The touch and zap method for in vivo whole-cell patch recording of intrinsic and visual responses of cortical neurons and Glial cells

Whole-cell patch recording is an essential tool for quantitatively establishing the biophysics of brain function, particularly in vivo. This method is of particular interest for studying the functional roles of cortical glial cells in the intact brain, which cannot be assessed with extracellular recordings. Nevertheless, a reasonable success rate remains a challenge because of stability, recording duration and electrical quality constraints, particularly for voltage clamp, dynamic clamp or conductance measurements. To address this, we describe "Touch and Zap", an alternative method for whole-cell patch clamp recordings, with the goal of being simpler, quicker and more gentle to brain tissue than previous approaches. Under current clamp mode with a continuous train of hyperpolarizing current pulses, seal formation is initiated immediately upon cell contact, thus the "Touch". By maintaining the current injection, whole-cell access is spontaneously achieved within seconds from the cell-attached configuration by a self-limited membrane electroporation, or "Zap", as seal resistance increases. We present examples of intrinsic and visual responses of neurons and putative glial cells obtained with the revised method from cat and rat cortices in vivo. Recording parameters and biophysical properties obtained with the Touch and Zap method compare favourably with those obtained with the traditional blind patch approach, demonstrating that the revised approach does not compromise the recorded cell. We find that the method is particularly well-suited for whole-cell patch recordings of cortical glial cells in vivo, targeting a wider population of this cell type than the standard method, with better access resistance. Overall, the gentler Touch and Zap method is promising for studying quantitative functional properties in the intact brain with minimal perturbation of the cell's intrinsic properties and local network. Because the Touch and Zap method is performed semi-automatically, this approach is more reproducible and less dependent on experimenter technique

A Numerical Approach to Ion Channel Modelling Using Whole-Cell Voltage-Clamp Recordings and a Genetic Algorithm

The activity of trans-membrane proteins such as ion channels is the essence of neuronal transmission. The currently most accurate method for determining ion channel kinetic mechanisms is single-channel recording and analysis. Yet, the limitations and complexities in interpreting single-channel recordings discourage many physiologists from using them. Here we show that a genetic search algorithm in combination with a gradient descent algorithm can be used to fit whole-cell voltage-clamp data to kinetic models with a high degree of accuracy. Previously, ion channel stimulation traces were analyzed one at a time, the results of these analyses being combined to produce a picture of channel kinetics. Here the entire set of traces from all stimulation protocols are analysed simultaneously. The algorithm was initially tested on simulated current traces produced by several Hodgkin-Huxley–like and Markov chain models of voltage-gated potassium and sodium channels. Currents were also produced by simulating levels of noise expected from actual patch recordings. Finally, the algorithm was used for finding the kinetic parameters of several voltage-gated sodium and potassium channels models by matching its results to data recorded from layer 5 pyramidal neurons of the rat cortex in the nucleated outside-out patch configuration. The minimization scheme gives electrophysiologists a tool for reproducing and simulating voltage-gated ion channel kinetics at the cellular level

Role of Mechanical Factors in the Morphology of the Primate Cerebral Cortex

The convoluted cortex of primates is instantly recognizable in its principal morphologic features, yet puzzling in its complex finer structure. Various hypotheses have been proposed about the mechanisms of its formation. Based on the analysis of databases of quantitative architectonic and connection data for primate prefrontal cortices, we offer support for the hypothesis that tension exerted by corticocortical connections is a significant factor in shaping the cerebral cortical landscape. Moreover, forces generated by cortical folding influence laminar morphology, and appear to have a previously unsuspected impact on cellular migration during cortical development. The evidence for a significant role of mechanical factors in cortical morphology opens the possibility of constructing computational models of cortical develoment based on physical principles. Such models are particularly relevant for understanding the relationship of cortical morphology to the connectivity of normal brains, and structurally altered brains in diseases of developmental origin, such as schizophrenia and autism

Neuronal Firing Sensitivity to Morphologic and Active Membrane Parameters

Both the excitability of a neuron's membrane, driven by active ion channels, and dendritic morphology contribute to neuronal firing dynamics, but the relative importance and interactions between these features remain poorly understood. Recent modeling studies have shown that different combinations of active conductances can evoke similar firing patterns, but have neglected how morphology might contribute to homeostasis. Parameterizing the morphology of a cylindrical dendrite, we introduce a novel application of mathematical sensitivity analysis that quantifies how dendritic length, diameter, and surface area influence neuronal firing, and compares these effects directly against those of active parameters. The method was applied to a model of neurons from goldfish Area II. These neurons exhibit, and likely contribute to, persistent activity in eye velocity storage, a simple model of working memory. We introduce sensitivity landscapes, defined by local sensitivity analyses of firing rate and gain to each parameter, performed globally across the parameter space. Principal directions over which sensitivity to all parameters varied most revealed intrinsic currents that most controlled model output. We found domains where different groups of parameters had the highest sensitivities, suggesting that interactions within each group shaped firing behaviors within each specific domain. Application of our method, and its characterization of which models were sensitive to general morphologic features, will lead to advances in understanding how realistic morphology participates in functional homeostasis. Significantly, we can predict which active conductances, and how many of them, will compensate for a given age- or development-related structural change, or will offset a morphologic perturbation resulting from trauma or neurodegenerative disorder, to restore normal function. Our method can be adapted to analyze any computational model. Thus, sensitivity landscapes, and the quantitative predictions they provide, can give new insight into mechanisms of homeostasis in any biological system

Twelve Tips for Just in Time Teaching of Communication Skills for Difficult Conversations in the Clinical Setting

The ability to communicate well with patients and other members of the healthcare team is a vital skill for physicians to have, but one that is often not emphasized in medical education. Learners of all levels can obtain and develop good communication skills regardless of their natural ability in this area, and the clinical setting represents an underutilized resource to accomplish this task. With this in mind, we have reviewed the growing body of literature on the subject and organized our findings into twelve tips to help educators capitalize on these missed opportunities. While our emphasis is helping learners with difficult discussions, these tips can be easily adapted to any other clinical encounter requiring clear communication. Teaching effective communication skills in the clinical setting requires some extra time, but the steps outlined should not take more than a few minutes to complete. Taking the time to develop these skills in our learners will make a significant difference not only their lives but also their patients and their families

Modelling the Somantic Electrical Response of Hippocampal Pyramidal Neurons

A modeling study of hippocampal pyramidal neurons is described. This study is based on simulations using HIPPO, a program which simulates the somatic electrical activity of these cells. HIPPO is based on a) descriptions of eleven non-linear conductances that have been either reported for this class of cell in the literature or postulated in the present study, and b) an approximation of the electrotonic structure of the cell that is derived in this thesis, based on data for the linear properties of these cells. HIPPO is used a) to integrate empirical data from a variety of sources on the electrical characteristics of this type of cell, b) to investigate the functional significance of the various elements that underly the electrical behavior, and c) to provide a tool for the electrophysiologist to supplement direct observation of these cells and provide a method of testing speculations regarding parameters that are not accessible

Modelling the somatic electrical response of hippocampal pyramidal neurons

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1987.MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING.Bibliography: leaves 291-295.by Lyle J. Borg-Graham.M.S

Integrated Mechanisms of Anticipation and Rate-of-Change Computations in Cortical Circuits

Local neocortical circuits are characterized by stereotypical physiological and structural features that subserve generic computational operations. These basic computations of the cortical microcircuit emerge through the interplay of neuronal connectivity, cellular intrinsic properties, and synaptic plasticity dynamics. How these interacting mechanisms generate specific computational operations in the cortical circuit remains largely unknown. Here, we identify the neurophysiological basis of both the rate of change and anticipation computations on synaptic inputs in a cortical circuit. Through biophysically realistic computer simulations and neuronal recordings, we show that the rate-of-change computation is operated robustly in cortical networks through the combination of two ubiquitous brain mechanisms: short-term synaptic depression and spike-frequency adaptation. We then show how this rate-of-change circuit can be embedded in a convergently connected network to anticipate temporally incoming synaptic inputs, in quantitative agreement with experimental findings on anticipatory responses to moving stimuli in the primary visual cortex. Given the robustness of the mechanism and the widespread nature of the physiological machinery involved, we suggest that rate-of-change computation and temporal anticipation are principal, hard-wired functions of neural information processing in the cortical microcircuit

