'Neurons in Action' in Action - Educational settings for simulations and tutorials using NEURON

Neurons in Action (NIA1, 2000; NIA1.5, 2004; NIA2, 2007), a set of tutorials and linked simulations, is designed to acquaint students with neuronal physiology through interactive, virtual laboratory experiments. Here we explore the uses of NIA in lecture, both interactive and didactic, as well as in the undergraduate laboratory, in the graduate seminar course, and as an examination tool through homework and problem set assignments. NIA, made with the simulator NEURON (http://www.neuron.yale.edu/neuron/), displays voltages, currents, and conductances in a membrane patch or signals moving within the dendrites, soma and/or axon of a neuron. Customized simulations start with the plain lipid bilayer and progress through equilibrium potentials; currents through single Na and K channels; Na and Ca action potentials; voltage clamp of a patch or a whole neuron; voltage spread and propagation in axons, motoneurons and nerve terminals; synaptic excitation and inhibition; and advanced topics such as channel kinetics and coincidence detection. The user asks and answers "what if" questions by specifying neuronal parameters, ion concentrations, and temperature, and the experimental results are then plotted as conductances, currents, and voltage changes. Such exercises provide immediate confirmation or refutation of the student's ideas to guide their learning. The tutorials are hyperlinked to explanatory information and to original research papers. Although the NIA tutorials were designed as a sequence to empower a student with a working knowledge of fundamental neuronal principles, we find that faculty are using the individual tutorials in a variety of educational situations, some of which are described here. Here we offer ideas to colleagues using interactive software, whether NIA or another tool, for educating students of differing backgrounds in the subject of neurophysiology.

A speech interface for air traffic control terminals

Several issues concerning the current use of speech interfaces are discussed and the design and development of a speech interface that enables air traffic controllers to command and control their terminals by voice is presented. A special emphasis is made in the comparison between laboratory experiments and field experiments in which a set of ergonomics-related effects are detected that cannot be observed in the controlled laboratory experiments. The paper presents both objective and subjective performance obtained in field evaluation of the system with student controllers at an air traffic control (ATC) training facility. The system exhibits high word recognition test rates (0.4% error in Spanish and 1.5% in English) and low command error (6% error in Spanish and 10.6% error in English in the field tests). Subjective impression has also been positive, encouraging future development and integration phases in the Spanish ATC terminals designed by Aeropuertos Españoles y Navegación Aérea (AENA).

Berth Management in Container Terminals: Quality of Service

This paper empirically evaluates container terminal service attributes. The methodology proposed focuses on statistical control. Based on the concept of service segmentation, the authors employed control charts to classify container terminal services. The purpose of control charts is to allow simple detection of events that are indicative of actual process change. This simple decision can be difficult where the process characteristic is continuously varying, the control chart provides statistically objective criteria of change. When change is detected and considered good its cause should be identified and possibly become the new way of working, where the change is bad then its cause should be identified and eliminated. Both theoretical and practical implications of the research findings are discussed in this paper.

Berth management in container terminals: quality of service

This paper empirically evaluates container terminal service attributes. The methodology proposed focuses on statistical control. Based on the concept of service segmentation, we employed control charts to classify container terminal services. The purpose of control charts is to allow simple detection of events that are indicative of actual process change. This simple decision can be difficult where the process characteristic is continuously varying; the control chart provides statistically objective criteria of change. When change is detected and considered good its cause should be identified and possibly become the new way of working, where the change is bad then its cause should be identified and eliminated. This paper is organized as follows: Section 1 is the introduction, Section 2 provides a brief note on other studies that inspired this research, section 3 focuses on the methodology used, and develops the results obtained and finally conclusions are shown in Section 4. Theoretical and practical implications of the research findings are discussed.

Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons

Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation.

Colocalization of α-actinin and Synaptopodin in the Pyramidal Cell Axon Initial Segment

The cisternal organelle that resides in the axon initial segment (AIS) of neocortical and hippocampal pyramidal cells is thought to be involved in regulating the Ca(2+) available to maintain AIS scaffolding proteins, thereby preserving normal AIS structure and function. Through immunocytochemistry and correlative light and electron microscopy, we show here that the actin-binding protein ?-actinin is present in the typical cistenal organelle of rodent pyramidal neurons as well as in a large structure in the AIS of a subpopulation of layer V pyramidal cells that we have called the "giant saccular organelle." Indeed, this localization of ?-actinin in the AIS is dependent on the integrity of the actin cytoskeleton. Moreover, in the cisternal organelle of cultured hippocampal neurons, ?-actinin colocalizes extensively with synaptopodin, a protein that interacts with both actin and ?-actinin, and they appear concomitantly during the development of these neurons. Together, these results indicate that ?-actinin and the actin cytoskeleton are important components of the cisternal organelle that are probably required to stabilize the AIS.

