Our findings provide optimized conditions for ex vivo ERGs from the murine retina and suggest that careful application of Ba supports reliable isolation of b-wave responses in mice. Under our recording conditions, murine retinas show reproducible ERGs for up to six hours.

SUMMARYPurpose: Lamotrigine (LTG) is a popular modern antiepileptic drug (AED); however, its mechanism of action has yet to be fully understood, as it is known to modulate many members of several ion channel families. In heterologous systems, LTG inhibits Ca v 2.3 (R-type) calcium currents, which contribute to kainic-acid (KA)-induced epilepsy in vivo. To gain insight into the role of R-type currents in LTG drug action in vivo, we compared the effects of LTG to two other AEDs in Ca v 2.3-deficient mice and controls on KA-induced seizures. Methods: Behavioral seizure rating and quantitative electrocorticography were performed after injection of 20 mg/kg (and 30 mg/kg) KA. One hour before KA injection, mice were pretreated with 30 mg/kg LTG, 50 mg/kg topiramate (TPM), or 30 mg/kg lacosamide (LSM). Key Findings: Ablation of Ca v 2.3 reduced total seizure scores by 28.6% (p = 0.0012), and pretreatment with LTG reduced seizure activity of control mice by 23.2% (p = 0.02). In Ca v 2.3-deficient mice, LTG pretreatment increased seizure activity by 22.1% (p = 0.018) and increased the percentage of degenerated CA1 pyramidal neurons (p = 0.02). All three AEDs reduced seizure activity in control mice; however, only the non-calcium channel modulating AED, LSM, had an anticonvulsive effect in Ca v 2.3-deficient mice. Furthermore, LTG altered electrocorticographic parameters differently in the two genotypes: decreasing relative power of ictal spikes in control mice but increasing relative power of high frequency fast ripple discharges during seizures in Ca v 2.3-deficient mice. Significance: These findings provided the first in vivo evidence for an essential role for Ca v 2.3 in LTG pharmacology and shed light on a paradoxical effect of LTG in their absence. Furthermore, LTG appears to promote ictal activity in Ca v 2.3-deficient mice by increasing high frequency components of seizures, resulting in increased neurotoxicity in the CA1. This paradoxical mechanism, possibly reflecting rebound hyperexcitation of pyramidal CA1 neurons after increased inhibition, may be key in understanding LTG-induced seizure aggravation observed in clinical practice.

Background/Aims: Lamotrigine (LTG) is a popular modern antiepileptic drug (AED), however, its mechanism of action has yet to be fully understood, as it is known to modulate many members of several ion channel families. In heterologous systems, LTG inhibits Ca_v2.3 (R-type) calcium currents, which contribute to kainic-acid- (KA) induced epilepsy in vivo. To gain insight into the role of R-type currents in LTG drug action in vivo, we compared the effects of LTG to topiramate and lacosamide in Ca_v2.3-deficient mice and controls on KA-induced seizures. Methods: Behavioral seizure rating and quantitative electrocorticography were performed after injection of 20 mg/kg [and 30 mg/kg] KA. One hour before KA injection, mice were pretreated with either 30 mg/kg LTG, 50 mg/kg topiramate (TPM) or 30 mg/kg lacosamide (LSM). Results: Ablation of Ca_v2.3 reduced total seizure scores by 28.6% (p=0.0012) and pretreatment with LTG reduced seizure activity of control mice by 23.2% (p=0.02). In Ca_v2.3-deficient mice LTG pretreatment increased seizure activity by 22.1% (p=0.018) and increased the percentage of degenerated CA1 pyramidal neurons (p=0.02). All three tested AEDs reduced seizure activity in control mice, however only the non-calcium channel modulating AED, LSM had an anticonvulsive effect in Ca_v2.3-deficient mice. Furthermore LTG altered electrocorticographic parameters differently in the two genotypes, decreasing relative power of ictal spikes in control mice compared to Ca_v2.3-defcient mice. Conclusion: These findings give first in vivo evidence for an essential role for Ca_v2.3 in LTG pharmacology and shed light on a paradoxical effect of LTG in their absence. Furthermore, LTG appears to promote ictal activity in Ca_v2.3-deficient mice resulting in increased neurotoxicity in the CA1 region. This paradoxical mechanism, possibly reflecting rebound hyperexcitation of pyramidal CA1 neurons after increased inhibition, may be key in understanding LTG-induced seizure aggravation, observed in clinical practice.

BackgroundImpairment of neurovascular coupling (NVC) was recently reported in the context of subarachnoid hemorrhage and may correlate with disease severity and outcome. However, previous techniques to evaluate NVC required invasive procedures. Retinal vessels may represent an alternative option for non-invasive assessment of NVC.MethodsA prototype of an adapted retinal vessel analyzer was used to assess retinal vessel diameter in mice. Dynamic vessel analysis (DVA) included an application of monochromatic flicker light impulses in predefined frequencies for evaluating NVC. All retinae were harvested after DVA and electroretinograms were performed.ResultsA total of 104 retinal scans were conducted in 21 male mice (90 scans). Quantitative arterial recordings were feasible only in a minority of animals, showing an emphasized reaction to flicker light impulses (8 mice; 14 scans). A characteristic venous response to flicker light, however, could observed in the majority of animals. Repeated measurements resulted in a significant decrease of baseline venous diameter (7 mice; 7 scans, p < 0.05). Ex-vivo electroretinograms, performed after in-vivo DVA, demonstrated a significant reduction of transretinal signaling in animals with repeated DVA (n = 6, p < 0.001).ConclusionsTo the best of our knowledge, this is the first non-invasive study assessing murine retinal vessel response to flicker light with characteristic changes in NVC. The imaging system can be used for basic research and enables the investigation of retinal vessel dimension and function in control mice and genetically modified animals.

