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Cleaning Patch Clamp Pipettes Enables their Reuse
Sunday, February 28, 2016 746-Pos Board B526 Cleaning Patch Clamp Pipettes Enables their Reuse Ilya Kolb1, William A. Stoy1, Erin Rousseau2, Olivia A. Moody3, Andrew Jenkins3,4, Craig R. Forest5. 1 Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA, 2College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, USA, 3 Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA, 4Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, USA, 5George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. Patch-clamp recording is a gold-standard technique for the measurement of membrane voltage and current fluctuations in electrically active cells. From the advent of the technique in the 1970s, it has been widely accepted that using a new, un-contaminated pipette to patch-clamp every cell is a critical requirement to form a high-quality seal with the cell and yield a successful recording. However, exchanging pipettes between each cell is a manual and timeconsuming task that requires dexterity and interrupts the otherwiseautomated flow of the experiment. We circumvent the need to manually exchange pipettes by instead creating a conceptually simple cleaning procedure that enables their reuse. This automated cleaning procedure consists of dipping used pipettes into a commercially available detergent, pneumatically forcing the detergent into the tip, and rinsing the tip in a non-cytotoxic solution. Pipettes cleaned in this fashion for 31 seconds yielded successful recordings at a rate similar to that of new pipettes (cleaned pipettes: 90%, n=50; new pipettes: 86%, n=50) in human embryonic kidney (HEK) cultures and mouse brain slice tissue (cleaned: 59%, n=46; new: 50%, n=18). The pipette tip was confirmed to be contamination-free using Scanning Electron Microscopy (SEM). A single pipette could be cleaned repeatedly; we measured no significant decreases in seal resistance or recording quality over 10 patch-clamp recordings and cleaning cycles with a single pipette (n=5 pipettes). Passive and active electrical characteristics of cells did not degrade over successive cleaning cycles. These findings suggest that pipettes that undergo the cleaning procedure are as effective as new pipettes and do not alter the characteristics of the recording. Aside from obvious advantages in time, effort and cost of replacing pipettes, reusing a single pipette reduces experimental variability and most critically, is a major step towards fully automating patchclamp electrophysiology experiments. 747-Pos Board B527 High Yield Subcortical Patch Clamping in Vivo William Stoy1, Bo Yang2, Thomas Capocasale1, Clarissa Whitmire1, Yi Liew1, Garrett Stanley1, Craig Forest3. 1 Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA, 2Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA, 3Mechanical Engineering, Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA. Whole cell recording from subcortical structures has revealed much about the membrane properties of sensory systems. Since its introduction by Margrie et al in 2002, however, patch clamp recordings deep in the intact brain have suffered from low yield and high access resistance, primarily due to pipette contamination during descent to subcortical nuclei. We describe a novel algorithm and best practice for reducing pipette tip contamination and improving access resistance when targeting deep, subcortical neurons in mice (e.g., 1-3 mm deep). In this work, we introduce an automated method for dodging obstructions such as neurons and blood vessels during descent. Traditionally, pipettes are localized in the intact brain by blind, linear rapid descent to the subcortical region of interest (e.g., hippocampus, thalamus). During this descent, pipettes are often contaminated by debris that prevents gigasealing and increases access resistance. Using this new algorithm, pipette resistance is measured at 128 Hz during descent for a high spatial resolution. If an obstruction is encountered prior to the region of interest (as indicated by an increase in resistance >1 Mohm threshold), the pipette is stopped, retracted, and moved laterally in 20 um steps. Lateral steps are repeated until resistance decreases below threshold, indicating that the obstruction has been avoided and descent is resumed. Pipettes inserted using this method arrived at the region of interest without a significant change in resistance (<300 kohms at 3000 um) 69% of the time (n=46), while pipettes inserted using traditional blind,
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linear methods were inserted successfully 32% of the time (n=31); Whole cell recordings following successful descent resulted in lower access resistance recordings and smaller holding currents than those inserted with the traditional algorithm. This pipette localization method therefore greatly improves the quality and accessibility of subcortical tissues in the intact brain. 748-Pos Board B528 Development of an in Vitro Model of Mild Traumatic Brain Injury Krishna P. Sheth, Kelly D. O’Connor, Tuan Nguyen. Physics Department, The College of New Jersey, Ewing, NJ, USA. Mild Traumatic Brain Injury (mTBI), otherwise known as concussion, occurs when mechanical forces cause brain dysfunction. Symptoms develop slowly and can be wide-ranging: from headaches and dizziness to loss of sleep, mood swings, and even depression. One does not fully heal from a concussion as a second similar concussion (even after all symptoms from the first have subsided) may bring upon even worse effects than the first. The mysteriousness of mTBI has led some to suggest that its pathogenesis comes from a global failure in neuronal network communication rather than a dysfunction of any specific type of neuron or brain nuclei. To test this hypothesis, we have developed a device that can induce injury to a cultured neuronal network while imaging its activity. The device is designed to injure only a subpopulation of neurons, thus allowing for the possibility of comparing activity from both injured and non-injured neurons within the same field-of-view both before and after injury is induced. The device is simple in design and works by rapidly stretching a silicone membrane on which neurons have been cultured. Localized, uniaxial stretching is produced by applying vacuum to rectangular ports situated beneath the membrane. To facilitate imaging, the device is relatively transparent and can be easily transported from the cell incubator to the microscope. We present here results characterizing the stretch and preliminary data showing neuronal activity before and after stretching. 