Theory of transient chimeras in finite Sakaguchi-Kuramoto networks

Chimera states are a phenomenon in which order and disorder can co-exist within a network that is fully homogeneous. Precisely how transient chimeras emerge in finite networks of Kuramoto oscillators with phase-lag remains unclear. Utilizing an operator-based framework to study nonlinear oscillator networks at finite scale, we reveal the spatiotemporal impact of the adjacency matrix eigenvectors on the Sakaguchi-Kuramoto dynamics. We identify a specific condition for the emergence of transient chimeras in these finite networks: the eigenvectors of the network adjacency matrix create a combination of a zero phase-offset mode and low spatial frequency waves traveling in opposite directions. This combination of eigenvectors leads directly to the coherent and incoherent clusters in the chimera. This approach provides two specific analytical predictions: (1) a precise formula predicting the combination of connectivity and phase-lag that creates transient chimeras, (2) a mathematical procedure for rewiring arbitrary networks to produce transient chimeras

The integration of postmodern values and rhetorical analysis : a case study

Both traditional preaching theory and the listening context of the hearers have undergone radical changes within the last thirty years. Contemporary preachers no longer can assume the authority inherent in their position or preaching methods, and postmodern listeners exhibit the desire for increased diversity and points of view in sermons. This thesis will address these challenges by advancing the notion that attention to rhetorical criticism in the exegesis of biblical texts sheds new light on the nature of preaching in terms of form and function. The resulting multi-vocal and non-hierarchical leadership orientation has application for postmodern audiences. The methodological structure of theological interpretation undergirding this thesis involves four tasks of the hermeneutical cycle adapted from Richard Osmer’s approach. This approach engages in the task of contextual interpretation that connects with both Christian tradition and Scripture, and furthermore leads to the construction of a pragmatic plan for future homiletics. Chapter 1 introduces the problem facing contemporary homileticians: the changed context of preacher and hearer. The chapter advocates that one way forward for preaching involves the use of rhetorical criticism as the exegetical basis for a values-based homiletic, and then finishes with an overview of the thesis chapters. Chapter 2 demonstrates the fourfold task of the hermeneutical cycle by establishing the provenance of the method, critiquing it and grounding the approach of the thesis in the contemporary postmodern setting. Chapter 3 engages in a contextual interpretation of historic shifts in the fields of rhetoric, biblical studies and homiletics, analyzing and evaluating these trends. The chapter concludes by constructing a pragmatic plan for future biblical studies, a rhetorical-critical-narratological methodology that will be applied to the text of Ezekiel. Chapter 4 demonstrates that a contextual interpretation, evaluation and analysis of the New Homiletic results in the formation of a values-based approach to preaching and leadership orientation that is appropriate to postmodernity. Chapter 5 builds upon a contextual interpretation of synchronic and diachronic methodologies and advances a complementary approach to exegesis. The chapter then applies the rhetorical-critical-narratological approach developed in Chapter 3 to the discourse of Ezekiel to establish its contextual and rhetorical situation. The chapter then engages in a close rhetorical-critical-narratological reading of the literary unit of Ezekiel 15. Chapter 6 engages in a contextual interpretation and evaluation of three Ezekiel commentaries and sermons from Ezekiel 15, locating them along the pendulum-like series of shifts identified within Chapter 3. Chapter 7 demonstrates the integration of biblical studies and homiletics with the production of a sample multiple point-of-view sermon based upon the exegesis of Ezekiel conducted in Chapter 5. The chapter critiques the sermon and provides an example of the rhetorical-critical method applied to a discursive genre from 1 Corinthians 4.18-5.13. Chapter 8 concludes the thesis by reviewing the contributions made by the study, proceeds to interpret contextually the challenge of postmodern homiletics, and finishes with recommendations for areas of future studies outside the scope of the thesis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo