines Agency and the American Food and Drug Administration. Its mechanism of action is thought to be inhibition of sodium-dependent action potentials in neurons, with possible membrane-stabilising effects. Absorption of the drug is significantly enhanced in the fed state. The drug is extensively metabolised by non-CYP450 systems with a half-life of 8-12 h. Most common adverse effects noted are somnolence, fatigue and tremor. Efficacy against partial seizures in adults and adolescents has been demonstrated in three randomised, placebo-controlled trials. Efficacy against seizures of Lennox-Gastaut syndrome, a severe, disabling childhood onset epilepsy syndrome, was shown in a single randomised, placebo-controlled trial. Efficacy against partial onset seizures in children has been suggested in an open-label trial. Should rufinamide become commercially available, reserving the drug as a second- or third-line agent should be considered.

Development of Functional Synaptic Connections in the Auditory System Visualized With Optical Recording: Afferent-Evoked Activity Is Present From Early Stages

A comprehensive survey of auditory network formation was performed in the brain stem of the chicken embryo using voltage-sensitive dye recording. Intact medulla/brain stem preparations with the auditory branch of the eighth nerve attached were dissected from 5.5- to 8-day chicken embryos, and responses evoked by nerve stimulation were recorded optically. In the medulla of 7- and 8-day embryos, we identified four response areas, corresponding to ipsilateral Nucleus magnocellularis (NM) and Nucleus angularis (NA), which receive the auditory afferents, and ipsi- and contralateral Nucleus laminaris (NL), which receive projections from NM. The optical responses consisted of a fast spikelike signal followed by a long-lasting slow signal, which reflected the sodium-dependent action potential and glutamatergic excitatory postsynaptic potential (EPSP), respectively. In NM, NA, and NL, the EPSP-related slow optical signals were detected from some 6-day and all 7- and 8-day preparations, indicating that functional synaptic connectivity in these nuclei arises by the 7-day stage. In the pons of 7- and 8-day embryos, we identified two additional response areas, which evidently correspond to ipsi- and contralateral Nucleus lemnisci lateralis (NLL), the higher-order nuclei of the auditory pathway. Furthermore, we detected optical responses from the contralateral cerebellum, which possibly correspond to transient projections observed only during embryogenesis. The present study demonstrates that functional auditory circuits are established in the chicken embryo at stages earlier than previously reported. We discuss the possible role of afferent-evoked activity with reference to auditory neural network formation.

Rufinamide

Rufinamide [E 2080, CGP 33101, RUF 331, Inovelon, Xilep] is the lead compound in a series of five-substituted phenylalkyl-3- carbamoyl-4H-1,2,4-triazoles with anticonvulsant activity that was discovered by Novartis AG. Although the precise mechanism of action for rufinamide is unknown, it is known that it limits the firing of sodium-dependent action potentials in neurons, hinting at a membrane-stabilising effect. Rufinamide is under regulatory review in Europe for epilepsy, and phase III trials in the same indication are underway with Eisai in the US. In February 2004, Eisai Co. Ltd announced that it had signed an in-licensing agreement with Novartis Pharma AG for the exclusive worldwide rights to manufacture, develop and market rufinamide for the treatment of epilepsy. Eisai submitted an MAA for rufinamide (Inovelon) as an adjunct therapy of seizures associated with Lennox-Gastaut Syndrome to the European Medicines Agency through the centralised procedure in March 2005. The drug also received orphan drug designation from the European commission in October 2004 for this indication. Eisai also plans to file for regulatory approval in the US. Rufinamide has demonstrated statistically significant efficacy as an adjunctive therapy in adult patients with epilepsy and in patients with seizures associated with Lennox-Gastaut syndrome. Novartis stated in June 2001, prior to the licensing agreement with Eisai, that it had stopped development of rufinamide in the US, Japan and Europe for neuropathic pain (at phase II) and epilepsy (at phase III).

Neuropeptide y and n-methyl--aspartic acid interact within the suprachiasmatic nuclei to alter circadian phase

Circadian rhythms are reset by exposure to photic stimuli and nonphotic stimuli. Glutamate appears to be the primary neurotransmitter that communicates photic stimuli to the circadian clock located in the suprachiasmatic nucleus. There is also substantial evidence that neuropeptide Y (NPY) mediates the effects of at least some nonphotic stimuli on the circadian clock. The purpose of this study was to investigate how NPY and glutamate receptor activation interact to reset the phase of the circadian clock. Microinjection of the glutamate agonist N -methyl- d -aspartic acid (NMDA) during the subjective day significantly decreased NPY-induced phase advances. During the late subjective night, NMDA induced light-like phase advances, which were significantly reduced by microinjection of NPY. Microinjection of NPY inhibited NMDA-induced phase advances during the late subjective night, even when sodium-dependent action potentials were inhibited by tetrodotoxin. These data support the hypothesis that, during the subjective night, NPY and NMDA act on the same clock cells or on cells that communicate with clock cells by mechanisms not requiring action potentials. Although NPY and NMDA appear to be mutually inhibitory during both the day and the night, the mechanisms of this inhibition appear to be different during the day versus the night.