Analytical aspects of diterpene alkaloid poisoning with monkshood.

A sensitive and specific method for aconitine extraction from biological samples was developed. Aconitine, the main toxic alkaloid from plants belonging to Aconitum species (family Ranunculaceae), was determined in plant material by an external standard method, and by a standard addition calibration method in biological fluids. Described here is one fatal case and five intoxications of accidental aconitine poisoning following the ingestion of aconite mistaken for an edible grass, Aruncus dioicus (Walt.) Fernald, "mountain asparagus", and Cicerbita alpina (L.) Wallroth. The aconitine content in urine was in the range 2.94 μg/mL (dead patient) – 0.20 μg/mL (surviving patients), which was almost two to four times higher than that in plasma

The role of activity in synaptic degeneration in a protein misfolding disease, prion disease

In chronic neurodegenerative diseases associated with aggregates of misfolded proteins (such as Alzheimer's, Parkinson's and prion disease), there is an early degeneration of presynaptic terminals prior to the loss of the neuronal somata. Identifying the mechanisms that govern synapse degeneration is of paramount importance, as cognitive decline is strongly correlated with loss of presynaptic terminals in these disorders. However, very little is known about the processes that link the presence of a misfolded protein to the degeneration of synapses. It has been suggested that the process follows a simple linear sequence in which terminals that become dysfunctional are targeted for death, but there is also evidence that high levels of activity can speed up degeneration. To dissect the role of activity in synapse degeneration, we infused the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of mice with prion disease and assessed synapse loss at the electron microscopy level. We found that injection of BoNT/A in naïve mice caused a significant enlargement of excitatory presynaptic terminals in the hippocampus, indicating transmission impairment. Long-lasting blockade of activity by BoNT/A caused only minimal synaptic pathology and no significant activation of microglia. In mice with prion disease infused with BoNT/A, rates of synaptic degeneration were indistinguishable from those observed in control diseased mice. We conclude that silencing synaptic activity neither prevents nor enhances the degree of synapse degeneration in prion disease. These results challenge the idea that dysfunction of synaptic terminals dictates their elimination during prion-induced neurodegeneration

Collaborative Interlaboratory Studies for the Validation of ELISA Methods for the Detection of Allergenic Fining Agents Used in Wine According to the Criteria of OIV Resolution 427–2010 Modified by OIV–Comex 502–2012

The clarification or fining of wine removes undesired substances (mainly proteins, phenols, and tannins), which would roil the wine and cause bitterness and astringency. A common fining agent, egg white, can be directly added to wine through the inlet of a circulating pump, but more typically egg white comes as commercial preparation in powdered form (commercially named egg albumin). Skimmed milk or more frequently purified caseinates are used to remove bitterness and hardness of white wine and sherry. Both egg white and caseinates are fining agents with optimal enological properties, but their residues could represent a risk for subjects suffering from food allergy. The rules for allergen labeling were detailed in Directives 2003/89/EC, and Directive 2005/26/EC established a list of food ingredients provisionally excluded from labeling, that included wine fining agents. Extended till June 2012, wine labeling exemption can be now maintained only if (1) egg and milk derivatives are not used and cross-contamination is under control; and (2) wine clarified with such products is negative for the presence of residues using techniques with detection and quantification limits of 0.25 and 0.5 ppm, respectively. Analytical requirements were defined in the OIV resolution 427–2010 (OIV 2010) modified by OIV/COMEX 502–2012 (OIV 2012). On the basis of a previous experience, an interlaboratory collaborative trial was organized to validate a commercial ELISA kit designed to measure allergenic residues in red wine fined with egg white proteins. In the meantime, the performance of the commercial caseinate ELISA kit for white wine was rechecked according to the new limit of detection and limit of quantification values, recommended by OIV in 2012. The collaborative interlaboratory studies showed that both ELISA kits had good reproducibility, repeatability, and robustness in detecting residues of allergenic fining agents in wine, in good agreement with the requirements of the OIV resolution 427–2010 modified by OIV/COMEX 502–2012

The synaptic blocker botulinum toxin A decreases the density and complexity of oligodendrocyte precursor cells in the adult mouse hippocampus

Oligodendrocyte progenitor cells (OPCs) are responsible for generating oligodendrocytes, the myelinating cells of the CNS. Life-long myelination is promoted by neuronal activity and is essential for neural network plasticity and learning. OPCs are known to contact synapses and it is proposed that neuronal synaptic activity in turn regulates their behavior. To examine this in the adult, we performed unilateral injection of the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of adult mice. We confirm BoNT/A cleaves SNAP-25 in the CA1 are of the hippocampus, which has been proven to block neurotransmission. Notably, BoNT/A significantly decreased OPC density and caused their shrinkage, as determined by immunolabeling for the OPC marker NG2. Furthermore, BoNT/A resulted in an overall decrease in the number of OPC processes, as well as a decrease in their lengths and branching frequency. These data indicate that synaptic activity is important for maintaining adult OPC numbers and cellular integrity, which is relevant to pathophysiological scenarios characterized by dysregulation of synaptic activity, such as age-related cognitive decline, Multiple Sclerosis and Alzheimer's disease