749-Pos Board B529 Experimental Multi-Physics Measurement of Neuronal Responses under Trauma Majid Malboubi1, Antoine Jerusalem2. 1 University of Oxford, Oxford, United Kingdom, 2Department of Engineering Science, University of Oxford, Oxford, United Kingdom. In traumatic brain injuries (TBIs) or spinal cord injuries (SCIs), neural tissue and thus axons are subjected to a range of damaging strains and strain rates. These injuring mechanical causes are translated into biochemical and biological alterations and/or responses. Despite extensive experimental campaigns, these mechanisms remain unclear. For instance, it is still not clear how injuring mechanical causes are transferred into electrophysiological alterations, affect action potential propagation, and ultimately change cell behaviour. Moreover the specific microscale mechanisms leading to brain or spinal cord injury are still unknown, and the role of the external loading conditions are yet to be identified. One major obstacle is that experimental campaigns on neurons remain highly challenging. Recent studies show that acute neurophysiological changes not only occur immediately after injury but can also be observable through secondary mechanisms up to a few days later. Therefore electrophysiological properties and mechanical deformations should be monitored simultaneously during and after injury at the cell level. To address these issues we have set up a multi-functional patch-clamp electrophysiology rig which not only enables measurement of electrophysiological properties in parallel with mechanical loading but also provides means for applying various, yet specific, loading conditions reliably and repeatedly. The proposed experimental approach will enhance our understanding of electrophysiologicalmechanical coupling in neurons, and more direct damages such as in traumatic brain injuries. Such approach will promote the technology transfer from basic and pre-clinical findings into clinical applications and consequently, medical treatment or prevention. 750-Pos Board B530 Measuring Hydraulic Conductance and Hydration Potential of Brain Extracellular Matrix by Osmotic Stress Maria P. McGee, Michael Morykwas, Louis Argenta. Plastic and Reconstructive Surgery, Wake Forest University Medical School, Winston-Salem, NC, USA.
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linear methods were inserted successfully 32% of the time (n=31); Whole cell recordings following successful descent resulted in lower access resistance recordings and smaller holding currents than those inserted with the traditional algorithm. This pipette localization method therefore greatly improves the quality and accessibility of subcortical tissues in the intact brain. 748-Pos Board B528 Development of an in Vitro Model of Mild Traumatic Brain Injury Krishna P. Sheth, Kelly D. O’Connor, Tuan Nguyen. Physics Department, The College of New Jersey, Ewing, NJ, USA. Mild Traumatic Brain Injury (mTBI), otherwise known as concussion, occurs when mechanical forces cause brain dysfunction. Symptoms develop slowly and can be wide-ranging: from headaches and dizziness to loss of sleep, mood swings, and even depression. One does not fully heal from a concussion as a second similar concussion (even after all symptoms from the first have subsided) may bring upon even worse effects than the first. The mysteriousness of mTBI has led some to suggest that its pathogenesis comes from a global failure in neuronal network communication rather than a dysfunction of any specific type of neuron or brain nuclei. To test this hypothesis, we have developed a device that can induce injury to a cultured neuronal network while imaging its activity. The device is designed to injure only a subpopulation of neurons, thus allowing for the possibility of comparing activity from both injured and non-injured neurons within the same field-of-view both before and after injury is induced. The device is simple in design and works by rapidly stretching a silicone membrane on which neurons have been cultured. Localized, uniaxial stretching is produced by applying vacuum to rectangular ports situated beneath the membrane. To facilitate imaging, the device is relatively transparent and can be easily transported from the cell incubator to the microscope. We present here results characterizing the stretch and preliminary data showing neuronal activity before and after stretching. 749-Pos Board B529 Experimental Multi-Physics Measurement of Neuronal Responses under Trauma Majid Malboubi1, Antoine Jerusalem2. 1 University of Oxford, Oxford, United Kingdom, 2Department of Engineering Science, University of Oxford, Oxford, United Kingdom. In traumatic brain injuries (TBIs) or spinal cord injuries (SCIs), neural tissue and thus axons are subjected to a range of damaging strains and strain rates. These injuring mechanical causes are translated into biochemical and biological alterations and/or responses. Despite extensive experimental campaigns, these mechanisms remain unclear. For instance, it is still not clear how injuring mechanical causes are transferred into electrophysiological alterations, affect action potential propagation, and ultimately change cell behaviour. Moreover the specific microscale mechanisms leading to brain or spinal cord injury are still unknown, and the role of the external loading conditions are yet to be identified. One major obstacle is that experimental campaigns on neurons remain highly challenging. Recent studies show that acute neurophysiological changes not only occur immediately after injury but can also be observable through secondary mechanisms up to a few days later. Therefore electrophysiological properties and mechanical deformations should be monitored simultaneously during and after injury at the cell level. To address these issues we have set up a multi-functional patch-clamp electrophysiology rig which not only enables measurement of electrophysiological properties in parallel with mechanical loading but also provides means for applying various, yet specific, loading conditions reliably and repeatedly. The proposed experimental approach will enhance our understanding of electrophysiologicalmechanical coupling in neurons, and more direct damages such as in traumatic brain injuries. Such approach will promote the technology transfer from basic and pre-clinical findings into clinical applications and consequently, medical treatment or prevention. 750-Pos Board B530 Measuring Hydraulic Conductance and Hydration Potential of Brain Extracellular Matrix by Osmotic Stress Maria P. McGee, Michael Morykwas, Louis Argenta. Plastic and Reconstructive Surgery, Wake Forest University Medical School, Winston-Salem, NC, USA.