Drug-Induced Seizures: Considerations for Underlying Molecular Mechanisms.

A broad spectrum of chemical entities have been associated with drug-induced seizure (DIS), emphasizing the importance of this potential liability across various drug classes (e.g., antidepressants, antipsychotics, antibiotics, and analgesics among others). Despite its importance within drug safety testing, an understanding of the molecular mechanisms associated with DIS is often lacking. The etiology of DIS is understood to be a result of either a deficit in inhibitory (e.g., gamma aminobutyric acid) or an elevated excitatory (e.g., glutamate) signaling, leading to synchronous neuronal depolarization affecting various brain regions and impairing normal neurological functions. How this altered neuronal signaling occurs and how these changes interact with other non-brain receptor driven DIS-associated changes such as metabolic disturbances, electrolyte imbalances, altered drug metabolism, and withdrawal effects are poorly understood. Herein, we discuss important molecular mechanisms identified in DIS for several drugs and/or drug classes. With a better understanding of the molecular mechanisms associated with DIS, in vivo or in vitro models may be applied to characterize and mitigate DIS risk during drug development. Susceptibility stratification for DIS presents species differences in the following order beagle dogs > rodents and cynomolgus monkeys > Göttingen minipigs with a more than 2-fold difference between canines and minipigs, which is important to consider during non-clinical species selection. While clinical signs such as myoclonus, severe muscle jerks, or convulsions are often associated with abnormal epileptiform EEG activity, tremors are most of the time physiological and rarely observed with concurrent epileptiform EEG activity which need to be considered during DIS risk evaluation.

The utilization of advance telemetry to investigate critical physiological parameters including electroencephalography in cynomolgus macaques following aerosol challenge with eastern equine encephalitis virus.

Most alphaviruses are mosquito-borne and can cause severe disease in humans and domesticated animals. In North America, eastern equine encephalitis virus (EEEV) is an important human pathogen with case fatality rates of 30-90%. Currently, there are no therapeutics or vaccines to treat and/or prevent human infection. One critical impediment in countermeasure development is the lack of insight into clinically relevant parameters in a susceptible animal model. This study examined the disease course of EEEV in a cynomolgus macaque model utilizing advanced telemetry technology to continuously and simultaneously measure temperature, respiration, activity, heart rate, blood pressure, electrocardiogram (ECG), and electroencephalography (EEG) following an aerosol challenge at 7.0 log10 PFU. Following challenge, all parameters were rapidly and substantially altered with peak alterations from baseline ranged as follows: temperature (+3.0-4.2°C), respiration rate (+56-128%), activity (-15-76% daytime and +5-22% nighttime), heart rate (+67-190%), systolic (+44-67%) and diastolic blood pressure (+45-80%). Cardiac abnormalities comprised of alterations in QRS and PR duration, QTc Bazett, T wave morphology, amplitude of the QRS complex, and sinoatrial arrest. An unexpected finding of the study was the first documented evidence of a critical cardiac event as an immediate cause of euthanasia in one NHP. All brain waves were rapidly (~12-24 hpi) and profoundly altered with increases of up to 6,800% and severe diffuse slowing of all waves with decreases of ~99%. Lastly, all NHPs exhibited disruption of the circadian rhythm, sleep, and food/fluid intake. Accordingly, all NHPs met the euthanasia criteria by ~106-140 hpi. This is the first of its kind study utilizing state of the art telemetry to investigate multiple clinical parameters relevant to human EEEV infection in a susceptible cynomolgus macaque model. The study provides critical insights into EEEV pathogenesis and the parameters identified will improve animal model development to facilitate rapid evaluation of vaccines and therapeutics.

Confounders and Pharmacological Characterization When Using the QT, JTp, and Tpe Intervals in Beagle Dogs.

