Patch-clamp recording in brain slices with improved slicer technology.

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.

Characterization of the driving force as a modulator of gating in cardiac ATP-sensitive K+ channels - evidence for specific elementary properties.

Single cardiac ATP-sensitive K+ channels and, comparatively, two other members of the inwardly rectifying K+ channel family, cardiac K+(ir) and K+(ACh) channels, were studied in the inside-out recording mode in order to analyze influence and significance of the electrochemical K+ gradient for open-state kinetics of these K+ channels. The conductive state of K+(ATP) channels was defined as a function of the electrochemical K+ gradient in that increased driving force correlates with shortened open-channel lifetime. Flux coupling of gating can be largely excluded as the underlying mechanism for two reasons: (i) tauopen proved identical in 23 pS, 56 pS and 80 pS channels; (ii) K+(ATP) channel protonation by an external pH shift from 9.5 to 5.5 reduced conductance without a concomitant detectable change of tauopen. Since gating continued to operate at EK, i.e., in the absence of K+ permeation through the pore, K+ driving force cannot be causally involved in gating. Rather the driving force acts to modulate the gating process similar to Rb+ whose interference with an externally located binding site stabilizes the open state. In K+(ir) and K+(ACh) channels, the open state is essentially independent on driving force meaning that their gating apparatus does not sense the electrochemical K+ gradient. Thus, K+(ATP) channels differ in an important functional aspect which may be tentatively explained by a structural peculiarity of their gating apparatus.

Thermodynamically specific gating kinetics of cardiac mammalian K+(ATP) channels in a physiological environment near 37 degrees C.

Elementary K+ currents through isolated ATP-sensitive K+ channels from neonatal rat cardiocytes were recorded to study their temperature dependence between 9 degrees C and 39 degrees C. Elementary current size and, thus, K+ permeation through the open pore varied monotonically with temperature with a Q10 of 1.25 corresponding to a low activation energy of 3.9 kcal/mol. Open-state kinetics showed a complicated temperature dependence with Q10 values of up to 2.94. Arrhenius anomalies of tau(open)(1) and tau(open)(2) indicate the occurrence of thermally-induced perturbations with a dominating influence on channel portions that are involved in gating but are obviously ineffective in altering pore-forming segments. At 39 degrees C, open-state exit reactions were associated with the highest activation energy (O2 exit reaction: 12.1 kcal/mol) and the largest amount of entropy. A transition from 19 degrees C to 9 degrees C elucidated a paradoxical kinetic response, shortening of both O-states, irrespective of the absence or presence of cAMP-dependent phosphorylation. Another member of the K+ channel family and also a constituent of neonatal rat cardiocyte membranes, 66 pS outwardly-rectifying channels, was found to react predictably since tau(open) increased on cooling. Obviously, cardiac K+(ATP) channels do not share this exceptional kinetic responsiveness to a temperature transition from 19 degrees C to 9 degrees C with other K+ channels and have a unique sensitivity to thermally-induced perturbations.

Electrophysiological interactions between carbachol and class I antiarrhythmic drugs (lidocaine, quinidine)--experimental studies in rabbit atrial myocardium.

UNLABELLED: Cholinergic agents exert no direct effect on the fast Na+ inward current but may influence the binding characteristic of class I antiarrhythmic drugs in atrial myocardium by shortening the action potential (AP) duration or by increasing the resting potential (RP). In order to examine such possible interactions we performed experiments using conventional intracellular microelectrodes on isolated preparations of rabbit atrial myocardium (Ke 2.7 mM, temperature 32 degrees C). At first the influence of the cholinergic agent carbachol (1 mg/l = 6.7 x 10(-6) M) on the RP and AP was examined at different stimulation rates (1.0, 2.5, and 3.3 Hz). Thereafter measurements were repeated under the influence of lidocaine (10 mg/l = 2.2 x 10(-5) M) or quinidine (5 mg/l = 2.2 x 10(-5) M) alone and in combination with carbachol (1 mg/l). RESULTS: (statistically significant differences, p less than 0.05): Carbachol increased the RP by about 10 mV and shortened the AP by about 60%. The maximal upstroke velocity of the AP (Vmax) was not significantly altered at 1.0 and 2.5 Hz, but increased under carbachol at 3.3 Hz. After addition of carbachol to the lidocaine-containing solution, Vmax increased to its control level at all stimulation rates. In experiments with quinidine, Vmax also increased after addition of carbachol but remained significantly below the control values. CONCLUSIONS: Carbachol effects on Vmax are most likely attributable to earlier recovery (caused by the shortening of the AP) and to faster recovery kinetics (due to hyperpolarization). The attenuation of the class I effect of lidocaine by carbachol can thus be considered mainly a consequence of the shortening of the inactivated state which results in a reduced affinity of lidocaine to its receptor and allows earlier dissociation of the drug. Minor binding of the drug due to hyperpolarization may play the major role in interactions between carbachol and quinidine.

Effects of polidocanol as a constituent of a venous sclerosing agent on cardiac electrical activity. Studies on the isolated perfused heart and papillary muscle of the guinea pig and on the isolated sinus node of the rabbit.

The effects of a frequently used sclerosing agent (Sotravarix 6%, main constituent polidocanol 60 mg/ml = 0.1 mol/l) on the electrical activity of the isolated perfused guinea pig heart and on transmembrane potential of isolated papillary muscle and sinus node were studied. Application of the sclerosing agent in a dilution of 1:40,000 (corresponding to polidocanol 2.6 x 10(-6) mol/l) caused a significant decrease in frequency (-27%) and (at constant frequency) a significant delay of the atrioventricular as well as of the intraventricular conduction (+105% or + 210%, resp. n = 4). These effects were only slightly reversible in a 30 min wash-out period. In the isolated papillary muscle, the sclerosing agent (dilution 1:10,000; polidocanol 1 x 10(-5) mol/l) decreased the maximum rate of rise of the action potential and shortened its duration, both by about 10%. In the isolated sinus node the same dilution decreased the spontaneous rate by about 8% mainly by reducing the diastolic depolarization rate (-18%). The effects of the sclerosing agent are attributable to the influence of polidocanol and consist in a blockade of the fast sodium channels (local anesthesia) and in additional inhibitory effects on calcium and/or potassium conductance.

Disturbances of neural conduction in isolated frog nerves following exposure to strong electric fields.

Frog sciatic nerves were isolated and the middle portion of each exposed to condenser discharges (field strength up to 1000 V/cm; time constants 0.2-8.0 ms) through the bathing fluid. The ability of the nerve to propagate action potentials (AP) was examined by stimulating the proximal end and recording the AP from the distal end of the exposed section. The fraction of the nerve fibers remaining propagative was estimated from the amplitude (or the area) of the compound AP. Strong discharges brought about a total block of propagation lasting for up to 30 minutes, followed by slow, but almost complete, restitution. The restitution was exponential against time and depended on the field strength and duration of the discharge. Discharges equal in energy but different in their voltage--condenser combinations had markedly different actions, with stronger effects being found at higher voltages and vice versa. Hence, the described effects are unlikely to be caused by dissipation of thermal energy only. Other mechanisms (ionic imbalance, dielectric breakdown, punch through) are discussed.

Electrophysiological profile of the antiarrhythmic compound asocainol studied on perfused guinea-pig hearts and on isolated cardiac preparations.

The studies deal with electrophysiological effects of asocainol [(+/-)-6,7,8,9-tetrahydro-2,12-dimethoxy-7-methyl-6-phenetyl-5 H-dibenz(d,f)azonine-1-ol] on isolated perfused guinea-pig hearts (Langendorff-preparation), on right ventricular papillary muscles, on Purkinje fibres from the guinea pig, and on isolated sinus nodes from the rabbit. In the perfused heart (n = 5) the lowest effective concentration of asocainol is about 0.2 mumol/l. At a concentration of 2 mumol/l the cardiac electrogram shows in spontaneously beating hearts a mean decrease in frequency of 15%, in electrically driven hearts (150/min at 32 degrees C) prolongation of PQ (+31%), of QRS (+24%) and of QT (+5%). In papillary muscles (32 degrees C; K+e 5.9 mmol/l; stimulation rate 0.5 Hz) asocainol (3-30 mumol/l) exerts the following effects: no change of the resting potential, concentration-dependent reduction of the maximum rate of rise (Vmax) of the action potential (AP) (-16 to -67%) as well as of the AP-amplitude (-4 to -16%), and shortening of the AP-duration at 50% repolarisation (-18 to -43%). The steady-state dependence of Vmax on the resting potential (RP) determined by variation of K+e (5.9-15 mmol/l) is shifted by asocainol to more negative potentials. The percentage deviation from controls of the Vmax-RP relationship is more pronounced at lower membrane potentials. The influence of asocainol on the recovery from inactivation of Vmax shows marked time-dependence. Slow response (Ca2+-mediated) APs elicited by strong stimuli in a K+e-rich solution (K+e 20-24 mmol/l) respond to asocainol (3-10 mumol/l) with a marked reduction in amplitude, Vmax and duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental study of partial liver resection with a combined CO2 and Nd:YAG laser.

The use of the CO2 laser in liver surgery is mainly limited by the lack of coagulation of the larger vessels. In an experimental study, partial liver resections were performed on pigs with a Nd:YAG as well as with a combined CO2 and Nd:YAG laser. The best cutting efficiency was obtained with the CO2 laser. On the other hand, the Nd:YAG laser and the combined laser sources showed excellent hemostasis at the cutting edge corresponding with a width of necrosis at about 5 mm in histomorphometric examination and zones with histologically different characteristics. Rebleedings from the resection lines were avoided in all cases using the combined CO2 and Nd:YAG laser.

Laser coagulation in the upper GI tract: a preliminary light and scanning electron microscopic study.

Experimentally intact as well as artificially damaged mucosa of porcine stomach was endoscopically coagulated with Nd:YAG laser beams. Energy, intensity pattern, and time of application of the irradiation was varied and supracellular changes of the coagulated mucosa were subsequently investigated with the scanning electron microscope. The mucosa, damaged by laser beams, showed three typical zones: 1. a superficial, central carbonisation zone protected with a thin layer of coagulated mucus; 2. an elevated hyperemic ring with complete loss of superficial epithelium, but intact basement membrane and a blood clotting of the mucosal capillaries; 3. normal mucosa. After coagulating artificially induced bleeding of the mucosa the irradiated centre showed a deep depression with a carbonized surface. Two weeks later regeneration had occurred and there was complete reepithelialisation of the mucosa to about two-thirds of normal thickness.