Optical probing of sodium dynamics in neurons and astrocytes.

Changes in intracellular Na(+) concentration underlie essential neurobiological processes, but few reliable tools exist for their measurement. Here we characterize a new synthetic Na(+)-sensitive fluorescent dye, Asante Natrium Green (ANG), with unique properties. This indicator was excitable in the visible spectrum and by two-photon illumination, suffered little photobleaching and located to the cytosol were it remained for long durations without noticeable unwanted effects on basic cell properties. When used in brain tissue, ANG yielded a bright fluorescent signal during physiological Na(+) responses both in neurons and astrocytes. Synchronous electrophysiological and fluorometric recordings showed that ANG produced accurate Na(+) measurement in situ. This new Na(+) indicator opens innovative ways of probing neuronal circuits.

Roles of astrocytic sodium dynamics in the neurometabolic coupling : a study by cellular imaging

RESUME LARGE PUBLIC Le système nerveux central est principalement composé de deux types de cellules :les neurones et les cellules gliales. Ces dernières, bien que l'emportant en nombre sur les neurones, ont longtemps été considérées comme des cellules sans intérêts par les neuroscientifiques. Hors, les connaissances modernes à leurs sujets indiquent qu'elles participent à la plupart des tâches physiologiques du cerveau. Plus particulièrement, elles prennent part aux processus énergétiques cérébraux. Ceux-ci, en plus d'être vitaux, sont particulièrement intrigants puisque le cerveau représente seulement 2 % de la masse corporelle mais consomme environ 25 % du glucose (substrat énergétique) corporel. Les astrocytes, un type de cellules gliales, jouent un rôle primordial dans cette formidable utilisation de glucose par le cerveau. En effet, l'activité neuronale (transmission de l'influx nerveux) est accompagnée d'une augmentation de la capture de glucose, issu de la circulation sanguine, par les astrocytes. Ce phénomène est appelé le «couplage neurométabolique » entre neurones et astrocytes. L'ion sodium fait partie des mécanismes cellulaires entrant en fonction lors de ces processus. Ainsi, dans le cadre de cette thèse, les aspects dynamiques de la régulation du sodium astrocytaire et leurs implications dans le couplage neurométabolique ont été étudiés par des techniques d'imagerie cellulaires. Ces études ont démontré que les mitochondries, machineries cellulaires convertissant l'énergie contenue dans le glucose, participent à la régulation du sodium astrocytaire. De plus, ce travail de thèse a permis de découvrir que les astrocytes sont capables de se transmettre, sous forme de vagues de sodium se propageant de cellules en cellules, un message donnant l'ordre d'accroître leur consommation d'énergie. Cette voie de signalisation leur permettrait de fournir de l'énergie aux neurones suite à leur activation. RESUME Le glutamate libéré dans la fente synaptique pendant l'activité neuronale, est éliminé par les astrocytes environnants. Le glutamate est co-transporté avec des ions sodiques, induisant une augmentation intracellulaire de sodium (Na+i) dans les astrocytes. Cette élévation de Na+i déclenche une cascade de mécanismes moléculaires qui aboutissent à la production de substrats énergétiques pouvant être utilisés par les neurones. Durant cette thèse, la mesure simultanée du sodium mitochondrial (Na+mit) et cytosolique par des techniques d'imagerie utilisant des sondes fluorescentes spécifiques, a indiqué que les variations de Na+i induites par le transport du glutamate sont transmises aux mitochondries. De plus, les voies d'entrée et de sortie du sodium mitochondrial ont été identifiées. L'échangeur de Na+ et de Ca2+ mitochondrial semble jouer un rôle primordial dans l'influx de Na+mit, alors que l'efflux de Na+mit est pris en charge par l'échangeur de Na+ et de H+ mitochondrial. L'étude du Na+mit a nécessité l'utilisation d'un système de photoactivation. Les sources de lumière ultraviolette (UV) classiques utilisées à cet effet (lasers, lampes à flash) ayant plusieurs désavantages, une alternative efficace et peu coûteuse a été développée. Il s'agit d'un système compact utilisant une diode électroluminescente (LED) à haute puissance et de longueur d'onde de 365nm. En plus de leurs rôles dans le couplage neurométabolique, les astrocytes participent à la signalisation multicellulaire en transmettant des vagues intercellulaires de calcium. Ce travail de thèse démontre également que des vagues intercellulaires de sodium peuvent être évoquées en parallèle à ces vagues calciques. Le glutamate, suite à sa libération par un mécanisme dépendent du calcium, est réabsorbé par les transporteurs au glutamate. Ce mécanisme a pour conséquence la génération de vagues sodiques se propageant de cellules en cellules. De plus, ces vagues sodiques sont corrélées spatialement avec une consommation accrue de glucose par les astrocytes. En conclusion, ce travail de thèse a permis de montrer que le signal sodique astrocytaire, déclenché en réponse au glutamate, se propage à la fois de façon intracellulaire aux mitochondries et de façon intercellulaire. Ces résultats suggèrent que les astrocytes fonctionnent comme un réseau de cellules nécessaire au couplage énergétique concerté entre neurones et astrocytes et que le sodium est un élément clé dans les mécanismes de signalisations cellulaires sous-jacents. SUMMARY Glutamate, released in the synaptic cleft during neuronal activity, is removed by surrounding astrocytes. Glutamate is taken-up with Na+ ions by specific transporters, inducing an intracellular Na+ (Na+i) elevation in astrocytes which triggers a cascade of molecular mechanisms that provides metabolic substrates to neurons. Thus, astrocytic Na+i homeostasis represents a key component of the so-called neurometabolic coupling. In this context, the first part of this thesis work was aimed at investigating whether cytosolic Na+ changes are transmitted to mitochondria, which could therefore influence their function and contribute to the overall intracellular Na+ regulation. Simultaneous monitoring of both mitochondrial Na+ (Na+mit) and cytosolic Na+ changes with fluorescent dyes revealed that glutamate-evoked cytosolic Na+ elevations are indeed transmitted to mitochondria. The mitochondrial Na+/Ca2+ exchangers have a prominent role in the regulation of Na+mit influx pathway, and Na+mit extrusion appears to be mediated by Na+/H+ exchangers. To demonstrate the implication of Na+/Ca2+ exchangers, this study has required the technical development of an UV-flash photolysis system. Because light sources for flash photolysis have to be powerful and in the near UV range, the use of UV lasers or flash lamps is usually required. As an alternative to these UV sources that have several drawbaks, we developped a compact, efficient and lowcost flash photolysis system which employs a high power 365nm light emitting diode. In addition to their role in neurometabolic coupling, astrocytes participate in multicellular signaling by transmitting intercellular Ca2+ waves. The third part of this thesis show that intercellular Na+ waves can be evoked in parallel to Ca2+ waves. Glutamate released by a Ca2+ wave-dependent mechanism is taken up by glutamate transporters, resulting in a regenerative propagation of cytosolic Na+ increases. Na+ waves in turn lead to a spatially correlated increase in glucose uptake. In conclusion, the present thesis demonstrates that glutamate-induced Na+ changes occurring in the cytosol of astrocytes propagate to both the mitochondrial matrix and the astrocytic network. These results furthermore support the view that astrocytic Na+ is a signal coupled to the brain energy metabolism.