INTRODUCTION: Corrected QT (QTc) interval is an essential proarrhythmic risk biomarker, but recent data have identified limitations to its use. The J to T-peak (JTp) interval is an alternative biomarker for evaluating drug-induced proarrhythmic risk. The aim of this study was to evaluate pharmacological effects using spatial magnitude leads and DII electrocardiogram (ECG) leads and common ECG confounders (ie, stress and body temperature changes) on covariate adjusted QT (QTca), covariate adjusted JTp (JTpca), and covariate adjusted T-peak to T-end (Tpeca) intervals. METHODS: Beagle dogs were exposed to body hyper- (42 °C) or hypothermic (33 °C) conditions or were administered epinephrine to assess confounding effects on heart rate corrected QTca, JTpca, and Tpeca intervals. Dofetilide (0.1, 0.3, 1.0 mg/kg), ranolazine (100, 140, 200 mg/kg), and verapamil (7, 15, 30, 43, 62.5 mg/kg) were administered to evaluate pharmacological effects. RESULTS: Covariate adjusted QT (slope -12.57 ms/°C) and JTpca (-14.79 ms/°C) were negatively correlated with body temperature but Tpeca was minimally affected. Epinephrine was associated with QTca and JTpca shortening, which could be related to undercorrection in the presence of tachycardia, while minimal effects were observed for Tpeca. There were no significant ECG change following ranolazine administration. Verapamil decreased QTca and JTpca intervals and increased Tpeca, whereas dofetilide increased QTca and JTpca intervals but had inconsistent effects on Tpeca. CONCLUSION: Results highlight potential confounders on QTc interval, but also on JTpca and Tpeca intervals in nonclinical studies. These potential confounding effects may be relevant to the interpretation of ECG data obtained from nonclinical drug safety studies with Beagle dogs.

Unraveling the mechanism and the risk behind seizure liability of lead compounds in a neuroscience project.

INTRODUCTION: Several compounds from a neuroscience project induced convulsions in animals, at low exposure levels via a hypothetical off-target mechanism. A set of in vitro and in vivo experiments were conducted in order to 1) identify the mechanism behind convulsions; 2) characterize the convulsions, 3) detect premonitory signs that could be monitored clinically, and 4) assess the development of tolerance after repeat dosing. METHODS: Patch clamp assays were conducted on 12 different ion channels (e.g. sodium, potassium, calcium, AMPA, NMDA, GABAA and purinergic receptors) known to be associated with seizures, to identify the off-target culprit. A multiphase study was conducted with UCB-A and UCB-B in Beagle dogs telemetered for video EEG/EMG monitoring to further characterize the convulsive pattern. First, both compounds were administered by intravenous constant infusion (dose: 5 mg/kg/h) over 2 h. Thereafter, the same dogs received a daily oral administration of UCB-A (8 mg/kg/day) for 7 days. RESULTS: Compounds inducing convulsions showed strong inhibitory activity on GABAA channels (IC50 values <10 µM), whereas compounds with partial or no inhibitory effect on these channels did not induce seizures. In EEG experiments, convulsions were preceded by premonitory clinical signs (e.g. tremors, myoclonic jerks) and morphological EEG abnormalities (e.g. sharp waves, spike and wave patterns), confirming their CNS origin. No attenuation of the seizurogenic effects was observed over the 7-day treatment period. DISCUSSION: A well-designed set of experiments including electrophysiological assays on seizure-related ion channels and EEG/EMG assessment in telemetered dogs allowed a proper seizure liability risk assessment, leading to a rapid no go decision for the two most advanced leads.

The application of electrophysiological methods to characterize AMPA receptors in dissociated adult rat and non-human primate cerebellar neurons for use in neuronal safety pharmacology assessments of the central nervous system.

