Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 μm diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca²⁺ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABA_A and GABA_C receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release ^14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal ²¹. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion ¹¹. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level ¹⁹. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced ^{20, 23, 18, 9}. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.     

A Comparison of Hydrocarbon Vapor Attenuation in the Field with Predictions from Vapor Diffusion Models

This study used field data from three sites in Southern California to evaluate vapor phase transport from: (1) free product (die-sel and gasoline spill) on groundwater; (2) dissolved benzene (gasoline spill) in groundwater; and (3) hydrocarbon-impacted soil (gasoline spill) in the vadose zone. A sampling program to evaluate the vapor pathway included the following: vertical profile data, minimal purging prior to sample collection, field analysis of data, confirmation of field data using a fixed laboratory analysis, and soil physical property data. Comparison of hydrocarbon vapor concentrations measured in this field study with those calculated using vapor diffusion models suggest that an additional attenuation factor of between 500 and 35,000 is needed to account for observed concentrations. Comparison of hydrocarbon profiles with oxygen, carbon dioxide, and methane values is consistent with the interpretation that biodegradation is primarily responsible for the observed attenuation. Therefore, vapor pathway models that do not account for bioattenuation will result in a large overesti-mation of the risk at spill sites and will not be consistent with field data.     

An automatic device to record the force necessary to compensate plant movements

A device, which automatically and continuously keeps moving plants in fixed positions, has been constructed. The apparatus is based on optical detection of any movements of the plant organ under study. The plant organ is kept in the desired position by means of wires, and the force necessary to apply to the wires to achieve this is recorded. The force reflects any tendency of the plant organ to move. The system is controlled by an Apple II computer. The device can work in one or two dimensions and record compensation forces in a wide range. In the experiments mentioned below, the magnitude of the forces necessary to keep the plants in a fixed position was of the order of 10^-4N.
Circadian leaf movements of Oxalis regnellii Mig. were studied in the device. The leaf rhythm continued, although the leaflets were clamped and the light input on the leaf therefore constant. Circumnutation of hypocotyls of Helianthus annus L. cv. Californicus were drastically reduced in amplitude when the hypocotyls were kept in vertical position by the wires. Since the gravitropic input signal to this system was zeroed by the equipment, the results demonstrated that in the absence of gravitropic inputs the circumnutation reactions drastically diminish. This confirmed that circumnutations of these hypocotyls are influenced by gravity. Finally, the apparatus was used to study phototropic reactions: By clamping phototropically stimulated coleoptiles of Avena sativa L. cv. Seger no gravitational stimulations were involved and the phototropic reaction without interference from gravity could be studied.     

AFM nano‐mechanical study of the beating profile of hiPSC‐derived cardiomyocytes beating bodies WT and DM1

Myotonic Dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterized by a variety of multisystemic features and associated with cardiac anomalies. Among cardiac phenomena, conduction defects, ventricular arrhythmias, and dilated cardiomyopathy represent the main cause of sudden death in DM1 patients. Patient‐specific induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) represent a powerful in vitro model for molecular, biochemical, and physiological studies of disease in the target cells. Here, we used an Atomic Force Microscope (AFM) to measure the beating profiles of a large number of cells, organized in CM clusters (Beating Bodies, BBs), obtained from wild type (WT) and DM1 patients. We monitored the evolution over time of the frequency and intensity of the beating. We determined the variations between different BBs and over various areas of a single BB, caused by morphological and biomechanical variations. We exploited the AFM tip to apply a controlled force over the BBs, to carefully assess the biomechanical reaction of the different cell clusters over time, both in terms of beating frequency and intensity. Our measurements demonstrated differences between the WT and DM1 clusters highlighting, for the DM1 samples, an instability which was not observed in WT cells. We measured differences in the cellular response to the applied mechanical stimulus in terms of beating synchronicity over time and cell tenacity, which are in good agreement with the cellular behavior in vivo. Overall, the combination of hiPSC‐CMs with AFM characterization can become a new tool to study the collective movements of cell clusters in different conditions and can be extended to the characterization of the BB response to chemical and pharmacological stimuli.     

Effect of 16 weeks diving practice at two different times of day on the pulmonary function,spirometry measurements and 6-minute walk test data of healthy professional Tunisian scuba divers

The aim of this study was to investigate the effect of time of the day (TOD) and 16 weeks diving practice (16WDP) on the spirometric parameters and 6-min walk test data (6MWT) on professional Tunisian scuba divers. In randomized order, 36 health males divided into 3 groups [morning practice group) (MPG): n = 12; evening practice group (EPG): n = 12; control group (CG) n = 12] participated voluntary in this study. They performed spirometry measurements and 6MWT during two periods: [before-season (June 05–10), and after-season (October 05–10)]. Our results revealed that assessment sessions comprised the following: FVC, FEV1, FEV1/FVC and PEF. Results were analyzed by applying repeated measures analysis of variance ANOVA. The spirometric parameters were similar upon two times of day on both EPG and MPG before the season (i.e. FEV1, FVC). Likewise, our finding revealed a significant decrease in lung functions following the 16 weeks practice of scuba diving upon two times of day. Thus, this period of hyperbaric scuba diving practice lead a significant alteration of lung function parameters with decrease of percent of variation in EPG vs. CEG compared to MPG vs. CMG: (i.e. FEV1, FVC, and PEF). In conclusion, 16 weeks of hyperbaric scuba diving lead a significant change in the spirometric and 6MWT values and respiratory problem with damage on lung function in healthy adult divers older than 40 years. Professional divers are recommended to have practice diving in the morning.