Loosely bound Zn2+ ions are increasingly recognized as potential modulators of synaptic plasticity and neuronal excitability under normal and pathophysiological conditions. Cav2.3 voltage-gated Ca2+ channels are among the most sensitive targets of Zn2+ and are therefore likely to be involved in the neuromodulatory actions of endogenous Zn2+. Although histidine residues on the external side of domain I have been implicated in the effects on Cav2.3 channel gating, the exact mechanisms involved in channel modulation remain incompletely understood. Here, we use a combination of electrophysiological recordings, modification of histidine residues, and computational modeling to analyze Zn2+-induced changes in Cav2.3 channel function. Our most important findings are that multiple high- and low-affinity mechanisms contribute to the net Zn2+ action, that Zn2+ can either inhibit or stimulate Ca2+ influx through Cav2.3 channels depending on resting membrane potential, and that Zn2+ effects may persist for some time even after cessation of the Zn2+ signal. Computer simulations show that (1) most salient features of Cav2.3 channel gating in the absence of trace metals can be reproduced by an obligatory model in which activation of two voltage sensors is necessary to open the pore; and (2) most, but not all, of the effects of Zn2+ can be accounted for by assuming that Zn2+ binding to a first site is associated with an electrostatic modification and mechanical slowing of one of the voltage sensors, whereas Zn2+ binding to a second, lower-affinity site blocks the channel and modifies the opening and closing transitions. While still far from complete, our model provides a first quantitative framework for understanding Zn2+ effects on Cav2.3 channel function and a step toward the application of computational approaches for predicting the complex actions of Zn2+ on neuronal excitability.

Recent recognition that mobile pools of Zn and Cu are involved in the regulation of neuronal, endocrine and other cells has stimulated the development of tools to visualize and quantify the level of free trace metal ions. Most of the methods used to measure or control loosely bound metals require reference media that contain exactly defined free concentrations of the target ions. Despite the central importance of proper metal ion buffering, there is still a lack of international standards and beginners in the field may have difficulties finding a coherent description of how to prepare trace metal ion buffers, especially when experiments are to be performed in multimetal systems. To close this gap, we provide a guide for the design, preparation and use of metal ion-buffered systems that facilitate immediate application under physiologically relevant ionic conditions. Thermodynamic and kinetic concepts of chemical speciation as well as general protocols and specific examples are outlined for the accurate preparation of single- and dual-metal ion buffers. In addition, experiments have been performed with FluoZin-3 to illustrate that metal ion-buffered systems are required for reliable preparation of nanomolar Zn solutions and that dual-metal ion buffers can be used to calibrate suitable fluorescent Zn sensors in the presence of millimolar Ca concentrations. Together, the information provided should sensitize readers to the many potential pitfalls and uncertainties that exist when working with physiologically relevant concentrations of trace metal ions and enable them to formulate their own metal ion buffers for most in vitro applications.

Robust preclinical models are inevitable for researchers to unravel pathomechanisms of subarachnoidal hemorrhage (SAH). For the mouse perforation model of SAH, the goal of this meta-review was the determination of variances in mortality, SAH severity grade, and vasospasm, and their experimental moderators, as many researchers are facing with incomparable results. We searched on the databases PubMed, Embase, and Web of Science for articles describing in vivo experiments using the SAH perforation mouse model and measuring mortality, SAH grade, and/or vasospasm. After screening, 42 articles (total of 1964 mice) were included into systematic review and meta-analysis. Certain model characteristics were insufficiently reported, e.g., perforation location (not reported in six articles), filament (material (n = 15) and tip texture (n = 25)), mouse age (n = 14), and weight (n = 10). Used injective anesthetics and location of perforation showed large variation. In a random-effects meta-analysis, the overall animal mortality following SAH was 21.3% [95% CI: 17.5%, 25.7%] and increased with longer observational periods. Filament material significantly correlated with animal mortality (p = 0.024) after exclusion of hyperacute studies (time after SAH induction < 24 h). Reported mean SAH grade was 10.7 [9.6, 11.7] on the scale of Sugawara (J Neurosci Methods 167:327–34, 2008). Furthermore, mean diameter of large cerebral arteries after SAH was reduced by 27.6% compared to sham-operated non-SAH mice. Uniforming standards of experimental procedures and their reporting are indispensable to increase overall comparability.

So-called pharmacoresistant (R-type) voltage-gated Ca 2+ channels are structurally only partially characterized. Most of them are encoded by the CACNA1E gene and are expressed as different Ca v 2.3 splice variants (variant Ca v 2.3a to Ca v 2.3e or f) as the ion conducting subunit. So far, no inherited disease is known for the CACNA1E gene but recently spontaneous mutations leading to early death were identified, which will be brought into focus. In addition, a short historical overview may highlight the development to understand that upregulation during aging, easier activation by spontaneous mutations or lack of bioavailable inorganic cations (Zn 2+ and Cu 2+) may lead to similar pathologies caused by cellular overexcitation.