INTRODUCTION: Pre-clinically, safety risk assessment of a drug is primarily tested in vivo using functional evaluation of adult animals while the mechanistic etiology of drug-induced CNS adverse effects is often uncharacterized. In vitro electrophysiology may provide a better understanding of drug effects without additional animal use. However, in vitro protocols are typically designed for using embryonic or juvenile animals. METHODS: We examined whether brain tissue isolated from adult rats (3-5 months old) and adult non-human primates (NHPs) (2-8 years old) can generate qualitatively equivalent readouts for electrophysiology to characterize AMPAR synaptic and single channel currents. We used a known positive AMPAR allosteric modulator (LY451395) to template a response profile and provide proof-of-concept data to assess responses of these native AMPARs in a drug context. RESULTS: Brain slices from adult animals provided a support to measure AMPAR-driven excitatory post-synaptic currents (EPSCs), and can be dissociated into primary neuronal cultures for AMPAR single channel characterization. Additionally, similarities and differences in AMPAR basal kinetics and responses to LY451395 were seen between the two animal species. DISCUSSION: Glutamatergic synaptic activity and AMPAR biophysical properties in adult animals may be used to characterize test-article-mediated alterations in CNS responses. The use of older animals opens the possibility for in vivo test-article administration, either acutely or repeatedly, before in vitro electrophysiological assessment in order to reveal cumulative or delayed-onset effects, adding versatility to safety pharmacology assessment of the CNS.

Nitric Oxide Signaling Strengthens Inhibitory Synapses of Cerebellar Molecular Layer Interneurons through a GABARAP-Dependent Mechanism.

Nitric oxide (NO) is an important signaling molecule that fulfills diverse functional roles as a neurotransmitter or diffusible second messenger in the developing and adult CNS. Although the impact of NO on different behaviors such as movement, sleep, learning, and memory has been well documented, the identity of its molecular and cellular targets is still an area of ongoing investigation. Here, we identify a novel role for NO in strengthening inhibitory GABAA receptor-mediated transmission in molecular layer interneurons of the mouse cerebellum. NO levels are elevated by the activity of neuronal NO synthase (nNOS) following Ca2+ entry through extrasynaptic NMDA-type ionotropic glutamate receptors (NMDARs). NO activates protein kinase G with the subsequent production of cGMP, which prompts the stimulation of NADPH oxidase and protein kinase C (PKC). The activation of PKC promotes the selective strengthening of α3-containing GABAARs synapses through a GΑΒΑ receptor-associated protein-dependent mechanism. Given the widespread but cell type-specific expression of the NMDAR/nNOS complex in the mammalian brain, our data suggest that NMDARs may uniquely strengthen inhibitory GABAergic transmission in these cells through a novel NO-mediated pathway.SIGNIFICANCE STATEMENT Long-term changes in the efficacy of GABAergic transmission is mediated by multiple presynaptic and postsynaptic mechanisms. A prominent pathway involves crosstalk between excitatory and inhibitory synapses whereby Ca2+-entering through postsynaptic NMDARs promotes the recruitment and strengthening of GABAA receptor synapses via Ca2+/calmodulin-dependent protein kinase II. Although Ca2+ transport by NMDARs is also tightly coupled to nNOS activity and NO production, it has yet to be determined whether this pathway affects inhibitory synapses. Here, we show that activation of NMDARs trigger a NO-dependent pathway that strengthens inhibitory GABAergic synapses of cerebellar molecular layer interneurons. Given the widespread expression of NMDARs and nNOS in the mammalian brain, we speculate that NO control of GABAergic synapse efficacy may be more widespread than has been appreciated.

Characterization of a partial-body irradiation model with oral cavity shielding in nonhuman primates.

Purpose: Characterization of a novel partial-body irradiation (PBI) shielding strategy in nonhuman primates (NHP; rhesus macaques), aimed at protecting the oral cavity, with respect to various gastrointestinal acute radiation syndrome (GI-ARS) syndrome parameters as well as buccal ulceration development.Materials and methods: NHPs were irradiated using a Cobalt-60 gamma source, in a single uniform dose, ranging from 9-13 Gy and delivered at 0.60-0.80 Gy min-1. Animals were either partially shielded via oral cavity shielding (PBIOS) or underwent total-body irradiation (TBI).Results: Clinical manifestations of GI-ARS, and also radiation-induced hematology and clinical chemistry changes, following PBIOS were comparable to the PBI NHP GI-ARS model utilizing shielding of the distal pelvic limbs and were significantly milder than TBI at similar radiation doses. Nadir citrulline levels were comparable between PBIOS and TBI but signs of recovery appeared earlier in PBIOS-treated animals. The PBIOS model prevented oral mucositis, whereas the TBI model presented buccal ulcerations at all tested radiation dose levels.Conclusions: Taken together, these results suggest that the PBIOS model is a suitable alternative to traditional PBI. For GI-ARS investigations requiring orally administered medical countermeasures, PBIOS confers added value due to the prevention of oral mucositis over traditional PBI.

