A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated p114a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. p114a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an R-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of p114a revealed a novel signal sequence, indicating that p114a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of p114a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, p114a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50) 1.59 mu M) and neuronal (IC50 = 8.7 mu M for alpha 3 beta 4) and neuromuscular (IC50 = 0.54 mu M for alpha 1 beta 1 is an element of delta) subtypes of the nicotinic acetylcholine receptor ( nAChR). Similarities in sequence and structure are apparent between the middle loop of p114a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

Documenting the heroin shortage in New South Wales

Australian heroin markets have recently undergone dramatic change, sparking debate about the nature of such markets. This study aimed to determine the onset, peak and decline of the heroin shortage in New South Wales (NSW), using the most appropriate available methods to detect market level changes. The parameters of the heroin shortage were determined by reviewing: reports of heroin users about availability and price (derived from the existing literature and the Illicit Drug Reporting System); qualitative interviews with injecting drug users, and health and law enforcement professionals working in the illicit drug field; and examining data on heroin seizures over the past decade. There was a marked reduction in heroin supply in NSW in early 2001. An increase in the price of heroin occurred in 2001, whereas it had decreased steadily since 1996. A reduction in purity also occurred, as reported by drug users and heroin seizures. The peak period of the shortage appears to have been January to April 2001. The market appears to have stabilised since that time, although it has not returned to pre- 2001 levels: heroin prices have decreased in NSW for street grams, but not to former levels, and the price of `caps' (street deals) remain elevated. Heroin purity in NSW has remained low, with perhaps a 10% increase above the lowest recorded levels. These data support the notion that the heroin market in NSW underwent significant changes, which appear to have involved a lasting shift in the nature of the market.

Seizure detection algorithm for neonates based on wave-sequence analysis

Objective: The description and evaluation of the performance of a new real-time seizure detection algorithm in the newborn infant. Methods: The algorithm includes parallel fragmentation of EEG signal into waves; wave-feature extraction and averaging; elementary, preliminary and final detection. The algorithm detects EEG waves with heightened regularity, using wave intervals, amplitudes and shapes. The performance of the algorithm was assessed with the use of event-based and liberal and conservative time-based approaches and compared with the performance of Gotman's and Liu's algorithms. Results: The algorithm was assessed on multi-channel EEG records of 55 neonates including 17 with seizures. The algorithm showed sensitivities ranging 83-95% with positive predictive values (PPV) 48-77%. There were 2.0 false positive detections per hour. In comparison, Gotman's algorithm (with 30 s gap-closing procedure) displayed sensitivities of 45-88% and PPV 29-56%; with 7.4 false positives per hour and Liu's algorithm displayed sensitivities of 96-99%, and PPV 10-25%; with 15.7 false positives per hour. Conclusions: The wave-sequence analysis based algorithm displayed higher sensitivity, higher PPV and a substantially lower level of false positives than two previously published algorithms. Significance: The proposed algorithm provides a basis for major improvements in neonatal seizure detection and monitoring. Published by Elsevier Ireland Ltd. on behalf of International Federation of Clinical Neurophysiology.

Heart rate variability characterization using a time-frequency based instantaneous frequency estimation technique

In this paper, a new method for characterizing the newborn heart rate variability (HRV) is proposed. The central of the method is the newly proposed technique for instantaneous frequency (IF) estimation specifically designed for nonstationary multicomponen signals such as HRV. The new method attempts to characterize the newborn HRV using features extracted from the time–frequency (TF) domain of the signal. These features comprise the IF, the instantaneous bandwidth (IB) and instantaneous energy (IE) of the different TF components of the HRV. Applied to the HRV of both normal and seizure suffering newborns, this method clearly reveals the locations of the spectral peaks and their time-varying nature. The total energy of HRV components, ET and ratio of energy concentrated in the low-frequency (LF) to that in high frequency (HF) components have been shown to be significant features in identifying the HRV of newborn with seizures.