MRS glucose mapping and PET joining forces: re-evaluation of the lumped constant in the rat brain under isoflurane anaesthesia.

Although numerous positron emission tomography (PET) studies with (18) F-fluoro-deoxyglucose (FDG) have reported quantitative results on cerebral glucose kinetics and consumption, there is a large variation between the absolute values found in the literature. One of the underlying causes is the inconsistent use of the lumped constants (LCs), the derivation of which is often based on multiple assumptions that render absolute numbers imprecise and errors hard to quantify. We combined a kinetic FDG-PET study with magnetic resonance spectroscopic imaging (MRSI) of glucose dynamics in Sprague-Dawley rats to obtain a more comprehensive view of brain glucose kinetics and determine a reliable value for the LC under isoflurane anaesthesia. Maps of Tmax /CMRglc derived from MRSI data and Tmax determined from PET kinetic modelling allowed to obtain an LC-independent CMRglc . The LC was estimated to range from 0.33 ± 0.07 in retrosplenial cortex to 0.44 ± 0.05 in hippocampus, yielding CMRglc between 62 ± 14 and 54 ± 11 μmol/min/100 g, respectively. These newly determined LCs for four distinct areas in the rat brain under isoflurane anaesthesia provide means of comparing the growing amount of FDG-PET data available from translational studies.

Temporal dynamics of auditory what and where processing in humans

Recent evidence suggests the human auditory system is organized,like the visual system, into a ventral 'what' pathway, devoted toidentifying objects and a dorsal 'where' pathway devoted to thelocalization of objects in space w1x. Several brain regions have beenidentified in these two different pathways, but until now little isknown about the temporal dynamics of these regions. We investigatedthis issue using 128-channel auditory evoked potentials(AEPs).Stimuli were stationary sounds created by varying interaural timedifferences and environmental real recorded sounds. Stimuli ofeach condition (localization, recognition) were presented throughearphones in a blocked design, while subjects determined theirposition or meaning, respectively.AEPs were analyzed in terms of their topographical scalp potentialdistributions (segmentation maps) and underlying neuronalgenerators (source estimation) w2x.Fourteen scalp potential distributions (maps) best explained theentire data set.Ten maps were nonspecific (associated with auditory stimulationin general), two were specific for sound localization and two werespecific for sound recognition (P-values ranging from 0.02 to0.045).Condition-specific maps appeared at two distinct time periods:;200 ms and ;375-550 ms post-stimulus.The brain sources associated with the maps specific for soundlocalization were mainly situated in the inferior frontal cortices,confirming previous findings w3x. The sources associated withsound recognition were predominantly located in the temporal cortices,with a weaker activation in the frontal cortex.The data show that sound localization and sound recognitionengage different brain networks that are apparent at two distincttime periods.References1. Maeder et al. Neuroimage 2001.2. Michel et al. Brain Research Review 2001.3. Ducommun et al. Neuroimage 2002.

The Munc13 proteins differentially regulate readily releasable pool dynamics and calcium-dependent recovery at a central synapse.

The Munc13 gene family encodes molecules located at the synaptic active zone that regulate the reliability of synapses to encode information over a wide range of frequencies in response to action potentials. In the CNS, proteins of the Munc13 family are critical in regulating neurotransmitter release and synaptic plasticity. Although Munc13-1 is essential for synaptic transmission, it is paradoxical that Munc13-2 and Munc13-3 are functionally dispensable at some synapses, although their loss in other synapses leads to increases in frequency-dependent facilitation. We addressed this issue at the calyx of Held synapse, a giant glutamatergic synapse that we found to express all these Munc13 isoforms. We studied their roles in the regulation of synaptic transmission and their impact on the reliability of information transfer. Through detailed electrophysiological analyses of Munc13-2, Munc13-3, and Munc13-2-3 knock-out and wild-type mice, we report that the combined loss of Munc13-2 and Munc13-3 led to an increase in the rate of calcium-dependent recovery and a change in kinetics of release of the readily releasable pool. Furthermore, viral-mediated overexpression of a dominant-negative form of Munc13-1 at the calyx demonstrated that these effects are Munc13-1 dependent. Quantitative immunohistochemistry using Munc13-fluorescent protein knock-in mice revealed that Munc13-1 is the most highly expressed Munc13 isoform at the calyx and the only one highly colocalized with Bassoon at the active zone. Based on these data, we conclude that Munc13-2 and Munc13-3 isoforms limit the ability of Munc13-1 to regulate calcium-dependent replenishment of readily releasable pool and slow pool to fast pool conversion in central synapses.

Using Pareto optimality to explore the topology and dynamics of the human connectome.

Graph theory has provided a key mathematical framework to analyse the architecture of human brain networks. This architecture embodies an inherently complex relationship between connection topology, the spatial arrangement of network elements, and the resulting network cost and functional performance. An exploration of these interacting factors and driving forces may reveal salient network features that are critically important for shaping and constraining the brain's topological organization and its evolvability. Several studies have pointed to an economic balance between network cost and network efficiency with networks organized in an 'economical' small-world favouring high communication efficiency at a low wiring cost. In this study, we define and explore a network morphospace in order to characterize different aspects of communication efficiency in human brain networks. Using a multi-objective evolutionary approach that approximates a Pareto-optimal set within the morphospace, we investigate the capacity of anatomical brain networks to evolve towards topologies that exhibit optimal information processing features while preserving network cost. This approach allows us to investigate network topologies that emerge under specific selection pressures, thus providing some insight into the selectional forces that may have shaped the network architecture of existing human brains.

Cooperative and Competitive Spreading Dynamics on the Human Connectome.

Increasingly detailed data on the network topology of neural circuits create a need for theoretical principles that explain how these networks shape neural communication. Here we use a model of cascade spreading to reveal architectural features of human brain networks that facilitate spreading. Using an anatomical brain network derived from high-resolution diffusion spectrum imaging (DSI), we investigate scenarios where perturbations initiated at seed nodes result in global cascades that interact either cooperatively or competitively. We find that hub regions and a backbone of pathways facilitate early spreading, while the shortest path structure of the connectome enables cooperative effects, accelerating the spread of cascades. Finally, competing cascades become integrated by converging on polysensory associative areas. These findings show that the organizational principles of brain networks shape global communication and facilitate integrative function.

Experience-based Auditory Predictions Modulate Brain Activity to Silence as Do Real Sounds.

