Toxoplasma gondii Actively Inhibits Neuronal Function in Chronically Infected Mice

Upon infection with the obligate intracellular parasite Toxoplasma gondii, fast replicating tachyzoites infect a broad spectrum of host cells including neurons. Under the pressure of the immune response, tachyzoites convert into slow-replicating bradyzoites, which persist as cysts in neurons. Currently, it is unclear whether T. gondii alters the functional activity of neurons, which may contribute to altered behaviour of T. gondii–infected mice and men. In the present study we demonstrate that upon oral infection with T. gondii cysts, chronically infected BALB/c mice lost over time their natural fear against cat urine which was paralleled by the persistence of the parasite in brain regions affecting behaviour and odor perception. Detailed immunohistochemistry showed that in infected neurons not only parasitic cysts but also the host cell cytoplasm and some axons stained positive for Toxoplasma antigen suggesting that parasitic proteins might directly interfere with neuronal function. In fact, in vitro live cell calcium (Ca2+) imaging studies revealed that tachyzoites actively manipulated Ca2+ signalling upon glutamate stimulation leading either to hyper- or hypo-responsive neurons. Experiments with the endoplasmatic reticulum Ca2+ uptake inhibitor thapsigargin indicate that tachyzoites deplete Ca2+ stores in the endoplasmatic reticulum. Furthermore in vivo studies revealed that the activity-dependent uptake of the potassium analogue thallium was reduced in cyst harbouring neurons indicating their functional impairment. The percentage of non-functional neurons increased over time In conclusion, both bradyzoites and tachyzoites functionally silence infected neurons, which may significantly contribute to the altered behaviour of the host

Cortical Inactivation by Cooling in Small Animals

Reversible inactivation of the cortex by surface cooling is a powerful method for studying the function of a particular area. Implanted cooling cryoloops have been used to study the role of individual cortical areas in auditory processing of awake-behaving cats. Cryoloops have also been used in rodents for reversible inactivation of the cortex, but recently there has been a concern that the cryoloop may also cool non-cortical structures either directly or via the perfusion of blood, cooled as it passed close to the cooling loop. In this study we have confirmed that the loop can inactivate most of the auditory cortex without causing a significant reduction in temperature of the auditory thalamus or other subcortical structures. We placed a cryoloop on the surface of the guinea pig cortex, cooled it to 2°C and measured thermal gradients across the neocortical surface. We found that the temperature dropped to 20–24°C among cells within a radius of about 2.5 mm away from the loop. This temperature drop was sufficient to reduce activity of most cortical cells and led to the inactivation of almost the entire auditory region. When the temperature of thalamus, midbrain, and middle ear were measured directly during cortical cooling, there was a small drop in temperature (about 4°C) but this was not sufficient to directly reduce neural activity. In an effort to visualize the extent of neural inactivation we measured the uptake of thallium ions following an intravenous injection. This confirmed that there was a large reduction of activity across much of the ipsilateral cortex and only a small reduction in subcortical structures

The large numbers of minicolumns in the primary visual cortex of humans, chimpanzees and gorillas are related to high visual acuity

Minicolumns are thought to be a fundamental neural unit in the neocortex and their replication may have formed the basis of the rapid cortical expansion that occurred during primate evolution. We sought evidence of minicolumns in the primary visual cortex (V-1) of three great apes, three rodents and representatives from three other mammalian orders: Eulipotyphla (European hedgehog), Artiodactyla (domestic pig) and Carnivora (ferret). Minicolumns, identified by the presence of a long bundle of radial, myelinated fibers stretching from layer III to the white matter of silver-stained sections, were found in the human, chimpanzee, gorilla and guinea pig V-1. Shorter bundles confined to one or two layers were found in the other species but represent modules rather than minicolumns. The inter-bundle distance, and hence density of minicolumns, varied systematically both within a local area that might represent a hypercolumn but also across the whole visual field. The distance between all bundles had a similar range for human, chimpanzee, gorilla, ferret and guinea pig: most bundles were 20–45 μm apart. By contrast, the space between bundles was greater for the hedgehog and pig (20–140 μm). The mean density of minicolumns was greater in tangential sections of the gorilla and chimpanzee (1,243–1,287 bundles/mm2) than in human (314–422 bundles/mm2) or guinea pig (643 bundles/mm2). The minicolumnar bundles did not form a hexagonal lattice but were arranged in thin curving and branched bands separated by thicker bands of neuropil/somata. Estimates of the total number of modules/minicolumns within V-1 were strongly correlated with visual acuity

