Liquid junction potentials and small cell effects in patch-clamp analysis.
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The behavior of the mitochondrial inner membrane multiple conductance channel (MCC) which has a peak conductance of 1-1.5 nS has been examined in rat heart mitochondria. MCC can display several unique characteristics: (a) prolonged open and closed times on the order of seconds to minutes, (b) a voltage dependence in which MCC opens (negative potential) or closes (positive potential) generally in steps, (c) a response to inhibitors such as amiodarone in steps corresponding at least approximately to those in (b), (d) a 'free-running mode' in which the current level rapidly fluctuates between a minimum of nine conductance levels but with a preferred occupation of the 0.5-0.7 nS levels, and (e) very large transitions (1-1.5 nS) resolved at 4 kHz bandwidth as single events with variable mean open time.
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Isolated neurons from mouse neonate dorsal root ganglia were analyzed using both whole-cell clamp and single-channel recording techniques and presented a complex repertoire of potassium (K) channels. Different types of potassium channels have been found: calcium-activated K channel presenting a large unit conductance of 260 pS in symmetrical K; voltage-dependent K channels of 130 pS without calcium-dependence; two types of inward rectifying K channels (90 and 120 pS in symmetrical K); low probability K channels; delayed rectifier channels and non-selective cationic channels.
Individual taste receptor cells were isolated from the tongue of the mouse by enzymatic treatment followed by mechanical dissociation. The cells were morphologically identical with taste cells from amphibians. Whole-cell voltage-clamp recordings indicated that the murine taste cells possess a variety of voltage-dependent inward and outward currents. Delayed rectifier currents were blocked by denatonium benzoate, one of the most bitter compounds known. This preparation should permit a detailed electrophysiologcal investigation of taste transduction in mammals at the level of taste receptor cells.
The purpose of this study was to develop a method for electrophysiological characterization of retrogradely labeled bulbospinal neurons in the specific cytoarchitectonic regions in the brainstem slices. Several days after the spinal cord was injected with the carbocyanine dye, DiI, retrogradely labeled bulbospinal neurons were visualized by epifluorescence optics in the brainstem slices with the aid of a silicon intensifier tube (SIT) camera. Labeled somata were routinely seen in the caudal raphe nuclei, rostroventral medial and lateral portions of the medulla, the A5 group and in other medullary sites known to project to the spinal cord. Electrophysiological properties of the DiI-labeled neurons were assessed by whole-cell recordings using micropipettes filled with biocytin. The slices were subsequently processed for dual visualization of biocytin and serotonin or a marker for noradrenergic neurons, tyrosine hydroxylase (TH). The electrophysiological properties of bulbospinal neurons were correlated with their morphology and neurochemical content. This technique may be useful in other areas of CNS for studying morphology, neurochemical content and physiology of retrogradely labeled neurons.
The PNSCROLL program has been specifically developed for interactively scrolling through binary data files under mouse control, in order to analyse single channel current records and printout, or plot, records and histograms of currents. PNSCROLL requires the use of a Microsoft Mouse and runs on an IBM or IBM-compatible microcomputer operating under MS DOS, ideally with 256K EGA or VGA graphics card and colour display. It will print on Epson dot matrix printers and plot records and histograms on HP plotters and LaserJet laser printers.
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It should be obvious that there are many ways to construct clamp set-ups that are either equivalent or sufficient for the experiments planned. The hardware and electronics can be obtained from several manufacturers, as can analysis software. What we have presented here are guidelines primarily meant to point a new experimenter in the right direction and, we hope, to guide more experienced investigators toward techniques that can improve the resolution of their measurements.
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The method we present instructs the computer to deliver sets of programmed membrane voltages and allows the acquisition of large amounts of digitized data, compact storage, ready identification of records and rapid, interactive analysis of channel current data. A typical cycle of analysis including amplitude determination and kinetic measurements for 10 s of continuous data digitized at 5 kHz requires 5-20 min depending on the complexity of the observed channel activity. This procedure is compatible with such storage protocols as that described by Bezanilla [Biophys. J., 47 (1985) 437-441], although there are obvious benefits to using standard computer storage devices such as hard disks, floppy disks, and Bernoulli Boxes. The programs described in this report are available from the authors.