Electrophysiological investigation of the properties of acetylcholine receptors of frog sympathetic ganglionic neurons

Changes in membrane potential and conductance were studied in neurons of isolated sympathetic ganglia ofRana ridibunda during perfusion with cholinomimetics and cholinolytics. Activation of nicotinic (N) acetylcholine receptors by carbachol, suberyldicholine, and tetramethylammonium led to depolarization with an increase in conductance, whereas activation of muscarinic (M) acetylcholine receptors by perfusion with carbachol or 5-methylfurmethide, led to depolarization with a decrease or (less frequently) an increase in conductance. The M-cholinolytic atropine was shown to cause depolarization with an increase in conductance if perfusion with atropine was preceded by perfusion with carbachol.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 475–482, September–October, 1979.     

Effect of d-sparteine on nicotinic acetylcholine receptors in rat superior cervical ganglion neurons

Effects of d-sparteine (d-SP), a ganglionic blocking agent, on membrane currents evoked by iontophoretic applications of acetylcholine to rat superior cervical ganglion neurons, were studied using a whole-cell patch-clamp recording technique. Blocking effects of d-SP were enhanced by membrane hyperpolarization to potentials more negative than –50 mV. Analysis of the d-SP effect on the dose—response relationship suggests that d-SP at concentrations of 0.5–5.0 µM exerts both voltage-independent and voltage-dependent competitive actions on nicotinic acetylcholine receptors. No use-dependence of the d-SP-induced blockade was found using paired ACh applications at interpulse intervals longer than 0.5 sec. Inhibitory constantK _i estimated by the Dixon method was equal to 0.62±0.15 and 0.28±0.08 µM at membrane potential levels –30 and –90 mV, respectively. These characteristics of the d-SP blocking effects are compatible with a voltage-dependent competitive blocking mechanism. The possibility remains that an open channel-blocking mechanism with a comparatively fast kinetics contributes to the d-SP-induced blockade, but its contribution is small.Neirofiziologiya/Neurophysiology, Vol. 25, No. 4, pp. 266–272, July–August, 1993.     

Functional properties and developmental regulation of nicotinic acetylcholine receptors on embryonic chicken sympathetic neurons

Measurement of acetylcholine (ACh)-induced currents indicates that the sensitivity of embryonic sympathetic neurons increases following innervation in vivo and in vitro. We have used single-channel recording to assess the contribution of changes in ACh receptor properties to this increase. Early in development (before synaptogenesis), we detect three classes of ACh-activated channels that differ in their conductance and kinetics. Molecular studies indicating a variety of neuronal receptor subunit clones suggest a similar diversity. Later in development (after innervation), changes in functional properties include increases in conductance and apparent mean open time, the addition of a new conductance class, as well as apparent clustering and segregation of channel types. These changes in channel function are compatible with the developmental increase in ACh sensitivity.     

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal reponses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuation of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.     

Voltage-dependent effect of tetraethylammonium on the nicotinic acetylcholine receptors of rat superior cervical ganglion neurons

The effect of tetraethylammonium (TEA) on the currents evoked in neurons of the rat superior cervical ganglion by iontophoretic application of acetylcholine (ACh) was studied using a whole-cell patch-clamp recording technique. Tetraethylammonium was used at a concentration of about 20 µM, providing no blocking effect on the ACh-induced membrane currents at a range of positive membrane potentials and reducing these currents recorded at a range of negative membrane potentials by about half. The blocking effect of TEA increased with hyperpolarization within the –50 to –90 mV membrane potential range, and did not depend on the membrane potential level within a range of 0 to –50 mV. The analysis of dose dependence showed that both the voltage-dependent and the voltage-independent blocking effects are due to TEA competitive action on the ganglionic nicotinic acetylcholine receptors (nAChR). The results suggest that the TEA-induced competitive blockade is voltage-dependent.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 63–66, January–February, 1995.     

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons.

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal responses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuations of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.     

Voltage dependence of acetylcholine-activated membrane conductance in sympathetic ganglion neurons

Acetylcholine-induced membrane conductance was investigated in superior cervical ganglion neurons using a patch-clamp technique. It was found that hyperpolarization and depolarization produce an increase and a reduction in acetylcholine (ACh) conductance. This reduction was unconnected with either reversal of the current induced by iontophoretic ACh application or the presence of Ca ions in the external solution. The time constant of relaxation (_r) of this current, produced by a jump in membrane potential, was found to increase e-fold when the membrane was hyperpolarized by 70 mV, matching the voltage dependence of ACh conductance. This led to the hypothesis that voltage-dependent ACh-induced conductance is entirely determined by the voltage dependence of nicotinic receptor channel gating kinetics.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 167–171, March–April, 1988.     

Time course of receptor-channel coupling in frog sympathetic neurons.

The M-type potassium current and the N-type calcium current are inhibited by several different neurotransmitters in frog sympathetic neurons. These effects seem to be mediated via G proteins, but it is not clear whether diffusible second messengers are involved. Using a rapid (approximately 100 ms) flow tube perfusion system to apply agonists, the inhibition of calcium current develops and recovers rapidly but not instantaneously (t1/2 = 1-2 s). M-current inhibition is considerably slower, with t1/2 approximately 30 s for recovery from inhibition. At least for M-current inhibition, there appears to be sufficient time for involvement of an enzymatic cascade in receptor-channel coupling.     

Cadmium block of calcium current in frog sympathetic neurons.

Cd2+ blocks whole-cell calcium currents in frog sympathetic neurons by 50% at approximately 300 nM. Strong depolarizations rapidly reverse that blockade (tau = 1.3 ms at +120 mV). Reblocking follows bimolecular kinetics (rate = 1.2 x 10(8) M-1 s-1) at voltages where channels are mostly open (0 to +30 mV). The unblocking rate is approximately 50 s-1, so the dissociation constant calculated from the rate constants is approximately 400 nM. Steady-state block is strong at -80 mV, so closed channels can also be blocked. However, reblocking is extremely slow (tau = 1-2 s) at voltages where the channels are mostly closed. The rates for Cd2+ entry and exit are greater than 100-fold lower for closed channels than for open channels, and closed channels appear to be closed at both ends.     

Compartmentalization of choline and acetylcholine metabolism in cultured sympathetic neurons

Although the cellular and molecular mechanisms underlying the delayed-type hypersensitivity (DTH) reaction have been investigated, the functions of infiltrating leukocytes and skin resident cells in the elicitation phase of the DTH reaction are not completely understood. To gain more insight into the role of these cells in the DTH reaction, we identified about 250 cDNA fragments showing elevated expression during the DNCB-induced guinea pig skin DTH reaction by differential display analysis. Characterization of 50 of them led to the identification of 28 genes whose expression was elevated in the DNCB-induced DTH reactive tissue. Sequencing of the 28 cDNA fragments and homology search analysis demonstrated that 10 of them represented known genes, some of which, in particular elafin (an elastase inhibitor) and ferritin, are considered to play roles in the DTH reaction. The other 18 fragments are probably derived from unknown genes. Cloning of the cDNAs of one of these genes indicated that it is that for guinea pig tryptophanyl-tRNA synthetase (WRS), a protein found to be induced by interferon-gamma and upregulated during the late stages of mononuclear phagocyte maturation in vitro. Strong induction of the WRS gene during the DTH reaction suggests its involvement in the in vivo immune response.     

On the resting potential of isolated frog sympathetic neurons

One of the oldest questions of electrophysiology, the origin of the resting potential, has yet to be answered satisfactorily for most cells. Isolated frog sympathetic neurons, studied with whole-cell recording, generally have resting potentials of approximately -75 mV with an input resistance of approximately 300 M omega. These properties are not expected from the M-type K+ current (IM) or from other ionic currents previously described in these cells. In the -60 to -110 M mV voltage region, at least three currents are present: an inwardly rectifying current (IQ), a resting current with little voltage sensitivity carried at least in part by K+, and a (Na+,K+)ATPase pump current. The resting K+ current, not IM or IQ is the primary ionic current near the resting potential under these conditions. The electrogenic pump contributes an additional approximately 10 mV of hyperpolarization.     

A calcium-permeable channel activated by muscarinic acetylcholine receptors and InsP3 in developing chick ciliary ganglion neurons

The electrical responses elicited by the muscarinic cholinergic pathway have been studied in cultured embryonic chick ciliary ganglion (CG) neurons. Neurons obtained from E7-E8 ganglia were maintained in serum-free medium for 1 to 3 days. Stimulation with 50 microM muscarine induced depolarizing responses in about 30% of the cells tested. In voltage clamp experiments at a holding potential of -50 mV, an inward current could be recorded in the same percentage of cells in response to muscarinic stimulation. In single channel experiments, with standard physiological solution in the pipette, muscarine transiently activated an inward conducting channel. Cell-attached recordings with 100 mM CaCl(2) in the pipette provided evidence that muscarinic agonists can activate a cationic calcium-permeable channel. Two main conductance levels could be detected, of 2.3+/-0.6 and 5.6+/-0.6 pS, respectively. In excised patches, addition of 5-20 microM inositol 1,4,5-trisphosphate (InsP(3)) to the bath reactivated a channel that could be blocked by heparin and whose characteristics were very similar to those of the channel seen in response to muscarinic stimulation. A channel with similar properties has been previously shown to be activated by basic fibroblast growth factor (bFGF) and InsP(3) in the same preparation.     

交感神经节细胞对P物质和5-羟色胺的反应

本工作旨在观察Ｐ物质（ＳＰ）受体与５－羟色胺（５－ＨＴ）受体是否分别还是同时存在于豚鼠腹腔神经节（ＣＧ）与肠系膜下神经节（ＩＭＧ）不同细胞,以及这两种递质之间是否存在相互作用。在１３３个ＣＧ细胞中,６６个（４９．６％）对ＳＰ及５－ＨＴ同时敏感,４０个（３０．１％）仅对其中一种递质敏感,此外２７个（２０．３％）对两都不敏感。     

Increased acetylcholine synthesis and release following presynaptic activity in a sympathetic ganglion

Abstract: The acetylcholine (ACh) content of sympathetic ganglia increases above its normal level following a period of preganglionic nerve stimulation. In the present experiments, this extra ACh that accumulates following activity was labeled radioactively from [³H]choline and its specific activity was compared with that of ACh subsequently released during preganglionic nerve stimulation. The specific activity of the released ACh was similar to that of the total tissue ACh, suggesting that the extra ACh mixes fully with endogenous stores. The present experiments also show that transmitter release during neuronal stimulation is necessary for the poststimulation increase in transmitter store. However, the increase was not evident when transmitter release was induced by K⁺. It is concluded that both transmitter release and impulse invasion of the nerve terminals are necessary for the adaptive phenomenon to manifest itself. The role of choline delivery and choline acetyltransferase activity in generating the poststimulation increase in transmitter store was tested. When choline transport activity measured as choline analogue (homocholine) accumulation increased, ACh synthesis was increased and when transport activity was not increased, neither was ACh synthesis. There was no poststimulation increase in measured choline acetyltransferase activity.     

The genesis and differentiation of neurons in a frog parasympathetic ganglion

The cellular mechanisms that underlie formation of an autonomic ganglion have been investigated by studying the formation of the cardiac ganglion of the frog. Analysis of the genesis of neurons with [³H]thymidine autoradiography revealed that neuronal precursors do not divide via a “stem cell lineage” but rather divide exponentially, such that both daughter cells either re-enter the mitotic cycle or differentiate. Neurogenesis in this autonomic ganglion is prolonged, beginning during the second day after fertilization and continuing for at least 2 weeks. The use of acetylcholinesterase (AChE) as a neuronal marker showed that differentiated neurons start condensing in their target 1.5 days after the first neurons are born. Neurons accumulate, concomitant with neurogenesis, at a constant rate of approximately six neurons per day. Transplantation and organ culture demonstrated that immature neurons are present well before definitive expression of the mature phenotype and that their initial expression does not depend upon maintained contact by preganglionic axons.     

Disulfide bonds within the alpha-subunit of insulin receptors in rat liver and brain membranes

We have characterized inter- and intrasubunit disulfide bonds of insulin receptors using reductant-treated rat liver and brain membranes. In autoradiograms of 125I-insulin cross-linked to both membranes pretreated with dithiothreitol, the intensity of affinity-labeled bands of the alpha beta-heterodimer and alpha-subunit was increased. Interestingly, labeled 120 and 110 kDa bands considered to be the alpha-subunit in partially reduced liver and brain membranes moved to 130 and 120 kDa bands under further reduced conditions, respectively. Double electrophoresis of each partially reduced band in the presence of reductants clearly demonstrates that the alpha-subunit of insulin receptors contains intrasubunit disulfide bonds.