Reprint of "EEG: Characteristics of drug-induced seizures in rats, dogs and non-human primates".

Seizures are amongst the most frequent neurological issues encountered in pre-clinical safety testing. The objective was to characterize EEG morphologies and premonitory signs in drug-induced seizures in preclinical species. A comparative (inter-species) retrospective analysis for drug-induced seizures recorded by video-telemetry was conducted in rats (n = 53), dogs (n = 195), and non-human primates (n = 234). The most frequent premonitory signs were, in rats, myoclonus (100%), tremors (93%), salivation (75%), partial ptosis (58%) and chewing/bruxism (58%); in dogs, tremors (77%), ataxia/uncoordination (60%), myoclonus (45%), salivation (43%), excessive licking (38%), high vocalization (38%) and decreased activity (34%); in non-human primates, tremors (79%), decreased activity (70%), myoclonus (57%), retching/emesis (37%), hunched posture (30%) and ataxia/uncoordination (27%). Seizure duration ranged from 3 s to 14 min with an average of 46 ±â€¯21 s, comparable across species. At seizure onset, spike frequency averaged 9.4 Hz for the three species compared to 4.3 Hz at seizure end. Peak average amplitudes were attained at mid-seizure and amplitudes at seizure end decreased from peak but remained higher than onset amplitudes. Spike duration was inversely correlated with frequency and presented a crescendo pattern. Morphological characteristics can serve to refine automated EEG analysis. From a regulatory perspective, the most common paradigm is to use the most sensitive species in seizure liability studies but translational potential and clinical relevance may be under represented in the decision making process in some cases. EEG morphologies during drug-induced seizures presented remarkable similarities between species and tremors were identified as a predominant premonitory clinical sign in all species.

Incidence of spontaneous arrhythmias in freely moving healthy untreated Sprague-Dawley rats.

Spontaneous arrhythmia characterization in healthy rats can support interpretation when studying novel therapies. Male (n = 55) and female (n = 40) Sprague-Dawley rats with telemetry transmitters for a derivation II ECG. Arrhythmias were assessed from continuous ECG monitoring over a period of 24-48 h, and data analyzed using an automated detection algorithm with 100% manual over-read. While a total of 1825 spontaneous ventricular premature beats (VPB) were identified, only 7 rats (or 7.4%) did not present with any over the recording period. Spontaneous episode(s) of ventricular tachycardia (VT) were noted in males (27%) and females (3%). The incidence of VPB was significantly higher (p < 0.01) during the night time (7 pm-7 am) compared to daytime, while males presented with significantly (p < 0.001) more VPB than females. Most VPB were observed as single ectopic beats, followed by salvos (2 or 3 consecutive VPBs), and VT (i.e. 4 consecutive VPBs). Most VPBs were single premature ventricular contractions (PVCs) (57%), while the remaining were escape complexes (43%). Spontaneous premature junctional complexes (PJC) were also observed and were significantly more frequent during the night, and in males. Lastly, 596 episodes of spontaneous 2nd-degree atrioventricular (AV) block were identified and were significantly more frequent during the day time in males. Most 2nd-degree AV block episodes were Mobitz type I (57%), with a significantly (p < 0.05) higher incidence in males. This work emphasizes the importance of obtaining sufficient baseline data when undertaking arrhythmia analysis in safety study and provides a better understanding of both sex- and time- dependent effects of spontaneous arrhythmias in rats.