Effet de la stimulation cholinergique sur la perception visuelle chez le rat et l'humain : études comportementales et électrophysiologiques

Le système cholinergique joue un rôle important dans de nombreuses fonctions cognitives telles que l'attention et l'apprentissage perceptuel. La stimulation pharmacologique du système cholinergique par le donépézil, un inhibiteur de l’acétylcholinestérase, est un moyen efficace pour améliorer les fonctions cognitives et le traitement cortical via les récepteurs muscariniques et nicotiniques. En effet, le donépézil permet l'accumulation d'acétylcholine dans la fente synaptique. Toutefois, l’effet de la stimulation pharmacologique du système cholinergique sur le traitement visuel complexe et l’apprentissage perceptuel n’est pas encore bien défini. L'objectif de cette thèse est d'étudier, d'une part, l'effet de la combinaison d’un entrainement visuel répétitif avec une stimulation cholinergique sur les capacités visuelles chez le rat et l’humain et, d'autre part, l’effet de la stimulation pharmacologique du système cholinergique sur la restauration des capacités visuelles dans un modèle de déficit visuel chez les rats. Nos résultats ont montré qu’un entrainement visuel/cholinergique entraînait : 1) une potentialisation à long terme de la réponse visuelle corticale chez le rat, 2) une récupération plus rapide des capacités visuelles chez la rat suite un écrasement du nerf optique 3) une amélioration de la performance dans une tâche perceptivo-cognitive de haut niveau plus rapide et conservée dans le temps chez les jeunes sujets sains. Le patron d’électroencéphalographie chez le sujet humain pratiquant une tâche d’attention visuelle n’est cependant pas modifié par l’administration d’une dose unique de donépézil. Ensembles, ces résultats soulignent le bénéfice considérable de la combinaison d’une stimulation du système cholinergique lors de l’entrainement visuel répétitif afin d'obtenir des améliorations de la perception visuelle. Cela présente une avenue très intéressante pour la réhabilitation chez les humains.The cholinergic system plays an important role in many cognitive functions such as attention and perceptual learning. Pharmacological stimulation of the cholinergic system via donepezil, an acetylcholinesterase inhibitor, is an efficient tool for enhancing cognitive functions and cortical processing via muscarinic and nicotinic receptors. In fact, donepezil allows the build-up of acetylcholine in the synaptic cleft. However, whether pharmacological manipulation of the cholinergic system has an effect on complex visual processing and perceptual learning remains unclear. The goal of this thesis is to investigate on the one hand the effect of combining repetitive visual training with cholinergic enhancement on visual capacities in rats and humans and on the other hand the effect of the pharmacological stimulation of the cholinergic system on visual restoration in a model of visual deficit in rats. Our results showed that cholinergic potentiation induces 1) a long-term potentiation of visual cortical response following repetitive visual stimulation, 2) a faster recovery of brightness discrimination in rats with an optic nerve crush, 3) a faster progression of and a sustained performance in a highly demanding perceptual-cognitive task for healthy young humans. However, the EEG pattern for subjects performing a visual attention task is not modified by a single administration of donepezil. Together these results underline the substantial benefice of combining cholinergic enhancement with visual training in order to obtain visual perception improvements, which presents an interesting avenue for visual rehabilitation paradigm in humans

Nitric oxide regulates the firing rate of neuronal subtypes in the guinea pig ventral cochlear nucleus

The gaseous free radical, nitric oxide (NO) acts as a ubiquitous neuromodulator, contributing to synaptic plasticity in a complex way that can involve either long term potentiation or depression. It is produced by neuronal nitric oxide synthase (nNOS) which is presynaptically expressed and also located postsynaptically in the membrane and cytoplasm of a sub-population of each major neuronal type in the ventral cochlear nucleus (VCN). We have used iontophoresis in vivo to study the effect of the NOS inhibitor L-NAME (L-NG-Nitroarginine methyl ester) and the NO donors SIN-1 (3-Morpholinosydnonimine hydrochloride) and SNOG (S-Nitrosoglutathione) on VCN units under urethane anaesthesia. Collectively, both donors produced increases and decreases in driven and spontaneous firing rates of some neurons. Inhibition of endogenous NO production with L-NAME evoked a consistent increase in driven firing rates in 18% of units without much effect on spontaneous rate. This reduction of gain produced by endogenous NO was mirrored when studying the effect of L-NAME on NMDA (N-Methyl-D-aspartic acid)-evoked excitation, with 30% of units showing enhanced NMDA-evoked excitation during L-NAME application (reduced NO levels). Approximately 25% of neurons contain nNOS and the NO produced can modulate the firing rate of the main principal cells: medium stellates (choppers), large stellates (onset responses) and bushy cells (primary like responses). The main endogenous role of NO seems to be to partly suppress driven firing rates associated with NMDA channel activity but there is scope for it to increase neural gain if there were a pathological increase in its production following hearing loss

Magnetic resonance imaging of appetite-induced hypothalamic activity

Tesis doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física de la Materia Condensada. Fecha de lectura: 26-10-2013La obesidad es un síndrome pandémico que subyace a las enfermedades más prevalentes y mórbidas en los países desarrollados. Es resultado de un desequilibrio en la regulación del apetito y del gasto energético; dos mecanismos que están fundamentalmente controlados por el hipotálamo. Las técnicas de imagen por resonancia magnética constituyen una herramienta excelente de evaluación anatómica y funcional del cerebro, en condiciones fisiológicas y patológicas, proporcionando un constante creciente tipo de aplicaciones. La tesis que aquí presento se ha centrado en el desarrollo de nuevas estrategias para evaluar de forma no invasiva los procesos de regulación cerebral del apetito en ratones y seres humanos, utilizando técnicas de resonancia magnética pesada en difusión. El Capítulo 1 proporciona una introducción a los principios básicos de la imagen por resonancia magnética, así como alguna de sus aplicaciones, recopilando las principales nociones fisiológicas de los mecanismos hipotalámicos de la regulación del apetito. También incluye s una breve compilación de las técnicas de neuroimagen más utilizadas en la evaluación de procesos relacionados con el apetito. En el Capítulo 2 describo el desarrollo y la implementación de una nueva técnica de imagen funcional basada en difusión para la detección de la actividad hipotalámica por apetito, en ratones y en seres humanos. El Capítulo 3 está dedicado a la aplicación de esta técnica a cuatro situaciones experimentales distintas, diseñadas para evaluar la respuesta específica de los núcleos hipotalámicos en procesos en los que la regulación del apetito y el balance energético están alterados. Finalmente, en el Capítulo 4 muestro la comparación del uso de la metodología analizada con distintos modelos de difusión- con los resultados obtenidos mediante la aplicación de una técnica funcional más convencional, ambas aplicadas al estudio de los efectos hipotalámicos de la administración de glucosa a ratones ayunados. En conclusión, mi Tesis doctoral demuestra que la técnica de imagen de resonancia magnética pesada en difusión proporciona un instrumento nuevo y robusto para el estudio de la regulación del apetito de forma no invasiva.Obesity is a pandemic syndrome that underlies the most morbid and prevalent diseases in developed countries. It results from an imbalance in appetite regulation and energy expenditure, two processes that are fundamentally controlled by the hypothalamus. Magnetic Resonance Imaging methods are excellently endowed to assess brain anatomy and function, under physiological and pathological conditions, providing an always increasing array of approaches. In this dissertation, I will introduce a collection of new strategies to evaluate non-invasively appetite regulation in the brain of mice and humans, based in the use of diffusion weighted magnetic resonance imaging methods. Chapter 1 introduces general concepts on the magnetic resonance imaging phenomenon and its applications, reviewing the key physiological mechanisms supporting hypothalamic appetite regulation. A short compilation of the most common neuroimaging techniques used to evaluate appetiterelated processes is also included. Chapter 2 describes the development and implementation of a new functional diffusion weighted imaging method applied to the detection of hypothalamic activity by fasting in mice and humans. Chapter 3 covers four different experimental manipulations designed to probe the role of specific hypothalamic nuclei in the regulation of appetite control and energy balance, under conditions where these are intentionally altered. Finally, Chapter 4 compares the use of the methodology analysed with different models of diffusion- with the results obtained with a more conventional functional imaging technique, both applied to the paradigm of glucose administration to fasting mice. In conclusion, this dissertation demonstrates that diffusion weighted magnetic resonance imaging methods provide a novel and useful approach to investigate appetite regulation non-invasively

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds