Comparative Studies of Calcium Transients Induced by Acetylcholine in Rat Chromaffin Cells

We used an intracellular fluorescent probe, FURA-2M, to examine the responses of isolated rat chromaffin cells to applications of 1 mM acetylcholine (ACh). Our data showed two different populations of the cell responses to such stimulation. Responses of the first type demonstrated fast rise and decay phases of the Ca²⁺ transients and no significant decrease in their amplitude during repetitive stimulation of the cell with ACh. Cell responses of the second type showed remarkably slower rise and decay phases of the Ca²⁺ transients and a noticeable drop of the cell responses during repetitive ACh stimulation that could be recovered after KCl depolarization. We find no significant differences in the amplitudes of the transients in these two populations of the cells. We conclude that there is heterogeneity of the chromaffin cells according to their ACh receptors: the first subpopulation predominantly expresses ionotropic (nicotinic) receptors (n cells), whereas the second cell population has mainly metabotropic (muscarinic) ones (m cells), which are associated with Ca²⁺ release from the intracellular stores.     

Conduction of hyperpolarization along hamster feed arteries: augmentation by acetylcholine

The conduction of vasodilation along resistance vessels has been presumed to reflect the electrotonic spread of hyperpolarization from cell to cell along the vessel wall through gap junction channels. However, the vasomotor response to acetylcholine (ACh) encompasses greater distances than can be explained by passive decay. To investigate the underlying mechanism for this behavior, we tested the hypothesis that ACh augments the conduction of hyperpolarization. Feed arteries (n = 23; diameter, 58 +/- 4 microm; segment length, 2-8 mm) were isolated from the hamster retractor muscle, cannulated at each end, and pressurized to 75 mmHg (at 37 degrees C). Vessels were impaled with one or two dye-containing microelectrodes simultaneously (separation distance, 50 microm to 3.5 mm). Membrane potential (E(m)) (rest, approximately -30 mV) and electrical responses were similar between endothelium and smooth muscle, as predicted for robust myoendothelial coupling. Current injection (-0.8 nA, 1.5 s) evoked hyperpolarization (-10 +/- 1 mV; membrane time constant, 240 ms) that conducted along the vessel with a length constant (lambda) = 1.2 +/- 0.1 mm; spontaneous E(m) oscillations (approximately 1 Hz) decayed with lambda = 1.2 + 0.1 mm. In contrast, ACh microiontophoresis (500 nA, 500 ms, 1 microm tip) evoked hyperpolarization (-14 +/- 2 mV) that conducted with lambda = 1.9 +/- 0.1 mm, 60% further (P < 0.05) than responses evoked by purely electrical stimuli. These findings indicate that ACh augments the conduction of hyperpolarization along the vessel wall.     

Electrical coupling and uncoupling of exocrine acinar cells

The electrical communication network in the mouse pancreatic acinar tissue has been investigated using simultaneous intracellular recording with two separate microelectrodes and direct microscopical control of the localizations of the microelectrode tips. All cells within one acinus were electrically coupled, and the coupling coefficient (the electrotonic potential change in a cell neighboring to the cell into which current is injected [V2] divided by the electrotonic potential change in the cell of current injection [V1]) between two cells near each other (less than 50 micron) was always close to 1. Cells farther apart (50-100 micron) were, in some cases, coupled; in other cases, there was no coupling at all. Coupling coefficients varied between 0 and 1. There was rarely electrical coupling over distances of more than 110 micron. Using microiontophoretic acetylcholine (ACh) application, it was possible to evoke almost complete electrical uncoupling of two previously coupled pancreatic or lacrimal acinar cells from different acini or within one acinus. The effects were fully and quickly reversible. While the ACh-evoked uncoupling in the pancreas was associated with membrane depolarization, ACh caused hyperpolarization in the lacrimal acinar cells. The uncoupling was associated with a very marked reduction in electrical time constant, indicating a reduction in input capacitance (effective surface cell membrane area). The concentrations of stimulants needed to evoke reduction in pancreatic cell-to-cell coupling were 1 micron for ACh, 0.14 nM for caerulein, and 3 nM for bombesin. These concentrations are smaller than those required to evoke maximal enzyme secretion.     