Monitoring Infants By Automatic Video Processing

This work has, as its objective, the development of non-invasive and low-cost systems for monitoring and automatic diagnosing specific neonatal diseases by means of the analysis of suitable video signals. We focus on monitoring infants potentially at risk of diseases characterized by the presence or absence of rhythmic movements of one or more body parts. Seizures and respiratory diseases are specifically considered, but the approach is general. Seizures are defined as sudden neurological and behavioural alterations. They are age-dependent phenomena and the most common sign of central nervous system dysfunction. Neonatal seizures have onset within the 28th day of life in newborns at term and within the 44th week of conceptional age in preterm infants. Their main causes are hypoxic-ischaemic encephalopathy, intracranial haemorrhage, and sepsis. Studies indicate an incidence rate of neonatal seizures of 0.2% live births, 1.1% for preterm neonates, and 1.3% for infants weighing less than 2500 g at birth. Neonatal seizures can be classified into four main categories: clonic, tonic, myoclonic, and subtle. Seizures in newborns have to be promptly and accurately recognized in order to establish timely treatments that could avoid an increase of the underlying brain damage. Respiratory diseases related to the occurrence of apnoea episodes may be caused by cerebrovascular events. Among the wide range of causes of apnoea, besides seizures, a relevant one is Congenital Central Hypoventilation Syndrome (CCHS) \cite{Healy}. With a reported prevalence of 1 in 200,000 live births, CCHS, formerly known as Ondine's curse, is a rare life-threatening disorder characterized by a failure of the automatic control of breathing, caused by mutations in a gene classified as PHOX2B. CCHS manifests itself, in the neonatal period, with episodes of cyanosis or apnoea, especially during quiet sleep. The reported mortality rates range from 8% to 38% of newborn with genetically confirmed CCHS. Nowadays, CCHS is considered a disorder of autonomic regulation, with related risk of sudden infant death syndrome (SIDS). Currently, the standard method of diagnosis, for both diseases, is based on polysomnography, a set of sensors such as ElectroEncephaloGram (EEG) sensors, ElectroMyoGraphy (EMG) sensors, ElectroCardioGraphy (ECG) sensors, elastic belt sensors, pulse-oximeter and nasal flow-meters. This monitoring system is very expensive, time-consuming, moderately invasive and requires particularly skilled medical personnel, not always available in a Neonatal Intensive Care Unit (NICU). Therefore, automatic, real-time and non-invasive monitoring equipments able to reliably recognize these diseases would be of significant value in the NICU. A very appealing monitoring tool to automatically detect neonatal seizures or breathing disorders may be based on acquiring, through a network of sensors, e.g., a set of video cameras, the movements of the newborn's body (e.g., limbs, chest) and properly processing the relevant signals. An automatic multi-sensor system could be used to permanently monitor every patient in the NICU or specific patients at home. Furthermore, a wire-free technique may be more user-friendly and highly desirable when used with infants, in particular with newborns. This work has focused on a reliable method to estimate the periodicity in pathological movements based on the use of the Maximum Likelihood (ML) criterion. In particular, average differential luminance signals from multiple Red, Green and Blue (RGB) cameras or depth-sensor devices are extracted and the presence or absence of a significant periodicity is analysed in order to detect possible pathological conditions. The efficacy of this monitoring system has been measured on the basis of video recordings provided by the Department of Neurosciences of the University of Parma. Concerning clonic seizures, a kinematic analysis was performed to establish a relationship between neonatal seizures and human inborn pattern of quadrupedal locomotion. Moreover, we have decided to realize simulators able to replicate the symptomatic movements characteristic of the diseases under consideration. The reasons is, essentially, the opportunity to have, at any time, a 'subject' on which to test the continuously evolving detection algorithms. Finally, we have developed a smartphone App, called 'Smartphone based contactless epilepsy detector' (SmartCED), able to detect neonatal clonic seizures and warn the user about the occurrence in real-time.

Induced visual illusions and gamma oscillations in human primary visual cortex

Using magnetoencephalography, we studied the spatiotemporal properties of cortical responses in terms of event-related synchronization and event-related desynchronization to a range of stripe patterns in subjects with no neurological disorders. These stripes are known for their tendency to induce a range of abnormal sensations, such as illusions, nausea, dizziness, headache and attacks of pattern-sensitive epilepsy. The optimal stimulus must have specific physical properties, and maximum abnormalities occur at specific spatial frequency and contrast. Despite individual differences in the severity of discomfort experienced, psychophysical studies have shown that most observers experience some degree of visual anomaly on viewing such patterns. In a separate experiment, subjects reported the incidence of illusions and discomfort to each pattern. We found maximal cortical power in the gamma range (30-60 Hz) confined to the region of the primary visual cortex in response to patterns of 2-4 cycles per degree, peaking at 3 cycles per degree. This coincides with the peak of mean illusions and discomfort, also maximal for patterns of 2-4 cycles per degree. We show that gamma band activity in V1 is a narrow band function of spatial frequency. We hypothesize that the intrinsic properties of gamma oscillations may underlie visual discomfort and play a role in the onset of seizures.

The effectiveness of lamotrigine in the treatment of photosensitive epilepsy

Photosensitive epilepsy and associated pattern sensitivity are more prevalent in females and are usually treated with sodium valproate. Sodium valproate has an adverse effect profile, which particularly affects females, including teratogenicity, association with the polycystic ovary syndrome and weight gain. It would be useful therefore if an alternative treatment for photosensitive epilepsy could be found. The principle aim of this study was to investigate the effectiveness of lamotrigine in the treatment of photosensitive epilepsy in adults and children. Patients were either drug-naive, commencing lamotrigine therapy or were transferring from other antiepileptic drugs to lamotrigine (primarily sodium valproate) due to lack of response, adverse effects or desired pregnancy. The photoparoxsymal response in the electroencephalograph was used as the primary measure of photo and pattern sensitivity. In addition the effects of lamotrigine on occipital spikes and normal responses in the EEG to visual stimuli were investigated. Secondary measures also included the resting EEG, seizures, body mass index, menstrual function, mood and cognitive function. The results suggest that in adult patients lamotrigine is efficacious in the treatment of photosensitive epilepsy, although it appears inferior to sodium valproate. Lamotrigine does however have a more favourable adverse effect profile than valproate. The results indicate that lamotrigine therapy is suitable for photosensitive epilepsy in women of childbearing age or in patients experiencing unacceptable adverse effects with valproate therapy. Patients are more likely to respond to lamotrigine treatment if they present with sensitivity to a limited number of frequencies. Lamotrigine does not seem to be as efficacious in the treatment of children, although against it may be considered a second line drug if the child does not respond to or will not tolerate sodium valproate.

Ambulatory EEG monitoring in the diagnosis and treatment of epilepsy and related disorders

Ambulatory EEG recording enables patients with epilepsy and related disorders to be monitored in an unrestricted environment for prolonged periods. Attacks can therefore be recorded and EEG changes at the time can aid diagnosis. The relevant Iiterature is reviewed and a study made of' 250 clinical investigations. A study was also made of the artefacts,encountered during ambulatory recording. Three quarters of referrals were for distinguishing between epileptic and non-epileptic attacks. Over 60% of patients showed no abnormality during attacks. In comparison with the basic EEG the ambulatory EEG provided about ten times as much information. A preliminary follow-up study showed that results, of ambulatory monitoring agreed with the final diagnosis in 8 of 12 patients studied. Of 10 patients referred, for monitoring the occurrence of absence seizures, 8 showed abnormality during the baslcJ EEG .and 10 during the ambulatory EEG. Other patients. were referred: for sleep recording and to clarify the seizure type. An investigation into once daily (OD) versus twice daily administration of sodium valproate in patients with absence seizures showed that an OD regime was equally as effective as a BD regime. Circadian variations in spike and wave activity in patients on and off treatment were also examined. There was significant agreement between subjects on the time of occurrence of abnormality during sleep only, This pattern was not ,affected with treatment nor was there any difference in the daily pattern of occurrence of abnormality between the two regimes. Overall findings suggested that ambulatory monitoring was a valuable tool in the diagnosis and treatment of epilepsy which with careful planning and patient selection could be used in any EEG department and would benefit a:wide range of patients.

Brain ictal state characterisation through multimodal information integration

This Thesis addresses the problem of automated false-positive free detection of epileptic events by the fusion of information extracted from simultaneously recorded electro-encephalographic (EEG) and the electrocardiographic (ECG) time-series. The approach relies on a biomedical case for the coupling of the Brain and Heart systems through the central autonomic network during temporal lobe epileptic events: neurovegetative manifestations associated with temporal lobe epileptic events consist of alterations to the cardiac rhythm. From a neurophysiological perspective, epileptic episodes are characterised by a loss of complexity of the state of the brain. The description of arrhythmias, from a probabilistic perspective, observed during temporal lobe epileptic events and the description of the complexity of the state of the brain, from an information theory perspective, are integrated in a fusion-of-information framework towards temporal lobe epileptic seizure detection. The main contributions of the Thesis include the introduction of a biomedical case for the coupling of the Brain and Heart systems during temporal lobe epileptic seizures, partially reported in the clinical literature; the investigation of measures for the characterisation of ictal events from the EEG time series towards their integration in a fusion-of-knowledge framework; the probabilistic description of arrhythmias observed during temporal lobe epileptic events towards their integration in a fusion-of-knowledge framework; and the investigation of the different levels of the fusion-of-information architecture at which to perform the combination of information extracted from the EEG and ECG time-series. The performance of the method designed in the Thesis for the false-positive free automated detection of epileptic events achieved a false-positives rate of zero on the dataset of long-term recordings used in the Thesis.

Ethosuximide modifies network excitability in the rat entorhinal cortex via an increase in GABA release

Ethosuximide is the drug of choice for treating generalized absence seizures, but its mechanism of action is still a matter of debate. It has long been thought to act by disrupting a thalamic focus via blockade of T-type channels and, thus, generation of spike-wave activity in thalamocortical pathways. However, there is now good evidence that generalized absence seizures may be initiated at a cortical focus and that ethosuximide may target this focus. In the present study we have looked at the effect ethosuximide on glutamate and GABA release at synapses in the rat entorhinal cortex in vitro, using two experimental approaches. Whole-cell patch-clamp studies revealed an increase in spontaneous GABA release by ethosuximide concurrent with no change in glutamate release. This was reflected in studies that estimated global background inhibition and excitation from intracellularly recorded membrane potential fluctuations, where there was a substantial rise in the ratio of network inhibition to excitation, and a concurrent decrease in excitability of neurones embedded in this network. These studies suggest that, in addition to well-characterised effects on ion channels, ethosuximide may directly elevate synaptic inhibition in the cortex and that this could contribute to its anti-absence effects. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Methodology of photic stimulation revisited:updated European algorithm for visual stimulation in the EEG laboratory

Intermittent photic stimulation (IPS) is a common procedure performed in the electroencephalography (EEG) laboratory in children and adults to detect abnormal epileptogenic sensitivity to flickering light (i.e., photosensitivity). In practice, substantial variability in outcome is anecdotally found due to the many different methods used per laboratory and country. We believe that standardization of procedure, based on scientific and clinical data, should permit reproducible identification and quantification of photosensitivity. We hope that the use of our new algorithm will help in standardizing the IPS procedure, which in turn may more clearly identify and assist monitoring of patients with epilepsy and photosensitivity. Our algorithm goes far beyond that published in 1999 (Epilepsia, 1999a, 40, 75; Neurophysiol Clin, 1999b, 29, 318): it has substantially increased content, detailing technical and logistical aspects of IPS testing and the rationale for many of the steps in the IPS procedure. Furthermore, our latest algorithm incorporates the consensus of repeated scientific meetings of European experts in this field over a period of 6 years with feedback from general neurologists and epileptologists to improve its validity and utility. Accordingly, our European group has provided herein updated algorithms for two different levels of methodology: (1) requirements for defining photosensitivity in patients and in family members of known photosensitive patients and (2) requirements for tailored studies in patients with a clear history of visually induced seizures or complaints, and in those already known to be photosensitive.

Psychiatric adverse effects of zonisamide in patients with epilepsy and mental disorder comorbidities

Over the last few years, zonisamide has been proposed as a potentially useful medication for patients with focal seizures, with or without secondary generalization. Since psychiatric adverse effects, including mania, psychosis, and suicidal ideation, have been associated with its use, it was suggested that the presence of antecedent psychiatric disorders is an important factor associated with the discontinuation of zonisamide therapy in patients with epilepsy. We, therefore, set out to assess the tolerability profile of zonisamide in a retrospective chart review of 23 patients with epilepsy and comorbid mental disorders, recruited from two specialist pediatric (n=11) and adult (n=12) neuropsychiatry clinics. All patients had a clinical diagnosis of treatment-refractory epilepsy after extensive neurophysiological and neuroimaging investigations. The vast majority of patients (n=22/23, 95.7%) had tried previous antiepileptic medications, and most adult patients (n=9/11, 81.8%) were on concomitant medication for epilepsy. In the majority of cases, the psychiatric adverse effects of zonisamide were not severe. Four patients (17.4%) discontinued zonisamide because of lack of efficacy, whereas only one patient (4.3%) discontinued it because of the severity of psychiatric adverse effects (major depressive disorder). The low discontinuation rate of zonisamide in a selected population of patients with epilepsy and neuropsychiatric comorbidity suggests that this medication is safe and reasonably well-tolerated for use in patients with treatment-refractory epilepsy. Given the limitations of the present study, including the relatively small sample size, further research is warranted to confirm this finding. © 2013 Elsevier Inc.

Magnetoencephalography in the study of epilepsy and consciousness

The neural bases of altered consciousness in patients with epilepsy during seizures and at rest have raised significant interest in the last decade. This exponential growth has been supported by the parallel development of techniques and methods to investigate brain function noninvasively with unprecedented spatial and temporal resolution. In this article, we review the contribution of magnetoencephalography to deconvolve the bioelectrical changes associated with impaired consciousness during seizures. We use data collected from a patient with refractory absence seizures to discuss how spike-wave discharges are associated with perturbations in optimal connectivity within and between brain regions and discuss indirect evidence to suggest that this phenomenon might explain the cognitive deficits experienced during prolonged 3/s spike-wave discharges. © 2013 Elsevier Inc.

Photosensitive benign myoclonic epilepsy in infancy

Purpose: To describe the electroclinical features of subjects who presented with a photosensitive benign myoclonic epilepsy in infancy (PBMEI). Methods: The patients were selected from a group of epileptic subjects with seizure onset in infancy or early childhood. Inclusion criteria were the presence of photic-induced myoclonic seizures and a favorable outcome. Cases with less than 24 month follow up were excluded from the analysis. Results: Eight patients were identified (4 males, 4 females). Personal history was uneventful. All of them had familial antecedents of epilepsy. Psychomotor development was normal in 6 cases, both before and after seizure onset. One patient showed a mild mental retardation and a further patient showed some behavioral disturbances. Neuroradiological investigations, when performed (5 cases), gave normal results. The clinical manifestations were typical and could vary from upward movements of the eyes to myoclonic jerks of the head and shoulders, isolated or briefly repetitive, never causing a fall. Age of onset was between 11 months and 3 years and 2 months. Characteristically, the seizures were always triggered by photic stimulation. Non photo-induced spontaneous myoclonic attacks were reported in 2 cases during the follow-up. Other types of seizures were present at follow-up in 2 cases. The outcome was favorable, even if, usually, seizure control required high AED plasma levels. Since the clinical symptoms were not recognized early, some patients were treated only many years after the onset of symptoms. Conclusion: Among BMEI patients, our cases constitute a subgroup in which myoclonic jerks were always triggered by photostimulation, in particular at onset of their epilepsy. © 2006 International League Against Epilepsy.

GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB.