Patch-clamp recordings of membrane currents evoked during natural synaptic activity in sympathetic neurons.

Membrane currents elicited by colonic distension and by electrical stimulation of the intermesenteric nerve containing colonic afferent nerve fibres were recorded from neurons of the mouse superior mesenteric ganglion at 20 degrees C with the whole-cell patch-clamp method. Electrically-evoked excitatory postsynaptic currents reversed at -3.5 mV. At membrane holding voltages of -70 mV and -110 mV, the excitatory postsynaptic currents were characterized by a single exponential decay with a mean (+/- S.E.M.) time-constant of 17.5 +/- 1.3 ms and 15.5 +/- 2.3 ms, respectively. Colonic distension evoked a series of the excitatory postsynaptic currents which ranged in amplitude from 10 to 700 pA (at a membrane holding voltage of -70 mV). Hexamethonium (100 microM) applied only to the ganglion abolished both electrically- and distension-evoked excitatory postsynaptic currents, suggesting activation of nicotinic acetylcholine receptors. The decay time-course of distension-evoked single excitatory postsynaptic currents was characterized by one, or, less commonly, by two exponentials. The decay time-constant histograms of distension-evoked single excitatory postsynaptic currents exhibited main kinetic components of 8.1 +/- 2.3 ms and 8.2 +/- 2.5 ms (peak +/- S.D.) at -70 and -110 mV membrane holding voltages, respectively. Longer time-constants ranging up to 51 ms were also observed. The number of the distension-evoked excitatory postsynaptic currents with a decay time-constant higher than 20 ms, as well as their mean amplitude, were significantly lower at -110 mV than at -70 mV membrane potential levels, in contrast to the currents with a decay time-constant lower than or equal to 20 ms. The results suggest that colonic afferent nerve fibres activate in the mouse superior mesenteric ganglion neurons a few populations of the postsynaptic nicotinic acetylcholine receptors with different channel kinetics, which are characterized by a lack of voltage sensitivity within -70 to -110 mV membrane potential range, except those with comparatively slow channel kinetics, which are possibly blocked by membrane hyperpolarization.

The ionic channel of neural nicotinic acetylcholine receptors is funnel-shaped.

Membrane currents evoked by iontophoretically applied acetylcholine were recorded from non-dissociated neurons of rat superior cervical ganglion using the whole-cell patch-clamp recording method. Blocking effects produced by a series of specially synthesized organic compounds, the blockers of the open channel of ganglionic nicotinic acetylcholine receptors, used as tools, were studied, and dimensions of the channel were deduced from correlation between the sizes of the blocking molecules and their blocking activities. Two channel cross-profiles were found, small and large, as approximated by the rectangles with the most probable dimensions 5.8 x 8.0 A and 7.0 x (8.4 - 9.0) A, at intra- and extracellular sides of the channel, correspondingly, in addition to the 6.1 x 8.3 A (medium) cross-profile found in our earlier work and localized between the two above. These findings indicate that the channel is funnel-shaped in the area where the open channel blockers are bound. The binding site is localized at the level of the medium cross-profile or between it and small cross-profile. The small cross-profile is probably a selectivity filter.

Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-alpha(181-192) peptide antibodies.

The subunit composition of nicotinic acetylcholine receptors of rat autonomic ganglia neurons was studied by means of antibodies, which differentiated between different alpha subunits and specifically blocked acetylcholine-induced membrane currents. Polyclonal rabbit antibodies and mouse monoclonal antibodies were raised against synthetic peptides matching in sequence the alpha(181-192) region of alpha3, alpha4, alpha5, and alpha7 subunits of rat neuronal nicotinic acetylcholine receptors. The antibodies discriminated among alpha3, alpha4, alpha5, and alpha7 peptides in enzyme-linked immunosorbent assay and bound to native acetylcholine receptors expressed in PC-12 cells. By means of immunoperoxidase staining of cultured rat autonomic neurons followed by transmission, dark-field and phase-contrast microscopy, it was found that all cells of the superior cervical ganglia expressed the alpha3, alpha5, and alpha7 nicotinic acetylcholine receptors, whereas approximately half of the cells were clearly alpha4-positive. In contrast, only about one-third of the intracardiac neurons were alpha3-positive, about 50% were alpha4-positive, one-seventh were alpha5-positive, and one-fifth were alpha7-positive. All antibodies tested blocked acetylcholine-induced currents in the neurons of the superior cervical ganglia as was demonstrated by whole-cell patch-clamp studies. Although each antibody could block up to 80% of the current, the degree of inhibition varied considerably from cell to cell. It is concluded that alpha3, alpha5, and alpha7 subunits are expressed in all neurons of the superior cervical ganglion and in some intracardiac neurons, whereas alpha4 subunits are expressed in some but not all neurons of both tissues. The neurons of the superior cervical ganglion express heterogeneous acetylcholine receptors and differ in relative amounts of acetylcholine receptor subtypes expressed.

The convergence of sympathetic preganglionic fibres on vasomotor neurones of the ganglia in lumbar sympathetic chain.

By means of intracellular recordings from the neurones of isolated L(3)-L(6) ganglia it was shown that B(1)-, B(2)- and C-preganglionic fibres widely converge on these neurones. Besides, a part of the ganglionic neurones is activated by slow-conducting C-fibres coming into the ganglia through the grey rami. Identified vasomotor neurones have the distinctive feature of preganglionic fibre convergence. Only B(2)- and C-preganglionic fibres were able to activate them, but the efficacy of the B(2)-fibre excitatory action is considerably higher.

Nicotinic acetylcholine receptor subtypes in rat superior cervical ganglion neurons as studied by sequential application of two alpha-subunit-specific antibodies.

All cultured neurons of rat superior cervical ganglion (SCG) were stained with nicotinic acetylcholine receptor (nAChR) alpha5- or alpha7-subunit-specific oligoclonal antibodies (Abs) and could additionally bind alpha3-subunit-specific monoclonal antibody (mAb). About 60% of the neurons were stained with alpha4-specific Ab and could not bind alpha3-specific mAb. The acetylcholine-induced membrane currents recorded with the whole-cell patch clamp method and partially blocked with alpha3-specific mAbs, could be additionally blocked with alpha5- and alpha7- specific Abs, and vice versa. The results suggest that: (1) each neuron of rat SCG expresses several nAChR subtypes with different alpha-subunits; (2) the alpha3-, alpha5- and alpha7-subunit-containing nAChRs are probably located far enough from each other thus enabling joint binding to the cell of the corresponding alpha-subunit specific Abs, in contrast to the alpha4-subunit-containing nAChRs which are probably located too close to the alpha3-containing ones to allow their joint binding.

Intracellular regulation of neuronal nicotinic cholinoreceptors.

Experiments on isolated superior cervical ganglia from rats were used to study the effects of substances affecting intracellular second messengers on membrane currents evoked by iontophoretic application of acetylcholine (ACh currents) and on excitatory postsynaptic currents (EPSC) induced by single discharges of preganglionic nerve fibers. These studies showed that the adenylate cyclase activator forskolin, the phosphodiesterase inhibitor isobutylmethylxanthine (IMBX), and the protein kinase C activator phorbol ester decreased the amplitude of the ACh current. Neither IMBX nor phorbol ester had any effect on the amplitude or decay time constant of EPSC, while forskolin increased the amplitude of EPSC without altering its decay time constant. Thapsigargin, which liberates intracellular calcium, not only decreased the amplitude of the ACh current, but also decreased EPSC amplitude without affecting its decay time constant. These results suggest that intracellular signaling via protein kinases A and C may affect neuronal nicotinic cholinoceptors (nAChR) only by altering receptor desensitization and not affecting receptor sensitivity to transmitters released from nerves or the kinetics of receptor ion channels. At the same time, neuronal nAChR are influenced by intracellular calcium, which decreases their ability to be activated by exogenous (perhaps acting via desensitization) and nerve-released acetylcholine without affecting the kinetics of ion channel function.

[Comparative sensitivity of the sympathetic and parasympathetic ganglia to ganglionic blockaders]. / Sravnenie chuvstvite'lnosti simpaticheskogo i parasimpaticheskogo gangliev k deistviiu ganglioblokatorov.

Pharmacological tests in vitro on the cat isolated superior cervical and ciliary ganglia identified the IEM-1194 as a selective parasympathetic ganglion-blocking agent. The ganglion-blocking activity of this drug was three times as high in parasympathetic ganglion (EC50 = 1.15 +/- 0.34 M/1 x 10(-5)) as in sympathetic one (EC50 = 3.2 +/- 0.2 M/1 x 10(-5)). I. v. administration of the IEM-1194 in a dose effectively (50%) blocking responses of the heart (bradycardia) and duodenum (peristaltics) to vagal stimulation, did not lower arterial blood pressure.

[Natural electrical activity of human sympathetic nerve fibers recorded from the body surface]. / Estestvennaia élektricheskaia aktivnost' simpaticheskikh nervnykh volokon cheloveka, zaregistrirovannaia ot poverkhnosti tela.

The electrical potentials recorded from human forearm were stored and averaged as synchronous with the cardiac rhythm. The R-wave of the ECG was used to synchronize the averaging system. The record obtained with this technique from human forearm during transient stop of blood flow to avoid mechanical artifacts was a sinusoid-like curve with peaks between the R-waves of the ECG. The nature of this electronegative wave was investigated by means of comparing electrograms and rheograms recorded from forearm of normal subjects and subjects after denervation or desympathization of their upper extremities. The data obtained suggest that the electronegative wave recorded with this technique is the averaged sum of tonic pulse-synchronous electrical activity of sympathetic nerve's fibers innervating vasoconstrictors of the skeletal muscle.

[The neuronal electrical responses of the superior cervical ganglion in the rabbit to nociceptive skin stimulation]. / Elektricheskie otvety neironov verkhnego sheinogo gangliia krolika na notsitseptivnoe razdrazhenie kozhi.

The patterns of tonic activity in the neurons of rabbit superior cervical ganglion at rest and during noxious stimulation of the skin were studied using intracellular recording. According to reflex changes in the activity patterns, all neurons studied were classified into three groups. Cardiac rhythmicity is more pronounced in the neurons of the second type than in those of the first type. The magnitude of the cardiac rhythmicity in both types of neurons was reduced after noxious stimulation of the skin. In the third type of neurons the cardiac rhythmicity was absent. In some neurons slow excitatory and inhibitory postsynaptic potentials appeared resulting from skin stimulation.