Interactions between stimuli's acoustic features and experience-based internal models of the environment enable listeners to compensate for the disruptions in auditory streams that are regularly encountered in noisy environments. However, whether auditory gaps are filled in predictively or restored a posteriori remains unclear. The current lack of positive statistical evidence that internal models can actually shape brain activity as would real sounds precludes accepting predictive accounts of filling-in phenomenon. We investigated the neurophysiological effects of internal models by testing whether single-trial electrophysiological responses to omitted sounds in a rule-based sequence of tones with varying pitch could be decoded from the responses to real sounds and by analyzing the ERPs to the omissions with data-driven electrical neuroimaging methods. The decoding of the brain responses to different expected, but omitted, tones in both passive and active listening conditions was above chance based on the responses to the real sound in active listening conditions. Topographic ERP analyses and electrical source estimations revealed that, in the absence of any stimulation, experience-based internal models elicit an electrophysiological activity different from noise and that the temporal dynamics of this activity depend on attention. We further found that the expected change in pitch direction of omitted tones modulated the activity of left posterior temporal areas 140-200 msec after the onset of omissions. Collectively, our results indicate that, even in the absence of any stimulation, internal models modulate brain activity as do real sounds, indicating that auditory filling in can be accounted for by predictive activity.

Neuroenergetic Response to Prolonged Cerebral Glucose Depletion after Severe Brain Injury and the Role of Lactate.

Lactate may represent a supplemental fuel for the brain. We examined cerebral lactate metabolism during prolonged brain glucose depletion (GD) in acute brain injury (ABI) patients monitored with cerebral microdialysis (CMD). Sixty episodes of GD (defined as spontaneous decreases of CMD glucose from normal to low [<1.0 mmol/L] for at least 2 h) were identified among 26 patients. During GD, we found a significant increase of CMD lactate (from 4±2.3 to 5.4±2.9 mmol/L), pyruvate (126.9±65.1 to 172.3±74.1 μmol/L), and lactate/pyruvate ratio (LPR; 27±6 to 35±9; all, p<0.005), while brain oxygen and blood lactate remained normal. Dynamics of lactate and glucose supply during GD were further studied by analyzing the relationships between blood and CMD samples. There was a strong correlation between blood and brain lactate when LPR was normal (r=0.56; p<0.0001), while an inverse correlation (r=-0.11; p=0.04) was observed at elevated LPR >25. The correlation between blood and brain glucose also decreased from r=0.62 to r=0.45. These findings in ABI patients suggest increased cerebral lactate delivery in the absence of brain hypoxia when glucose availability is limited and support the concept that lactate acts as alternative fuel.

Mdx myotubes have normal excitability but show reduced contraction-relaxation dynamics.

The pathogenesis of Duchenne muscular dystrophy (DMD), characterised by lack of the cytoskeletal protein dystrophin, is not completely understood. An early event in the degenerative process of DMD muscle could be a rise in cytosolic calcium concentration. In order to investigate whether this leads to alterations of contractile behaviour, we studied the excitability and contractile properties of cultured myotubes from control (C57BL/10) and mdx mice, an animal model for DMD. The myotubes were stimulated electrically and their motion was recorded photometrically. No significant differences were found between control and mdx myotubes with respect to the following parameters: chronaxy and rheobase (0.33 +/- 0.03 ms and 23 +/- 4 V vs. 0.39 +/- 0.07 ms and 22 +/- 2 V for C57 and mdx myotubes, respectively), tetanisation frequency (a similar distribution pattern was found between 5 and 30 Hz), fatigue during tetanus (found in 35% of both types of myotubes) and post-tetanic contracture. In contrast, contraction and relaxation times were longer (P < 0.005) in mdx (36 +/- 2 and 142 +/- 13 ms, respectively) than in control myotubes (26 +/- 1 and 85 +/- 9 ms, respectively). Together with our earlier findings, these results suggest a decreased capacity for calcium removal in mdx cells leading, in particular, to alterations of muscle relaxation.

The dynamics of interfaces: Seven authors in search of encounters across levels of description of an event involving a mother, father, and baby