Inhibition of the Electrically Evoked Release of [3H]Acetylcholine in Rat Striatal Slices: An Experimental Model for Drugs that Enhance Dopaminergic Neurotransmission

The activation by endogenous dopamine of the inhibitory 3,4-dihydroxyphenylethylamine (dopamine) receptors modulating the electrically evoked release of [3H]acetylcholine [( 3H]ACh) and [3H]dopamine in rat striatal slices is a function of the concentration of dopamine accumulated in the synaptic cleft during electrical stimulation. When the release of 3H-neurotransmitters was elicited with a 2-min period of stimulation at a frequency of 1 Hz, neither dopamine autoreceptors nor dopamine receptors modulating [3H]ACh were activated by endogenously released dopamine. On the other hand, exposure to (S)-sulpiride facilitated the release of [3H]dopamine and [3H]ACh elicited when the 2-min stimulation was carried out at a frequency of 3 Hz but this effect was not observed at a lower frequency of stimulation (1 Hz). In the presence of amphetamine the dopamine receptors modulating the electrically evoked release of [3H]ACh can be activated by endogenous dopamine even at the lower frequency of stimulation (1 Hz). Similar effects can be obtained if the neuronal uptake of dopamine is inhibited by cocaine or nomifensine. The inhibition by amphetamine of the release of [3H]ACh elicited by electrical stimulation at 1 Hz involves dopamine receptors and can be fully antagonized by clozapine, haloperidol, chlorpromazine, or pimozide. The stereoselectivity of this antagonism can be demonstrated with the optical enantiomers of sulpiride and butaclamol. This inhibitory effect of amphetamine on cholinergic neurotransmission appears to be the result of the stimulation of dopamine receptors of the D2 subtype, as they were resistant to blockade by the preferential D1 receptor antagonist SCH 23390.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine modulation of the In vivo acetylcholine response in the Drosophila mushroom body

Olfactory sensory information in Drosophila is transmitted through antennal lobe projections to Mushroom Body neurons (Kenyon cells) by means of cholinergic synapses. Application of acetylcholine (ACh) and odors produce significant increases in intracellular calcium ([Ca²⁺]_i) in these neurons. Behavioral studies show that Kenyon cell activity is modulated by dopaminergic inputs and this modulation is thought to be the basis for an olfactory conditioned response. However, quantitative assessment of the synaptic inputs to Kenyon cells is currently lacking. To assess neuronal activity under in vivo conditions, we have used the endogenously‐expressed camgaroo reporter to measure [Ca²⁺]_i in these neurons. We report here the dose‐response relationship of Kenyon cells for ACh and dopamine (DA). Importantly, we also show that simultaneous application of ACh and DA results in a significant decrease in the response to ACh alone. In addition, we show inhibition of the ACh response by cyclic adenosine monophosphate. This is the first quantitative assessment of the effects of these two important transmitters in this system, and it provides an important basis for future analysis of the cellular mechanisms of this well established model for associative olfactory learning. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

硝苯吡啶对腹腔神经节突触传递的阻断作用

本文应用细胞内记录技术,观察了钙通道阻滞剂硝苯吡啶（nifedipine)对离体豚鼠腹腔神经节突触传递的影响,硝苯吡啶（0.1-10umol/L)不影响所检细胞的静息膜电位,膜电阻及细胞内刺激引起的动作电位,但能显著阻断N-型胆碱能的突触传递,并且这种作用可被低钙模拟、高钙拮抗,硝苯吡啶（10umol/L)也不影响突触后膜对乙酰胆碱（ACh)的敏感性;但在高钾克氏液中,能减少微小兴奋性突触后电位（mEPSPs)的频率;在低钙和高镁克氏液中,能减少量子含量,而对量子大小无影响。结果表明,治疗量的硝苯吡啶(0.1umol/L)通过阻滞突触前膜钙内流及ACh的量子性释放,产生突触阻断作用。这可能是硝苯吡啶降压机理的一个组成部分。     

Neural regulation of slow-wave frequency in the murine gastric antrum

Gastric peristaltic contractions are driven by electrical slow waves modulated by neural and humoral inputs. Excitatory neural input comes primarily from cholinergic motor neurons, but ACh causes depolarization and chronotropic effects that might disrupt the normal proximal-to-distal spread of gastric slow waves. We used intracellular electrical recording techniques to study cholinergic responses in stomach tissues from wild-type and W/W(V) mice. Electrical field stimulation (5 Hz) enhanced slow-wave frequency. These effects were abolished by atropine and the muscarinic M(3)-receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide. ACh released from nerves did not depolarize antral muscles. At higher rates of stimulation (10 Hz), chronotropic effects were mediated by ACh and a noncholinergic transmitter and blocked by muscarinic antagonists and neurokinin (NK(1) and NK(2))-receptor antagonists. Neostigmine enhanced slow-wave frequency, suggesting that the frequency of antral pacemakers is kept low by efficient metabolism of ACh. Neostigmine had no effect on slow-wave frequency in muscles of W/W(v) mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). These muscles also showed no significant chronotropic response to 5-Hz electrical field stimulation or the cholinergic agonist carbachol. The data suggest that the chronotropic effects of cholinergic nerve stimulation occur via ICC-IM in the murine stomach. The capacity of gastric muscles to metabolize ACh released from enteric motor neurons contributes to the maintenance of the proximal-to-distal slow-wave frequency gradient in the murine stomach. ICC-IM play a critical role in neural regulation of gastric motility, and ICC-IM become the dominant pacemaker cells during sustained cholinergic drive.     

Mechanisms of heterosynaptic facilitation in molluscan neurons

This review is focused on the analysis of research data obtained in one of the models of conditioned reflex, heterosynaptic facilitation (HSF), in the molluscan nervous system. Our experiments were performed on identified giant command neurons LS1 and PS1 of the freshwater snail Planorbarius corneus. HSF was elicited during the electrical stimulation of two nerves: pallial (the analog of unconditioned stimulation — US) and one of the cerebral nerves (the analog of the conditioned stimulation — CS). The degree of HSF manifestation depended not on the intensity of the synaptic response of the giant neuron to US, but the efficacy of the connection between the pallial nerve and neurosecretory neurons surrounding the command neuron of the mesocerebrum. It is demonstrated that HSF develops due to the diffuse neurohumoral action of serotonin (5-hydroxytryptamine — 5-HT) on the postsynaptic structures, but not as a result of local synaptic action on the presynaptic mechanism. Approximately 70% of US cases of 5-HT application induced a four- to six-fold increase in amplitude of the excitatory postsynaptic potential (EPSP) and acetylcholine (ACh) response. Both responses are N-cholinergic and depend on the membrane permeability to Na⁺ and K⁺. In 30% of the cases, ACh response diminished simultaneously with EPSP increase. The 5-HT effect on EPSP and ACh responses were mimicked by the action of phosphodiersterase blockers and adenylate cyclase activators. Thus, the activation of the adenylate cyclase system following 5-HT action facilitates the postsynaptic mechanism underlying HSF formation in command neurons of Planorbarius corneus. Dopamine (DA) and noradrenaline (NA) blocked EPSP and simultaneously increased the amplitude of ACh response. These monoamines were also blocked HSF. The wash-out of catecholamines following HSF blockade enhanced the restoration and subsequent prolongation of synaptic facilitation. It is thus concluded that DA or NA may control the HSF intensity and duration under natural conditions of the nervous system in the molluscs.Neirofiziologiya/Neurophysiology, Vol. 25, No. 3, pp. 224–232, May–June, 1993.     

Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.     

Endothelial cell signaling during conducted vasomotor responses

ACh and KCl stimulate vasomotor responses that spread rapidly and bidirectionally along arteriole walls, most likely via spread of electric current or Ca2+ through gap junctions. We examined these possibilities with isolated, cannulated, and perfused hamster cheek pouch arterioles (50- to 80-microm resting diameter). After intraluminal loading of 2 microM fluo 3 to measure Ca2+ or 1 microM di-8-ANEPPS to measure membrane potential, photometric techniques were used to selectively measure changes in intracellular Ca2+ concentration ([Ca2+]i) or membrane potential in endothelial cells. Activation of the endothelium by micropipette application of ACh (10-4 M, 1.0-s pulse) to a short segment of arteriole (100-200 microm) increased endothelial cell [Ca2+]i and caused hyperpolarization at the site of stimulation. This response was followed rapidly by vasodilation of the entire arteriole ( approximately 2-mm length). Change in membrane potential always preceded dilation, both at the site of stimulation and at distant sites along the arteriole. In contrast, an increase in endothelial cell [Ca2+]i was observed only at the application site. Micropipette application of KCl, which can depolarize both smooth muscle and endothelial cells (250 mM, 2.5-s pulse), also caused a rapid, spreading response consisting of depolarization followed by vasoconstriction. With KCl stimulation, in addition to changes in membrane potential, increases in endothelial cell [Ca2+]i were observed at distant sites not directly exposed to KCl. The rapid longitudinal spread of both hyperpolarizing and depolarizing responses support electrical coupling as the mode of signal transmission along the arteriolar length. In addition, the relatively short distance between heterologous cell types enables the superimposed radial Ca2+ signaling between smooth muscle and endothelial cells to modulate vasomotor responses.     

Fasciculin II,a protein inhibitor of acetylcholinesterase,tested on central synapses ofAplysia

Fasciculin II, a potential inhibitor of acetylcholinesterase (AChE), was tested on two types of Aplysia cholinergic receptors: H type, opening Cl- channels; and D type, opening cationic channels. Evoked postsynaptic inhibitory responses and responses to ionophoretic application of acetylcholine (ACh) or carbachol onto H-type receptors were potentiated in the presence of fasciculin II at 10(-9) M, whereas the same concentration of this drug was without effect on the evoked postsynaptic excitatory responses or on the application of ACh or carbachol on D-type receptors. The observed effects of fasciculin II were identical to those obtained with other inhibitors of AChE on the same preparation. The facilitatory effect on the carbachol response in H-type cells indicates that fasciculin II, as other AChE inhibitors, does not act on H-type synapses solely by blocking the hydrolysis of ACh. We concluded that fasciculin II was a good inhibitor of acetylcholinesterase on neuronal preparations in vivo. The results are further discussed as a new element in favor of a previously proposed hypothesis of a molecular interaction between AChE and ACh H-type receptors.     

Intestinal secretagogues increase cytosolic free Ca2+ concentration and K+ conductance in a human intestinal epithelial cell line

Summary A human intestinal epithelial cell line (Intestine 407) is known to retain receptors for intestinal secretagogues such as acetylcholine (ACh), histamine, serotonin (5-HT) and vasoactive intestinal peptide (VIP). The cells were also found to possess separate receptors for secretin and ATP, the stimulation of which elicited transient hyperpolarizations coupled to decreased membrane resistances. These responses were reversed in polarity at the K⁺ equilibrium potential. The hyperpolarizing responses to six agonists were reversibly inhibited by quinine or quinidine. By means of Ca²⁺-selective microelectrodes, increases in the cytosolic free Ca²⁺ concentration were observed in response to individual secretagogues. The time course of Ca²⁺ responses coincided with that of hyperpolarizing responses. The responses to ACh and 5-HT were abolished by a reduction in the extracellular Ca²⁺ concentration down to pCa 7 or by application of Co²⁺. Thus, in Intestine 407 cells, not only the intestinal secretagogues, which are believed to act via increased cytosolic Ca²⁺ (ACh, 5-HT and histamine), but also those which elevate cyclic AMP (VIP, secretin and ATP) induce increases in cytosolic Ca²⁺, thereby activating the K⁺ conductance. It is likely that the origin of increased cytosolic Ca²⁺ is mainly extracellular for ACh- and 5-HT-induced responses, whereas histamine, VIP, secretin and ATP mobilize Ca²⁺ from the internal compartment.     

Development of a new coulter counter system: Measurement of the volume,internal conductivity,and dielectric breakdown voltage of a single guard cell protoplast ofVicia faba

Summary Two intracellular microelectrodes were used to study electrotonic interaction between cultured embryonic (16- to 20-day-old) chick myocardial cells reaggregated into small spheresin vitro. Under different culture conditions, reaggregates with two types of functional membrane properties were produced: (i) highly differentiated reaggregates, and (ii) reverted reaggregates. In the highly differentiated state, the cells had high stable resting potentials and produced rapidly-rising tetrodotoxin (TTX)-sensitive action potentials in response to electric field stimulation. In the reverted state, the cells exhibited slowrising spontaneous action potentials having prominent pacemaker potentials and TTX-insensitive upstrokes. These states resemble electrophysiological properties of the highly differentiated (18 daysin ovo) and less fully differentiated (3 daysin ovo) intact embryonic chick heart, respectively. Both types of reaggregates had similar ultrastructural appearance, with many elongated cells and intercalated disc-like structures; gap-like junctions were not seen. The highly differentiated cells had input resistances of about 5 M, and exhibited only little electrotonic interaction in response to intracellular current injection either when the cells were at rest or during the action potential plateau. Intracellular stimulation produced propagating action potentials which triggered contraction of the entire reaggregate. Large hyperpolarizing current pulses applied during the action potential plateau caused premature repolarization which also propagated to the other impaled cell. In the reverted reaggregates, electrotonic interaction was weak or absent in about 52% of the impaled cell pairs, moderate in 30%, and strong in 18% (encountered only at interelectrode distances of less than 100 m). The difference in degree of electrotonic interaction may be due to the state of differentiation with respect to the membrane electrical properties.     

Evoked Acetylcholine Release by Immortalized Brain Endothelial Cells Genetically Modified to Express Choline Acetyltransferase and/or the Vesicular Acetylcholine Transporter

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.     

Remote stimulation by heat induces characteristic membrane-potential responses in the veins of wild-type and abscisic acid-deficient tomato plants

The systemic induction of proteinase inhibitor genes in tomato plants is either mediated by fast electrical signals or alternatively by chemical messengers. In the present study we analyzed the pathway of the electrical signal. The question of which cell types are involved in this pathway of long-distance signaling within plants is still controversial. To identify these we inserted microelectrodes into the veins of tomato leaves (Lycopersicon esculentum Mill. cv. Moneymaker). A newly developed computer program and microcomputer interface enabled us to position these microelectrodes inside the vein with an accuracy of 1 μm. Due to this precision in positioning we were able to demonstrate that the pathway of the electrical signal is not restricted to a specific tissue type, e.g. the phloem. In particular, the entire vein contributes to the propagation of the electrical wave along the plant. Therefore, an apoplastic contribution to the long-distance signal transduction mechanism appears most likely. To furthermore investigate the involvement of cis-abscisic acid (ABA) in this long-distance signal transduction pathway, ABA-deficient tomato mutants (Lycopersicon esculentum cv. Sitiens) were used in comparison to the wild type. Significant differences between the membrane-potential relaxation kinetics of the wild type and the mutants could be detected. Wild-type tomato plants exhibited six characteristic classes of membrane-potential relaxation kinetics following heat treatment. In contrast, the ABA-deficient mutants were more restricted in terms of their relaxation upon heat stimulation. The responses in the membrane potential of all cells within a vein consisted of only three categories. In conclusion, ABA did not affect all cells within the vein in a similar manner. Single cells exhibited different response patterns to systemic heat application in the presence of ABA. Moreover, ABA had a pronounced effect on the resting potentials of individual cells within the veins of tomato. Received: 1 July 1997 / Accepted: 16 January 1998     

Prevention of diminished parasympathetic control of the heart in experimental heart failure

Decreased synaptic transmission in parasympathetic ganglia contributes to abnormal parasympathetic function in heart failure (HF). Because nicotinic ACh receptors (nAChR) mediate synaptic transmission at the ganglion and upregulate in response to chronic exposure to agonist in vitro, we tested the hypothesis that repeated exposures of ganglionic neurons to a nAChR agonist can prevent a loss of parasympathetic control in HF. Two sets of experiments were performed. In set 1, unpaced control dogs and dogs undergoing pacing-induced HF were treated with a repeated intravenous nicotinic agonist during the development of HF. Under conditions of sympathetic blockade, R-R responses to a bolus injection of 200 microg 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; nicotinic agonist) were found to be increased five times over the untreated group after 6 wk. In experimental set 2, dogs treated with weekly DMPP injections and in HF were anesthetized and underwent electrical stimulation of the right vagus nerve, which showed sinus cycle length responses >10 times that of controls (P < 0.05). Complete ganglionic blockade with hexamethonium abolished all responses, confirming that synaptic transmission was mediated entirely by nAChRs in both controls and HF. Despite decreased ganglionic function leading to reduced parasympathetic control of the heart in HF, repeated exposure with a nicotinic agonist during the development of HF results in not only preserved but also supranormal effects of parasympathetic stimulation on the sinus node.     

Acetylcholine elongates neuronal growth cone filopodia via activation of nicotinic acetylcholine receptors

In addition to acting as a classical neurotransmitter in synaptic transmission, acetylcholine (ACh) has been shown to play a role in axonal growth and growth cone guidance. What is not well understood is how ACh acts on growth cones to affect growth cone filopodia, structures known to be important for neuronal pathfinding. We addressed this question using an identified neuron (B5) from the buccal ganglion of the pond snail Helisoma trivolvis in cell culture. ACh treatment caused pronounced filopodial elongation within minutes, an effect that required calcium influx and resulted in the elevation of the intracellular calcium concentration ([Ca]_i). Whole‐cell patch clamp recordings showed that ACh caused a reduction in input resistance, a depolarization of the membrane potential, and an increase in firing frequency in B5 neurons. These effects were mediated via the activation of nicotinic acetylcholine receptors (nAChRs), as the nAChR agonist dimethylphenylpiperazinium (DMPP) mimicked the effects of ACh on filopodial elongation, [Ca]_i elevation, and changes in electrical activity. Moreover, the nAChR antagonist tubucurarine blocked all DMPP‐induced effects. Lastly, ACh acted locally at the growth cone, because growth cones that were physically isolated from their parent neuron responded to ACh by filopodial elongation with a similar time course as growth cones that remained connected to their parent neuron. Our data revealed a critical role for ACh as a modulator of growth cone filopodial dynamics. ACh signaling was mediated via nAChRs and resulted in Ca influx, which, in turn, caused filopodial elongation. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 487–501, 2013     

Learning-related long-term synaptic facilitation in snails: Possible mechanisms of long-term memory formation

The long-term changes in the electrical activity of command neurons related to sensitization and elaboration of associative defensive behavioral habit (food rejection) were studied inHelix snail. The long-term effects consisted of facilitation of synaptic components in neuronal responses to the test stimulations. Variations were found in the dynamics of long-term synaptic facilitation of responses to the applied chemical and tactile stimuli in the course of sensitization, as well as dependence of the degree of long-term facilitation of responses to the test stimulation at the site of its application with respect to the site of the sensitizing stimulation (site-specific sensitization). After conditioning, the synaptic response of command neurons to the conditioning stimulation appeared approximately 30 min later than did the long-term sensitization in these cells. The minimum duration of long-term synaptic facilitation of responses to the test stimulation varied from 1 h (for tactile stimulation) to 3 h (for chemical stimulation). The maximum duration of effects exceeded 4 h. It is suggested that the observed features of the synaptic plasticity in command neurons during learning are based on the selective regulation of synaptic inputs by specific protein regulators, whose lifespan does not exceed 1 h to 3 h.Neirofiziologiya/Neurophysiology, Vol. 25, No. 5, pp. 383–389, September–October, 1993.     

Conductance Change Associated with Receptor Potentials of Gustatory Cells in Rat

The electrical properties of gustatory cells and cells which do not respond to chemical stimuli in the taste bud of fungiform papillae in rats were studied by means of intracellular microelectrodes. Neither of these cell types showed spike electrogenesis. Gustatory cells showed a depolarization, the receptor potential, associated with an increase in the membrane conductance in response to NaCl, sucrose, and HCl, whereas quinine produced a decrease in the conductance together with an increase in the receptor potential magnitude. The reversal point of the receptor potential in response to NaCl or KCl was close to zero membrane potential, but in the case of quinine it was at a more negative potential level than the resting potential. From these results two receptive processes are postulated in the gustatory cell membrane. When the gustatory cells were stimulated for a long duration by concentrated NaCl or sucrose, receptor potentials showed adaptation with decrease in magnitude, but adaptation of the responses to HCl and quinine were hardly detected. Adaptation of the receptor potential was not correlated with conductance change.     

Intracellular injection of dopamine enhances acetylcholine responses of neuron R2 in the Aplysia abdominal ganglion

1. At different levels of the holding potential on neuron R2 membrane in the Aplysia depilans abdominal ganglion, dopamine injected intracellularly increases the amplitude of both inward and outward currents recorded in response to the application of acetylcholine (ACh) to the ganglion surface. 2. The addition of dopamine to the external perfused solution produces generation of inward currents and a decrease in the cell response to the ACh. 3. The enhancing effect of injected dopamine on ACh responses is retained after inhibition of acetylcholinesterase (AChE) by a specific organophosphorous inhibitor, compound Gd-42. 4. The modulating effect of injected dopamine on ACh responses is discussed in terms of the existence of intracellular receptors of neurotransmitters in the differentiated cells.