A model of the spatially dependent mechanical properties of the axon during its growth

Neuronal growth is a complex process involving many intra- and extracellular mechanisms which are collaborating conjointly to participate to the development of the nervous system. More particularly, the early neocortical development involves the creation of a multilayered structure constituted by neuronal growth (driven by axonal or dendritic guidance cues) as well as cell migration. The underlying mechanisms of such structural lamination not only implies important biochemical changes at the intracellular level through axonal microtubule (de)polymerization and growth cone advance, but also through the directly dependent stress/stretch coupling mechanisms driving them. Efforts have recently focused on modeling approaches aimed at accounting for the effect of mechanical tension or compression on the axonal growth and subsequent soma migration. However, the reciprocal influence of the biochemical structural evolution on the mechanical properties has been mostly disregarded. We thus propose a new model aimed at providing the spatially dependent mechanical properties of the axon during its growth. Our in-house finite difference solver Neurite is used to describe the guanosine triphosphate (GTP) transport through the axon, its dephosphorylation in guanosine diphosphate (GDP), and thus the microtubules polymerization. The model is calibrated against experimental results and the tensile and bending mechanical stiffnesses are ultimately inferred from the spatially dependent microtubule occupancy. Such additional information is believed to be of drastic relevance in the growth cone vicinity, where biomechanical mechanisms are driving axonal growth and pathfinding. More specifically, the confirmation of a lower stiffness in the distal axon ultimately participates in explaining the controversy associated to the tensile role of the growth cone.

A methodology proposal to obtain operational parameter forecasts and operational risk assessment in container terminals

Las terminales de contenedores son sistemas complejos en los que un elevado número de actores económicos interactúan para ofrecer servicios de alta calidad bajo una estricta planificación y objetivos económicos. Las conocidas como "terminales de nueva generación" están diseñadas para prestar servicio a los mega-buques, que requieren tasas de productividad que alcanzan los 300 movimientos/ hora. Estas terminales han de satisfacer altos estándares dado que la competitividad entre terminales es elevada. Asegurar la fiabilidad de las planificaciones del atraque es clave para atraer clientes, así como reducir al mínimo el tiempo que el buque permanece en el puerto. La planificación de las operaciones es más compleja que antaño, y las tolerancias para posibles errores, menores. En este contexto, las interrupciones operativas deben reducirse al mínimo. Las principales causas de dichas perturbaciones operacionales, y por lo tanto de incertidumbre, se identifican y caracterizan en esta investigación. Existen una serie de factores que al interactuar con la infraestructura y/o las operaciones desencadenan modos de fallo o parada operativa. Los primeros pueden derivar no solo en retrasos en el servicio sino que además puede tener efectos colaterales sobre la reputación de la terminal, o incluso gasto de tiempo de gestión, todo lo cual supone un impacto para la terminal. En el futuro inmediato, la monitorización de las variables operativas presenta gran potencial de cara a mejorar cualitativamente la gestión de las operaciones y los modelos de planificación de las terminales, cuyo nivel de automatización va en aumento. La combinación del criterio experto con instrumentos que proporcionen datos a corto y largo plazo es fundamental para el desarrollo de herramientas que ayuden en la toma de decisiones, ya que de este modo estarán adaptadas a las auténticas condiciones climáticas y operativas que existen en cada emplazamiento. Para el corto plazo se propone una metodología con la que obtener predicciones de parámetros operativos en terminales de contenedores. Adicionalmente se ha desarrollado un caso de estudio en el que se aplica el modelo propuesto para obtener predicciones de la productividad del buque. Este trabajo se ha basado íntegramente en datos proporcionados por una terminal semi-automatizada española. Por otro lado, se analiza cómo gestionar, evaluar y mitigar el efecto de las interrupciones operativas a largo plazo a través de la evaluación del riesgo, una forma interesante de evaluar el effecto que eventos inciertos pero probables pueden generar sobre la productividad a largo plazo de la terminal. Además se propone una definición de riesgo operativo junto con una discusión de los términos que representan con mayor fidelidad la naturaleza de las actividades y finalmente, se proporcionan directrices para gestionar los resultados obtenidos. Container terminals are complex systems where a large number of factors and stakeholders interact to provide high-quality services under rigid planning schedules and economic objectives. The socalled next generation terminals are conceived to serve the new mega-vessels, which are demanding productivity rates up to 300 moves/hour. These terminals need to satisfy high standards because competition among terminals is fierce. Ensuring reliability in berth scheduling is key to attract clients, as well as to reduce at a minimum the time that vessels stay the port. Because of the aforementioned, operations planning is becoming more complex, and the tolerances for errors are smaller. In this context, operational disturbances must be reduced at a minimum. The main sources of operational disruptions and thus, of uncertainty, are identified and characterized in this study. External drivers interact with the infrastructure and/or the activities resulting in failure or stoppage modes. The later may derive not only in operational delays but in collateral and reputation damage or loss of time (especially management times), all what implies an impact for the terminal. In the near future, the monitoring of operational variables has great potential to make a qualitative improvement in the operations management and planning models of terminals that use increasing levels of automation. The combination of expert criteria with instruments that provide short- and long-run data is fundamental for the development of tools to guide decision-making, since they will be adapted to the real climatic and operational conditions that exist on site. For the short-term a method to obtain operational parameter forecasts in container terminals. To this end, a case study is presented, in which forecasts of vessel performance are obtained. This research has been entirely been based on data gathered from a semi-automated container terminal from Spain. In the other hand it is analyzed how to manage, evaluate and mitigate disruptions in the long-term by means of the risk assessment, an interesting approach to evaluate the effect of uncertain but likely events on the long-term throughput of the terminal. In addition, a definition for operational risk evaluation in port facilities is proposed along with a discussion of the terms that better represent the nature of the activities involved and finally, guidelines to manage the results obtained are provided.

Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.

Axon pathology in Parkinson’s disease and Lewy body dementia hippocampus contains α-, β-, and γ-synuclein

Pathogenic α-synuclein (αS) gene mutations occur in rare familial Parkinson’s disease (PD) kindreds, and wild-type αS is a major component of Lewy bodies (LBs) in sporadic PD, dementia with LBs (DLB), and the LB variant of Alzheimer’s disease, but β-synuclein (βS) and γ-synuclein (γS) have not yet been implicated in neurological disorders. Here we show that in PD and DLB, but not normal brains, antibodies to αS and βS reveal novel presynaptic axon terminal pathology in the hippocampal dentate, hilar, and CA2/3 regions, whereas antibodies to γS detect previously unrecognized axonal spheroid-like lesions in the hippocampal dentate molecular layer. The aggregation of other synaptic proteins and synaptic vesicle-like structures in the αS- and βS-labeled hilar dystrophic neurites suggests that synaptic dysfunction may result from these lesions. Our findings broaden the concept of neurodegenerative “synucleinopathies” by implicating βS and γS, in addition to αS, in the onset/progression of PD and DLB.

Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury

The purine nucleoside inosine has been shown to induce axon outgrowth from primary neurons in culture through a direct intracellular mechanism. For this study, we investigated the effects of inosine in vivo by examining whether it would stimulate axon growth after a unilateral transection of the corticospinal tract. Inosine applied with a minipump to the rat sensorimotor cortex stimulated intact pyramidal cells to undergo extensive sprouting of their axons into the denervated spinal cord white matter and adjacent neuropil. Axon growth was visualized by anterograde tracing with biotinylated dextran amine and by immunohistochemistry with antibodies to GAP-43. Thus, inosine, a naturally occurring metabolite without known side effects, might help to restore essential circuitry after injury to the central nervous system.

Operant conditioning of H-reflex changes synaptic terminals on primate motoneurons.

Operant conditioning of the primate triceps surae H-reflex, the electrical analog of the spinal stretch reflex, creates a memory trace that includes changes in the spinal cord. To define the morphological correlates of this plasticity, we analyzed the synaptic terminal coverage of triceps surae motoneurons from animals in which the triceps surae H-reflex in one leg had been increased (HRup mode) or decreased (HRdown mode) by conditioning and compared them to each other and to motoneurons from unconditioned animals. Motoneurons were labeled by intramuscular injection of cholera toxin-horseradish peroxidase. A total of 5055 terminals on the cell bodies and proximal dendrites of 114 motoneurons from 14 animals were studied by electron microscopy. Significant differences were found between HRup and HRdown animals and between HRup and naive (i.e., unconditioned) animals. F terminals (i.e., putative inhibitory terminals) were smaller and their active zone coverage on the cell body was lower on motoneurons from the conditioned side of HRup animals than on motoneurons from the conditioned side of HRdown animals. C terminals (i.e., terminals associated with postsynaptic cisterns and rough endoplasmic reticulum) were smaller and the number of C terminals in each C complex (i.e., a group of contiguous C terminals) was larger on motoneurons from the conditioned side of HRup animals than on motoneurons either from the conditioned side of HRdown animals or from naive animals. Because the treatment of HRup and HRdown animals differed only in the reward contingency, the results imply that the two contingencies had different effects on motoneuron synaptic terminals. In combination with other recent data, they show that H-reflex conditioning produces a complex pattern of spinal cord plasticity that includes changes in motoneuron physiological properties as well as in synaptic terminals. Further delineation of this pattern should reveal the contribution of the structural changes described here to the learned change in behavior.

Spatial localization of calcium channels in giant fiber lobe neurons of the squid (Loligo opalescens).

Whole-cell voltage clamp was used to investigate the properties and spatial distribution of fast-deactivating (FD) Ca channels in squid giant fiber lobe (GFL) neurons. Squid FD Ca channels are reversibly blocked by the spider toxin omega-Agatoxin IVA with an IC50 of 240-420 nM with no effect on the kinetics of Ca channel gating. Channels with very similar properties are expressed in both somatic and axonal domains of cultured GFL neurons, but FD Ca channel conductance density is higher in axonal bulbs than in cell bodies at all times in culture. Channels presumably synthesized during culture are preferentially expressed in the growing bulbs, but bulbar Ca conductance density remains constant while Na conductance density increases, suggesting that processes determining the densities of Ca and Na channels in this extrasomatic domain are largely independent. These observations suggest that growing axonal bulbs in cultured GFL neurons are not composed entirely of "axonal" membranes because FD Ca channels are absent from the giant axon in situ but, rather, suggest a potential role for FD Ca channels in mediating neurotransmitter release at the motor terminals of the giant axon.