The interfaces between the intrapsychic, interactional, and intergenerational domains are a new frontier. As a pilot, we exposed ourselves to a complex but controllable situation as viewed by people whose main interest is in one of the three interfaces; we also fully integrated the subjects in the team, to learn about their subjective perspectives and to provide them with an enriching experience. We started with a brief "triadification" sequence (i.e., moving from a "two plus one" to a "three together" family organization). Considering this sequence as representing at a micro level many larger family transitions, we proceeded with a microanalytic interview, a psychodynamic investigation, and a family interview. As expected, larger patterns of correspondences are emerging. Central questions under debate are: What are the most appropriate units at each level of description and what are their articulations between these levels? What is the status of "triadification"? Les interfaces entre les domaines intrapsychiques, interactionnels et intergénérationnels représentent une nouvelle frontiére. A titre exploratoire, nous nous sommes exposés à une situation complexe mais contrǒlable ainsi que le voient ceux dont I'intérět principal se porte sur l'une de ces trois interfaces. Nous avons aussi entièrement intégré les sujets dans l'équipe, de facon à comprendre leur perspective subjective et à leur offrir une expérience enrichissante. Nous avons commencé avec une brève séquence de "triadification," c'est-à-dire passer d'une organisation familiale "deux plus un" à Ltne organisation familiale "trois (add sentenc)ensemble." Considérant cette séquence comme representative à un niveau microscopique de transitions familiales bien plus larges, nous avons procedé à l'entretien microanalytique, à une enquěte psychodynamique et à un entretien familial. Comme prévu, de grands patterns de correspondances émergent. Les questions essentielles sur lesquelles portent le débat sont: quelles les unités les plus appropiées à chaque niveau de description et quelles sont les articulations entre ces niveaux? Quel est le statut de la "triadification"?

Ab initio modeling and molecular dynamics simulation of the alpha 1b-adrenergic receptor activation.

This work describes the ab initio procedure employed to build an activation model for the alpha 1b-adrenergic receptor (alpha 1b-AR). The first version of the model was progressively modified and complicated by means of a many-step iterative procedure characterized by the employment of experimental validations of the model in each upgrading step. A combined simulated (molecular dynamics) and experimental mutagenesis approach was used to determine the structural and dynamic features characterizing the inactive and active states of alpha 1b-AR. The latest version of the model has been successfully challenged with respect to its ability to interpret and predict the functional properties of a large number of mutants. The iterative approach employed to describe alpha 1b-AR activation in terms of molecular structure and dynamics allows further complications of the model to allow prediction and interpretation of an ever-increasing number of experimental data.

La dépendance à l'égard de l'environnement lors de lésions cérébrales: conduites d'imitation, de préhension et d'utilisation [Environmental dependence in brain lesions: imitative behavior, prehension and utilization].

This paper reviews the literature on clinical signs such as imitation behavior, grasp reaction, manipulation of tools, utilization behavior, environmental dependency, hyperlexia, hypergraphia and echolalia. Some aspects of this semiology are of special interest because they refer to essential notions such as free-will and autonomy.

Long-term follow-up of high-dose chemotherapy with autologous stem-cell transplantation and response-adapted whole-brain radiotherapy for newly diagnosed primary CNS lymphoma: results of the multicenter Ostdeutsche Studiengruppe Hamato-Onkologie OSHO-53 phase II study

Introduction: We previously reported the results of a phase II study for patients with newly diagnosed primary CNS lymphoma (PCNSL) treated with autologous peripheral blood stem-cell transplantation (aPBSCT) and responseadapted whole brain radiotherapy (WBRT). The purpose of this report is to update the initial results and provide long-term data regarding overall survival, prognostic factors, and the risk of treatment-related neurotoxicity.Methods: A long-term follow-up was conducted on surviving primary central nervous system lymphoma patients having been treated according to the ,,OSHO-53 study", which was initiated by the Ostdeutsche Studiengruppe Hamatologie-Onkologie. Between August 1999 and October 2004 twentythree patients with an average age of 55 and median Karnofsky performance score of 70% were enrolled and received high-dose mthotrexate (HD-MTX) on days 1 and 10. In case of at least a partial remission (PR), high-dose busulfan/ thiotepa (HD-BuTT) followed by aPBSCT was performed. Patients without response to induction or without complete remission (CR) after HD-BuTT received WBRT. All patients (n=8), who are alive in 2011, were contacted and Mini Mental State examination (MMSE) and the EORTC QLQ-C30 were performed.Results: Eight patients are still alive with a median follow-up of 116,9 months (79 - 141, range). One of them suffered from a late relapse eight and a half years after initial diagnosis of PCNSL, another one suffers from a gall bladder carcinoma. Both patients are alive, the one with the relapse of PCNSL has finished rescue therapy and is further observed, the one with gall baldder carcinoma is still under therapy. MMSE and QlQ-C30 showed impressive results in the patients, who were not irradiated. Only one of the irradiated patients is still alive with a clear neurologic deficit but acceptable quality of life.Conclusions: Long-term follow-up of our patients, who were included in the OSHO-53 study show an overall survival of 30 percent. If WBRT can be avoided no long-term neurotoxicity has been observed and the patients benefit from excellent Quality of Life. Induction chemotherapy with two cycles of HD-MTX should be intensified to improve the unsatisfactory OAS of 30 percent.