Application of near infra-red laser light increases current threshold in optic nerve consistent with increased Na+-dependent transport.

Increases in the current threshold occur in optic nerve axons with the application of infra-red laser light, whose mechanism is only partly understood. In isolated rat optic nerve, laser light was applied near the site of electrical stimulation, via a flexible fibre optic. Paired applications of light produced increases in threshold that were reduced on the second application, the response recovering with increasing delays, with a time constant of 24 s. 3-min duration single applications of laser light gave rise to a rapid increase in threshold followed by a fade, whose time-constant was between 40 and 50 s. After-effects were sometimes apparent following the light application, where the resting threshold was reduced. The increase in threshold was partially blocked by 38.6 mM Li+ in combination with 5  µ M bumetanide, a manoeuvre increasing refractoriness and consistent with axonal depolarization. Assessing the effect of laser light on the nerve input resistance ruled out a previously suggested fall in myelin resistance as contributing to threshold changes. These data appear consistent with an axonal membrane potential that partly relies on temperature-dependent electroneutral Na+ influx, and where fade in the response to the laser may be caused by a gradually diminishing Na+ pump-induced hyperpolarization, in response to falling intracellular [Na+].

Changing the firing threshold for normal optic nerve axons by the application of infra-red laser light.

Normal optic nerve axons exhibit a temperature dependence, previously explained by a membrane potential hyperpolarization on warming. We now report that near infra-red laser light, delivered via a fibre optic light guide, also affects axonal membrane potential and threshold, at least partly through a photo-thermal effect. Application of light to optic nerve, at the recording site, gave rise to a local membrane potential hyperpolarization over a period of about a minute, and increased the size of the depolarizing after potential. Application near the site of electrical stimulation reversibly raised current-threshold, and the change in threshold recorded over minutes of irradiation was significantly increased by the application of the Ih blocker, ZD7288 (50 µM), indicating Ih limits the hyperpolarizing effect of light. Light application also had fast effects on nerve behaviour, increasing threshold without appreciable delay (within seconds), probably by a mechanism independent of kinetically fast K+ channels and Na+ channel inactivation, and hypothesized to be caused by reversible changes in myelin function.

Painful and painless mutations of SCN9A and SCN11A voltage-gated sodium channels.

Chronic pain is a global problem affecting up to 20% of the world's population and has a significant economic, social and personal cost to society. Sensory neurons of the dorsal root ganglia (DRG) detect noxious stimuli and transmit this sensory information to regions of the central nervous system (CNS) where activity is perceived as pain. DRG neurons express multiple voltage-gated sodium channels that underlie their excitability. Research over the last 20 years has provided valuable insights into the critical roles that two channels, NaV1.7 and NaV1.9, play in pain signalling in man. Gain of function mutations in NaV1.7 cause painful conditions while loss of function mutations cause complete insensitivity to pain. Only gain of function mutations have been reported for NaV1.9. However, while most NaV1.9 mutations lead to painful conditions, a few are reported to cause insensitivity to pain. The critical roles these channels play in pain along with their low expression in the CNS and heart muscle suggest they are valid targets for novel analgesic drugs.

The effects of temperature on the biophysical properties of optic nerve F-fibres.

In multiple sclerosis, exacerbation of symptoms with rising body temperature is associated with impulse conduction failure. The mechanism is not fully understood. Remarkably, normal optic nerve axons also show temperature dependent effects, with a fall in excitability with warming. Here we show two properties of optic nerve axons, accommodation and inward rectification (Ih), respond to temperature changes in a manner consistent with a temperature dependent membrane potential. As we could find no evidence for the functional expression of KV7.2 in the axons, using the K+ channel blocker tetraethylammonium ions, we suggest this may explain the membrane potential lability. In order to understand how the axonal membrane potential may show temperature dependence, we have developed a hypothesis involving the electroneutral movement of Na+ ions across the axon membrane, that increases with increasing temperature with an appropriate Q10. Part, but probably not all, of the electroneutral Na+ movement is eliminated by removing extracellular Cl- or exposure to bumetanide, consistent with the involvement of the transporter NKCC1. Numerical simulation suggests a change in membrane potential of - 15-20 mV mimics altering temperature between room and physiological in the largest axons.

The dichotomous effects of caffeine on homologous recombination in mammalian cells.

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR) in mammalian cells. A 5 mM caffeine treatment caused the cell cycle to stall at G2/M and cells eventually underwent apoptosis. Caffeine exposure also induced a strong DDR along with subsequent activation of wildtype p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Additionally, caffeine treatment increased chromatin accessibility and elevated the efficiency of illegitimate recombination. Conversely, the increase in nascent DNA synthesis did not translate into a higher number of gene targeting events. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and for reasons discussed, stimulates early steps of HR, but not the formation of complete recombination products.

Lacosamide efficacy and tolerability in clinical practice - Post marketing analysis from a single dedicated epilepsy center.

OBJECTIVES: Post marketing analysis of anti-epileptic drug (AED) efficacy and tolerability is of great value to the clinician since it is more representative of clinical practice than clinical trial data. We analyzed our experience with lacosamide (LCM) in patients treated after marketing. PATIENTS AND METHODS: We identified all patients who were treated with LCM during the four year period after marketing, excluding patients who were in clinical trials. We recorded demographic data and analyzed efficacy and tolerability in patients who had at least one follow up visit or telephone call 3 months after the initiation of LCM. RESULTS: A total of 165 patients met our inclusion criteria. The mean age was 41 years. The majority of the cohort had focal epilepsy (146 patients) (88.4%). The mean duration of treatment was 31.2 months. Eighty one patients (49.1%) were continuing LCM at last follow up. Adverse effects (AEs) and discontinuation were significantly more common when LCM was added to one or more Na-channel blocking agents (NCB) (p = 0.0003 and 0.17). The 50% responder rate was 26% at 3 months and increased to 49% at 36 months. Patients were more likely to continue the drug and less likely to have AEs with slower titration over >4 weeks (p = 0.02 for each). Four or more previously failed AEDs predicted poorer response rate compared to three or less AEDs (p = 0.001). CONCLUSION: LCM use in clinical practice was associated with greater rate of seizure freedom than in clinical trials. Discontinuation and occurrence of AEs were significantly more likely with faster titration and adding LCM to NCB agents.

Serum Albumin's Protective Inhibition of Amyloid-ß Fiber Formation Is Suppressed by Cholesterol, Fatty Acids and Warfarin.

Central to Alzheimer's disease (AD) pathology is the assembly of monomeric amyloid-ß peptide (Aß) into oligomers and fibers. The most abundant protein in the blood plasma and cerebrospinal fluid is human serum albumin. Albumin can bind to Aß and is capable of inhibiting the fibrillization of Aß at physiological (µM) concentrations. The ability of albumin to bind Aß has recently been exploited in a phase II clinical trial, which showed a reduction in cognitive decline in AD patients undergoing albumin-plasma exchange. Here we explore the equilibrium between Aß monomer, oligomer and fiber in the presence of albumin. Using transmission electron microscopy and thioflavin-T fluorescent dye, we have shown that albumin traps Aß as oligomers, 9 nm in diameter. We show that albumin-trapped Aß oligomeric assemblies are not capable of forming ion channels, which suggests a mechanism by which albumin is protective in Aß-exposed neuronal cells. In vivo albumin binds a variety of endogenous and therapeutic exogenous hydrophobic molecules, including cholesterol, fatty acids and warfarin. We show that these molecules bind to albumin and suppress its ability to inhibit Aß fiber formation. The interplay between Aß, albumin and endogenous hydrophobic molecules impacts Aß assembly; thus, changes in cholesterol and fatty acid levels in vivo may impact Aß fibrillization, by altering the capacity of albumin to bind Aß. These observations are particularly intriguing given that high cholesterol or fatty acid diets are well-established risk factors for late-onset AD.

Big conductance calcium-activated potassium channel openers control spasticity without sedation.

BACKGROUND AND PURPOSE: Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti-metabolite approach to identify drugs that target spasticity. EXPERIMENTAL APPROACH: Following the initial chemistry, a variety of biochemical, pharmacological and electrophysiological approaches, using isolated cells, tissue-based assays and in vivo animal models, were used to demonstrate the activity, efficacy, pharmacokinetics and mechanism of action of VSN16R. Toxicological and safety studies were performed in animals and humans. KEY RESULTS: VSN16R had nanomolar activity in tissue-based, functional assays and dose-dependently inhibited spasticity in a mouse experimental encephalomyelitis model of MS. This effect occurred with over 1000-fold therapeutic window, without affecting normal muscle tone. Efficacy was achieved at plasma levels that are feasible and safe in humans. VSN16R did not bind to known CB1 /CB2 /GPPR55 cannabinoid-related receptors in receptor-based assays but acted on a vascular cannabinoid target. This was identified as the major neuronal form of the big conductance, calcium-activated potassium (BKCa ) channel. Drug-induced opening of neuronal BKCa channels induced membrane hyperpolarization, limiting excessive neural-excitability and controlling spasticity. CONCLUSIONS AND IMPLICATIONS: We identified the neuronal form of the BKCa channel as the target for VSN16R and demonstrated that its activation alleviates neuronal excitability and spasticity in an experimental model of MS, revealing a novel mechanism to control spasticity. VSN16R is a potential, safe and selective ligand for controlling neural hyper-excitability in spasticity.

Diuretic-sensitive electroneutral Na+ movement and temperature effects on central axons.

KEY POINTS: Optic nerve axons get less excitable with warming. F-fibre latency does not shorten at temperatures above 30°C. Action potential amplitude falls when the Na+ -pump is blocked, an effect speeded by warming. Diuretics reduce the rate of action potential fall in the presence of ouabain. Our data are consistent with electroneutral entry of Na+ occurring in axons and contributing to setting the resting potential. ABSTRACT: Raising the temperature of optic nerve from room temperature to near physiological has effects on the threshold, refractoriness and superexcitability of the shortest latency (fast, F) nerve fibres, consistent with hyperpolarization. The temperature dependence of peak impulse latency was weakened at temperatures above 30°C suggesting a temperature-sensitive process that slows impulse propagation. The amplitude of the supramaximal compound action potential gets larger on warming, whereas in the presence of bumetanide and amiloride (blockers of electroneutral Na+ movement), the action potential amplitude consistently falls. This suggests a warming-induced hyperpolarization that is reduced by blocking electroneutral Na+ movement. In the presence of ouabain, the action potential collapses. This collapse is speeded by warming, and exposure to bumetanide and amiloride slows the temperature-dependent amplitude decline, consistent with a warming-induced increase in electroneutral Na+ entry. Blocking electroneutral Na+ movement is predicted to be useful in the treatment of temperature-dependent symptoms under conditions with reduced safety factor (Uhthoff's phenomenon) and provide a route to neuroprotection.