Spontaneous postsynaptic currents (PSCs) provide key information about the mechanisms of synaptic transmission and the activity modes of neuronal networks. However, detecting spontaneous PSCs in vitro and in vivo has been challenging, because of the small amplitude, the variable kinetics, and the undefined time of generation of these events. Here, we describe a, to our knowledge, new method for detecting spontaneous synaptic events by deconvolution, using a template that approximates the average time course of spontaneous PSCs. A recorded PSC trace is deconvolved from the template, resulting in a series of delta-like functions. The maxima of these delta-like events are reliably detected, revealing the precise onset times of the spontaneous PSCs. Among all detection methods, the deconvolution-based method has a unique temporal resolution, allowing the detection of individual events in high-frequency bursts. Furthermore, the deconvolution-based method has a high amplitude resolution, because deconvolution can substantially increase the signal/noise ratio. When tested against previously published methods using experimental data, the deconvolution-based method was superior for spontaneous PSCs recorded in vivo. Using the high-resolution deconvolution-based detection algorithm, we show that the frequency of spontaneous excitatory postsynaptic currents in dentate gyrus granule cells is 4.5 times higher in vivo than in vitro.

The use of advanced patch-clamp recording techniques in brain slices, such as simultaneous recording from multiple neurons and recording from dendrites or presynaptic terminals, demands slices of the highest quality. In this context the mechanics of the tissue slicer are an important factor. Ideally, a tissue slicer should generate large-amplitude and high-frequency movements of the cutting blade in a horizontal axis, with minimal vibrations in the vertical axis. We developed a vibroslicer that fulfils these in part conflicting requirements. The oscillator is a permanent-magnet-coil-leaf-spring system. Using an auto-resonant mechano-electrical feedback circuit, large horizontal oscillations (up to 3 mm peak-to-peak) with high frequency ( approximately 90 Hz) are generated. To minimize vertical vibrations, an adjustment mechanism was employed that allowed alignment of the cutting edge of the blade with the major axis of the oscillation. A vibroprobe device was used to monitor vertical vibrations during adjustment. The system is based on the shading of the light path between a light-emitting diode (LED) and a photodiode. Vibroprobe monitoring revealed that the vibroslicer, after appropriate adjustment, generated vertical vibrations of <1 microm, significantly less than many commercial tissue slicers. Light- and electron-microscopic analysis of surface layers of slices cut with the vibroslicer showed that cellular elements, dendritic processes and presynaptic terminals are well preserved under these conditions, as required for patch-clamp recording from these structures.

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca(2+)-dependent and a small Ca(2+)-independent K+ channel have been described. Whereas the latter most likely is responsible for the K+ secretion in this nephron segment, the function of the large-conductance K+ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K+ channel was strongly voltage- and calcium-dependent. At 3 mumol/l cytosolic Ca2+ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mumol/l Ca2+ activity the open probability (Po) of this channel was pH-dependent. At pH 7.0 Po was decreased to 4 +/- 2% (n = 9) and at pH 8.5 it was increased to 425 +/- 52% (n = 9) of the control. At this low Ca2+ activity the Po of the channel was reduced by 1 mmol/l ATP to 8 +/- 4% (n = 6). Cell swelling activated the large-conductance K+ channel (n = 14) and hyperpolarized the membrane potential of the cells by 9 +/- 1 mV (n = 23). Intracellular Ca2+ activity increased after hypotonic stress. This increase depended on the extracellular Ca2+ activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of antidiuretic hormone [( Arg]vasopressin, ADH) on intracellular calcium activity [Ca2+]i of isolated perfused rabbit cortical thick ascending limb (cTAL) segments was investigated with the calcium fluorescent dye fura-2. The fluorescence emission ratio at 500-530 nm (R) was monitored as a measure of [Ca2+]i after excitation at 335 nm and 380 nm. In addition the transepithelial potential difference (PDte) and transepithelial resistance (Rte) of the tubule were measured simultaneously. After addition of ADH (1-4 nmol/l) to the basolateral side of the cTAL R increased rapidly, but transiently, from 0.84 +/- 0.05 to 1.36 +/- 0.08 (n = 46). Subsequently, within 7-12 min R fell to control values even in the continued presence of ADH. The increase in R evoked by the ADH application corresponded to a rise of [Ca2+]i from a basal level of 155 +/- 23 nmol/l [Ca2+]i up to 429 +/- 53 nmol/l [Ca2+]i at the peak of the transient, as estimated by intra- or extracellular calibration procedures. The electrical parameters (PDte and Rte) of the tubules were not changed by ADH. The ADH-induced Ca2+ transient was dependent on the presence of Ca2+ on the basolateral side, whereas luminal Ca2+ had no effect. d(CH2)5[Tyr(Me)2]2,Arg8vasopressin, a V1 antagonist (Manning compound, 10 nmol/l), blocked the ADH effect on [Ca2+]i completely (n = 5). The V2 agonist 1-desamino-[D-Arg8]vasopressin (10 nmol/l, n = 4), and the cAMP analogues, dibutyryl-cAMP (400 mumol/l, n = 4), 8-(4-chlorophenylthio)-cAMP (100 mumol/l, n = 1) or 8-bromo-cAMP (200 mumol/l, n = 4) had no influence on [Ca2+]i. The ADH-induced [Ca2+]i increase was not sensitive to the calcium-channel blockers nifedipine and verapamil (100 mumol/l, n = 4). We conclude that ADH acts via V1 receptors to increase cytosolic calcium activity transiently in rabbit cortical thick ascending limb segments, possibly by an initial Ca2+ release from intracellular stores and by further Ca2+ influx through Ca2+ channels in the basolateral membrane. These channels are insensitive to L-type Ca2+ channel blockers, e.g. nifedipine and verapamil.

In inside-out patches from cultured neonatal rat heart cells, single Na+ channel currents were analyzed under the influence of the cardiotonic compound DPI 201-106 (DPI), a putative novel channel modifier. In absence of DPI, normal cardiac single Na+ channels studied at -30 mV have one open state which is rapidly left with a rate constant of 826.5 sec -1 at 20 degrees C during sustained depolarization. Reconstructed macroscopic currents relax completely with 7 to 10 msec. The current decay fits a single exponential. A considerable percentage of openings may occur during relaxation of the macroscopic current. In patches treated with 3 X 10(-6) m DPI in the pipette solution, stepping to -30 mV results in drastically prolonged and usually repetitive openings. This channel activity mostly persists over the whole depolarization (usually 160 msec in duration) but is abruptly terminated on clamping back the patch to the holding potential. Besides these modified events, apparently normal openings occur. The open time distribution of DPI-treated Na+ channels is the sum of two exponentials characterized by time constants of 0.85 msec (which is close to the time constant found in the control patches, 1.21 msec) and 12 msec. Moreover, DPI-modified Na+ channels exhibit a sustained high, time-independent open probability. Similar to normal Na+ channels, is voltage-dependent and increases on shifting the holding potential in the hyperpolarizing direction. These kinetic changes suggest an elimination of Na+ channel inactivation as it may follow from an interaction of DPI with Na+ channels.

Epithelial chloride channels can be blocked by various inhibitors, which show considerable differences in their molecular structure. In the present patch-clamp study, we compared different blockers of one type of epithelial Cl- channel with respect to their inhibitory potency. We applied the blockers to excised inside-out-or outside-out-oriented membrane patches of cultured HT29 colon carcinoma and respiratory epithelial cells (REC) containing the outwardly rectifying intermediate-conductance (ICOR) chloride channel. Four types of inhibitory compounds were tested: stilbene disulphonate derivatives, indanyloxyacetic acid, amidine, and arylaminobenzoates. The concentrations for half-maximal inhibition (IC50) for the different channel blockers were (mumol/l): 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid 100; 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid 80; indanyloxyacetic acid 9; 4,4'-dinitrostilbene-2,2'-disulphonic acid 8; amidine 8 and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) 0.9. All compounds, when applied to the cytosolic side of the channel, induced a flicker-type block of the ICOR Cl- channel at lower concentrations and a complete channel inhibition at higher concentrations. The inhibitory potency of NPPB was much higher when it was added to the external surface of the channel in outside-out-oriented membrane patches. At 1 mumol/l the inhibition was complete. All blocker effects were fully reversible. The probe with the highest affinity (NPPB) and a closely related compound 5-nitro-2-(3-phenylethylamino)-benzoate (NPEB) were used to construct macromolecular probes by linking these blockers to aminopolyethyleneglycol (PEG) or amino-ethyl-O-dextran (5 kDa).2+ These macromolecular NPPB and NPEB derivatives inhibited the ICOR Cl- channels only from the outside but had no effect on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)

The luminal membrane of principal cells of rat cortical collecting duct (CCD) is dominated by a K+ conductance. Two different K+ channels are described for this membrane. K+ secretion probably occurs via a small-conductance Ca(2+)-independent channel. The function of the second, large-conductance Ca(2+)-dependent channel is unclear. This study examines properties of this channel to allow a comparison of this K+ channel with the macroscopic K+ conductance of the CCD and with similar K+ channels from other preparations. The channel is poorly active on the cell. It has a conductance of 263 +/- 11 pS (n = 36, symmetrical K+ concentrations) and of 139 +/- 3 pS (n = 91) with 145 mmol/l K+ on one side and 3.6 mmol/l K+ on the other side of the membrane. Its open probability is high after excision (0.71 +/- 0.03, n = 85). The channel flickers rapidly between open and closed states. Its permeability in the cell-free configuration was 7.0 +/- 0.2 x 10(-13) cm3/s (n = 85). It is inhibited by several typical blockers of K+ channels such as Ba2+, tetraethylammonium, quinine, and quinidine and high concentrations of Mg2+. The Ca2+ antagonist verapamil and diltiazem also inhibit this K+ channel. As is typical for the maxi K+ channel, it is inhibited by charybdotoxin but not by apamin. The selectivity of this large-conductance K+ channel demonstrates significant differences between the permeability sequence (pK > pRb > pNH4 > pCs = pLi = pNa = pcholine = 0) and the conductance sequence (gK > gNH4 > gRb > gLi = gcholine > gCs = gNa = 0). The only other cations that are significantly conducted by this channel besides K+ (gK at Vc = infinity is 279 +/- 8 pS, n = 88) re NH+4 (gNH4 = 127 +/- 22 pS, n = 10) and Rb+ (gRb = 36 +/- 5 pS, n = 6). The K+ currents through this channel are reduced by high concentrations of choline+, Cs+, Rb+, and NH+4. These properties and the dependence of this channel on Ca2+ and voltage classify it as a "maxi" K+ channel. A possible physiological function of this channel is discussed in the accompanying paper.

Increases of cytosolic Ca2+, as occur with agonists such as ATP, neurotensin (NT), hypotonic cell swelling and ionomycin, enhance the membrane conductance (GM) and hence the input conductance (GI) of HT29 cells. In the present study we have examined whether these increases in GM are paralleled by exocytosis. To this end the membrane capacitance (CM) of HT29 cells was measured by patch clamp techniques. Two methods to monitor CM were used: a direct method (DM) and a phase tracking method (PTM). With the DM the following results were obtained. NT (10(-8) mol/l, n = 9) increased GM and CM significantly from 2.4 +/- 0.3 nS and 23.5 +/- 3 pF to 32 +/- 8 nS and 27.3 +/- 3.1 pF respectively. ATP (10(-4) mol/l, n = 29) had a very similar effect. GM and CM were increased from 5.7 +/- 1 nS and 36 +/- 4.4 pF to 111 +/- 21 nS and 44 +/- 5.4 pF respectively. Hypotonic cell swelling (160 mosmol/l, n = 18) had a comparable effect: GM and CM were increased from 4.9 +/- 1 nS and 30 +/- 4.1 pF to 46 +/- 10 nS and 37 +/- 4.9 pF respectively. Ionomycin (10(-7) mol/l, n = 4) gave similar results. With the PTM it was possible to monitor the rapid changes in GM and CM, as they were induced by ATP (n = 42) and NT (n = 29), with high time resolution.(ABSTRACT TRUNCATED AT 250 WORDS)