Unilateral Application of Cathodal tDCS Reduces Transcallosal Inhibition and Improves Visual Acuity in Amblyopic Patients

Objective: Amblyopia is a neurodevelopmental disorder characterized by visual acuity and contrast sensitivity loss, refractory to pharmacological and optical treatments in adulthood. In animals, the corpus callosum (CC) contributes to suppression of visual responses of the amblyopic eye. To investigate the role of interhemispheric pathways in amblyopic patients, we studied the response of the visual cortex to transcranial Direct Current Stimulation (tDCS) applied over the primary visual area (V1) contralateral to the "lazy eye." Methods: Visual acuity (logMAR) was assessed before (T0), immediately after (T1) and 60' following the application of cathodal tDCS (2.0 mA, 20') in 12 amblyopic patients. At each time point, Visual Evoked Potentials (VEPs) triggered by grating stimuli of different contrasts (K90%, K20%) were recorded in both hemispheres and compared to those obtained in healthy volunteers. Results: Cathodal tDCS improved visual acuity respect to baseline (p < 0.0001), whereas sham polarization had no significant effect. At T1, tDCS induced an inhibitory effect on VEPs amplitudes at all contrasts in the targeted side and a facilitation of responses in the hemisphere ipsilateral to the amblyopic eye; compared with controls, the facilitation persisted at T2 for high contrasts (K90%; Holm-Sidak post hoc method, p < 0.001), while the stimulated hemisphere recovered more quickly from inhibition (Holm-Sidak post hoc method, p < 0.001). Conclusions: tDCS is a promising treatment for amblyopia in adults. The rapid recovery of excitability and the concurrent transcallosal disinhibition following perturbation of cortical activity may support a critical role of interhemispheric balance in the pathophysiology of amblyopia

Re-Assembled Botulinum Neurotoxin Inhibits CNS Functions without Systemic Toxicity

The therapeutic potential of botulinum neurotoxin type A (BoNT/A) has recently been widely recognized. BoNT/A acts to silence synaptic transmission via specific proteolytic cleavage of an essential neuronal protein, SNAP25. The advantages of BoNT/A-mediated synaptic silencing include very long duration, high potency and localized action. However, there is a fear of possible side-effects of BoNT/A due to its diffusible nature which may lead to neuromuscular blockade away from the injection site. We recently developed a “protein-stapling” technology which allows re-assembly of BoNT/A from two separate fragments. This technology allowed, for the first time, safe production of this popular neuronal silencing agent. Here we evaluated the re-assembled toxin in several CNS assays and assessed its systemic effects in an animal model. Our results show that the re-assembled toxin is potent in inhibiting CNS function at 1 nM concentration but surprisingly does not exhibit systemic toxicity after intraperitoneal injection even at 200 ng/kg dose. This shows that the re-assembled toxin represents a uniquely safe tool for neuroscience research and future medical applications

Exploiting botulinum neurotoxins for the study of brain physiology and pathology

Botulinum neurotoxins are metalloproteases that specifically cleave N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in synaptic terminals, resulting in a potent inhibition of vesicle fusion and transmitter release. The family comprises different serotypes (BoNT/A to BoNT/G). The natural target of these toxins is represented by the neuromuscular junction, where BoNTs block acetylcholine release. In this review, we describe the actions of botulinum toxins after direct delivery to the central nervous system (CNS), where BoNTs block exocytosis of several transmitters, with near-complete silencing of neural networks. The use of clostridial neurotoxins in the CNS has allowed us to investigate specifically the role of synaptic activity in different physiological and pathological processes. The silencing properties of BoNTs can be exploited for therapeutic purposes, for example to counteract pathological hyperactivity and seizures in epileptogenic brain foci, or to investigate the role of activity in degenerative diseases like prion disease. Altogether, clostridial neurotoxins and their derivatives hold promise as powerful tools for both the basic understanding of brain function and the dissection and treatment of activity-dependent pathogenic pathways

The Corpus Callosum and the Visual Cortex: Plasticity Is a Game for Two

Throughout life, experience shapes and selects the most appropriate brain functional connectivity to adapt to a changing environment. An ideal system to study experience-dependent plasticity is the visual cortex, because visual experience can be easily manipulated. In this paper, we focus on the role of interhemispheric, transcallosal projections in experience-dependent plasticity of the visual cortex. We review data showing that deprivation of sensory experience can modify the morphology of callosal fibres, thus altering the communication between the two hemispheres. More importantly, manipulation of callosal input activity during an early critical period alters developmental maturation of functional properties in visual cortex and modifies its ability to remodel in response to experience. We also discuss recent data in rat visual cortex, demonstrating that the corpus callosum plays a role in binocularity of cortical neurons and is involved in the plastic shift of eye preference that follows a period of monocular eyelid suture (monocular deprivation) in early age. Thus, experience can modify the fine connectivity of the corpus callosum, and callosal connections represent a major pathway through which experience can mediate functional maturation and plastic rearrangements in the visual cortex