EEG: Characteristics of drug-induced seizures in rats, dogs and non-human primates.

Seizures are amongst the most frequent neurological issues encountered in pre-clinical safety testing. The objective was to characterize EEG morphologies and premonitory signs in drug-induced seizures in preclinical species. A comparative (inter-species) retrospective analysis for drug-induced seizures recorded by video-telemetry was conducted in rats (n = 53), dogs (n = 195), and non-human primates (n = 234). The most frequent premonitory signs were, in rats, myoclonus (100%), tremors (93%), salivation (75%), partial ptosis (58%) and chewing/bruxism (58%); in dogs, tremors (77%), ataxia/uncoordination (60%), myoclonus (45%), salivation (43%), excessive licking (38%), high vocalization (38%) and decreased activity (34%); in non-human primates, tremors (79%), decreased activity (70%), myoclonus (57%), retching/emesis (37%), hunched posture (30%) and ataxia/uncoordination (27%). Seizure duration ranged from 3 s to 14 min with an average of 46 ±â€¯21 s, comparable across species. At seizure onset, spike frequency averaged 9.4 Hz for the three species compared to 4.3 Hz at seizure end. Peak average amplitudes were attained at mid-seizure and amplitudes at seizure end decreased from peak but remained higher than onset amplitudes. Spike duration was inversely correlated with frequency and presented a crescendo pattern. Morphological characteristics can serve to refine automated EEG analysis. From a regulatory perspective, the most common paradigm is to use the most sensitive species in seizure liability studies but translational potential and clinical relevance may be under represented in the decision making process in some cases. EEG morphologies during drug-induced seizures presented remarkable similarities between species and tremors were identified as a predominant premonitory clinical sign in all species.

Seizure liability assessments using the hippocampal tissue slice: Comparison of non-clinical species.

INTRODUCTION: Traditionally, rat hippocampal tissue slice models are used as an in vitro electrophysiology assay to assess seizurogenic potential in early drug development despite non-clinical species-specific differences noted during in vivo seizure studies. METHODS: Hippocampal tissue slices were acutely isolated from rats, minipigs, dogs and nonhuman primates (NHP). Population spikes (PS) were evoked through stimulation of the CA3 Schaffer collateral pathway and recorded using in vitro electrophysiological techniques via an extracellular electrode placed within the CA1 stratum pyramidale cell body layer. RESULTS: Hippocampal slices, across all species, displayed a concentration-dependent increase in PS area and number with the pro-convulsant pentylenetetrazol (PTZ; 0.1-10mM). Beagle dogs exhibited higher sensitivities to PTZ-induced changes in PS area and number compared to both rats and NHPs which presented nuanced differences in their responsiveness to PTZ modulation. Minipigs were comparatively resistant to PTZ-induced changes in both PS area and number. Rat and NHP hippocampal tissues were further characterized with the pro-convulsant agents 4-aminopyradine (4-AP; 1-100µM) and cefazolin (0.001-10mM). Rats possessed higher sensitivities to 4-AP- and cefazolin-induced changes to both PS area and number whereas NHP displayed greater modulation in PS duration. The anti-convulsant agents, diazepam (10-500µM) and lidocaine (1-500µM), were also tested on either rat and/or NHP tissue with both drugs repressing PS activation at high concentrations. DISCUSSION: Hippocampal tissue slices, across all species, possessed distinct sensitivities to pro- and anti-convulsant agents which may benefit the design of non-clinical seizure liability studies and their associated data interpretation.

Safety pharmacology methods and models in an evolving regulatory environment.

This editorial prefaces the annual themed issue on safety pharmacology (SP) methods published in the Journal of Pharmacological and Toxicological Methods (JPTM). We highlight here the content derived from the recent 2016 Safety Pharmacology Society (SPS), Canadian Society of Pharmacology and Therapeutics (CSPT), and Japanese Safety Pharmacology Society (JSPS) joint meeting held in Vancouver, B.C., Canada. This issue of JPTM continues the tradition of providing a publication summary of articles primarily presented at the joint meeting with direct bearing on the discipline of SP. As the regulatory landscape is expected to evolve with revision announced for the existing guidance document on non-clinical proarrhythmia risk assessment (ICHS7B) there is also imminent inception of the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative. Thus, the field of SP is dynamically progressing with characterization and implementation of numerous alternative non-clinical safety models. Novel method development and refinement in all areas of the discipline are reflected in the content.

Cardiac contractility: Correction strategies applied to telemetry data from a HESI-sponsored consortium.

INTRODUCTION: QT has a long history of heart rate (HR) correction but limited investigations have been undertaken to assess the impact of cardiovascular parameters on left ventricular (LV) contractility in drug safety testing. Cardiac contractility is affected by preload (Cyon-Frank-Starling law), afterload (Anrep effect) and HR (Bowditch effect). We evaluated multi-parameter correction methods to help with dP/dtmax interpretation. METHODOLOGY: Modeling was undertaken using data from dogs in single or double 4×4 Latin square studies. Correction models (16 fitting formulas×2 modeling approaches (universal and individualized)×2 correction approaches (linear or proportional)) were evaluated. 3D/2D cloud analysis of the beat-to-beat data for the control, pimobendan, and either itraconazole or atenolol groups were used to evaluate correlations between parameters and derive an optimal correction method. RESULTS: Cardiac contractility (i.e., dP/dtmax) was best correlated to HR and systolic LV pressure with a correlation coefficient of 0.8. In decreasing order, dP/dtmin, mean arterial blood pressure (BP), systolic BP, diastolic BP, arterial pulse pressure and LV end diastolic pressure (LVEDP) showed a reduced correlation to dP/dtmax. Subject-specific models improved the correction by up to 14% when compared to universal correction models. The non-linear correction model was superior to the linear model. DISCUSSION: Results suggest that the optimal correction formula for dP/dtmax would be subject-specific, non-linear and would include HR and LV systolic pressure. Correcting contractility for HR and systolic LV pressure may enhance data interpretation in non-clinical drug safety assessments. Similar correction methods could be evaluated for other species used in safety pharmacology.

Recalibration of nonclinical safety pharmacology assessment to anticipate evolving regulatory expectations.

Safety pharmacology (SP) has evolved in terms of architecture and content since the inception of the SP Society (SPS). SP was initially focused on the issue of drug-induced QT prolongation, but has now become a broad spectrum discipline with expanding expectations for evaluation of drug adverse effect liability in all organ systems, not merely the narrow consideration of torsades de pointes (TdP) liability testing. An important part of the evolution of SP has been the elaboration of architecture for interrogation of non-clinical models in terms of model development, model validation and model implementation. While SP has been defined by mandatory cardiovascular, central nervous system (CNS) and respiratory system studies ever since the core battery was elaborated, it also involves evaluation of drug effects on other physiological systems. The current state of SP evolution is the incorporation of emerging new technologies in a wide range of non-clinical drug safety testing models. This will refine the SP process, while potentially expanding the core battery. The continued refinement of automated technologies (e.g., automated patch clamp systems) is enhancing the scope for detection of adverse effect liability (i.e., for more than just IKr blockade), while introducing a potential for speed and accuracy in cardiovascular and CNS SP by providing rapid, high throughput ion channel screening methods for implementation in early drug development. A variety of CNS liability assays, which exploit isolated brain tissue, and in vitro electrophysiological techniques, have provided an additional level of complimentary preclinical safety screens aimed at establishing the seizurogenic potential and risk for memory dysfunction of new chemical entities (NCEs). As with previous editorials that preface the annual themed issue on SP methods published in the Journal of Pharmacological and Toxicological Methods (JPTM), we highlight here the content derived from the most recent (2015) SPS meeting held in Prague, Czech Republic. This issue of JPTM continues the tradition of providing a publication summary of articles primarily presented at the SPS meeting with direct bearing on the discipline of SP. Novel method development and refinement in all areas of the discipline are reflected in the content.

The emerging role of in vitro electrophysiological methods in CNS safety pharmacology.

Adverse CNS effects account for a sizeable proportion of all drug attrition cases. These adverse CNS effects are mediated predominately by off-target drug activity on neuronal ion-channels, receptors, transporters and enzymes - altering neuronal function and network communication. In response to these concerns, there is growing support within the pharmaceutical industry for the requirement to perform more comprehensive CNS safety testing prior to first-in-human trials. Accordingly, CNS safety pharmacology commonly integrates several in vitro assay methods for screening neuronal targets in order to properly assess therapeutic safety. One essential assay method is the in vitro electrophysiological technique - the 'gold standard' ion channel assay. The in vitro electrophysiological method is a useful technique, amenable to a variety of different tissues and cell configurations, capable of assessing minute changes in ion channel activity from the level of a single receptor to a complex neuronal network. Recent advances in automated technology have further expanded the usefulness of in vitro electrophysiological methods into the realm of high-throughput, addressing the bottleneck imposed by the manual conduct of the technique. However, despite a large range of applications, manual and automated in vitro electrophysiological techniques have had a slow penetrance into the field of safety pharmacology. Nevertheless, developments in throughput capabilities and in vivo applicability have led to a renewed interest in in vitro electrophysiological techniques that, when complimented by more traditional safety pharmacology methods, often increase the preclinical predictability of potential CNS liabilities.

Functional neurotoxicity evaluation of noribogaine using video-EEG in cynomolgus monkeys.

INTRODUCTION: Continuous video-electroencephalographic (EEG) monitoring remains the gold standard for seizure liability assessments in preclinical drug safety assessments. EEG monitored by telemetry was used to assess the behavioral and EEG effects of noribogaine hydrochloride (noribogaine) in cynomolgus monkeys. Noribogaine is an iboga alkaloid being studied for the treatment of opioid dependence. METHODS: Six cynomolgus monkeys (3 per gender) were instrumented with EEG telemetry transmitters. Noribogaine was administered to each monkey at both doses (i.e., 160 and 320mg/kg, PO) with an interval between dosing of at least 6days, and the resulting behavioral and EEG effects were evaluated. IV pentylenetetrazol (PTZ), served as a positive control for induced seizures. RESULTS: The administration of noribogaine at either of the doses evaluated was not associated with EEG evidence of seizure or with EEG signals known to be premonitory signs of increased seizure risk (e.g., sharp waves, unusual synchrony, shifts to high-frequency patterns). Noribogaine was associated with a mild reduction in activity levels, increased scratching, licking and chewing, and some degree of poor coordination and related clinical signs. A single monkey exhibited brief myoclonic movements that increased in frequency at the high dose, but which did not appear to generalize, cluster or to be linked with EEG abnormalities. Noribogaine was also associated with emesis and partial anorexia. In contrast, PTZ was associated with substantial pre-ictal EEG patterns including large amplitude, repetitive sharp waves leading to generalized seizures and to typical post-ictal EEG frequency attenuation. INTERPRETATION: EEG patterns were within normal limits following administration of noribogaine at doses up to 320mg/kg with concurrent clinical signs that correlated with plasma exposures and resolved by the end of the monitoring period. PTZ was invariably associated with EEG paroxysmal activity leading to ictal EEG. In the current study, a noribogaine dose of 320mg/kg was considered to be the EEG no observed adverse effect level (NOAEL) in conscious freely moving cynomolgus monkeys.

α6-Containing GABAA Receptors Are the Principal Mediators of Inhibitory Synapse Strengthening by Insulin in Cerebellar Granule Cells.

Activity-dependent strengthening of central synapses is a key factor driving neuronal circuit behavior in the vertebrate CNS. At fast inhibitory synapses, strengthening is thought to occur by increasing the number of GABAA receptors (GABARs) of the same subunit composition to preexisting synapses. Here, we show that strengthening of mouse cerebellar granule cell GABAergic synapses occurs by a different mechanism. Specifically, we show that the neuropeptide hormone, insulin, strengthens inhibitory synapses by recruiting α6-containing GABARs rather than accumulating more α1-containing receptors that are resident to the synapse. Because α6-receptors are targeted to functionally distinct postsynaptic sites from α1-receptors, we conclude that only a subset of all inhibitory synapses are strengthened. Together with our recent findings on stellate cells, we propose a general mechanism by which mature inhibitory synapses are strengthened. In this scenario, α1-GABARs resident to inhibitory synapses form the hardwiring of neuronal circuits with receptors of a different composition fulfilling a fundamental, but unappreciated, role in synapse strengthening.

Mitochondrial reactive oxygen species regulate the strength of inhibitory GABA-mediated synaptic transmission.

Neuronal communication imposes a heavy metabolic burden in maintaining ionic gradients essential for action potential firing and synaptic signalling. Although cellular metabolism is known to regulate excitatory neurotransmission, it is still unclear whether the brain's energy supply affects inhibitory signalling. Here we show that mitochondrial-derived reactive oxygen species (mROS) regulate the strength of postsynaptic GABA(A) receptors at inhibitory synapses of cerebellar stellate cells. Inhibition is strengthened through a mechanism that selectively recruits α3-containing GABA(A) receptors into synapses with no discernible effect on resident α1-containing receptors. Since mROS promotes the emergence of postsynaptic events with unique kinetic properties, we conclude that newly recruited α3-containing GABA(A) receptors are activated by neurotransmitter released onto discrete postsynaptic sites. Although traditionally associated with oxidative stress in neurodegenerative disease, our data identify mROS as a putative homeostatic signalling molecule coupling cellular metabolism to the strength of inhibitory transmission.

Crosslinking the ligand-binding domain dimer interface locks kainate receptors out of the main open state.

Kainate-selective ionotropic glutamate receptors (iGluRs) fulfil key roles in the CNS, making them the subject of detailed structural and functional analyses. Although they are known to gate a channel pore with high and low ion-permeation rates, it is still not clear how switches between these gating modes are achieved at the structural level. Here, we uncover an unexpected role for the ligand-binding domain (LBD) dimer assembly in this process. Covalent crosslinking of the dimer interface keeps kainate receptors out of the main open state but permits access to lower conductance states suggesting that significant rearrangements of the dimer interface are required for the receptor to achieve full activation. These observations differ from NMDA-selective iGluRs where constraining dimer movement reduces open-channel probability. In contrast, our data show that restricting movement of the dimer interface interferes with conformational changes that underlie both activation and desensitization. Working within the limits of a common architectural design, we propose functionally diverse iGluR families were able to emerge during evolution by re-deploying existing gating structures to fulfil different tasks.

Hco-LGC-38 is novel nematode cys-loop GABA receptor subunit.

We have identified and characterized a novel cys-loop GABA receptor subunit (Hco-LGC-38) from the parasitic nematode Haemonchus contortus. This subunit is present in parasitic and free-living nematodes and shares similarity to both the UNC-49 group of GABA receptor subunits from nematodes and the resistant to dieldrin (RDL) receptors of insects. Expression of the Hco-lgc-38 gene in Xenopus oocytes and subsequent electrophysiological analysis has revealed that the gene encodes a homomeric channel sensitive to GABA (EC(50) 19 µM) and the GABA analogue muscimol. The sensitivity of the Hco-LGC-38 channel to GABA is similar to reported values for the Drosophila RDL receptor whereas its lower sensitivity to muscimol is similar to nematode GABA receptors. Hco-LGC-38 is also highly sensitive to the channel blocker picrotoxin and moderately sensitive to fipronil and dieldrin. Homology modeling of Hco-LGC-38 and subsequent docking of GABA and muscimol into the binding site has uncovered several types of potential interactions with binding-site residues and overall appears to share similarity with models of other invertebrate GABA receptors.