Rab3A deletion selectively reduces spontaneous neurotransmitter release at the mouse neuromuscular synapse

Rab3A is a synaptic vesicle-associated GTP-binding protein thought to be involved in modulation of presynaptic transmitter release through regulation of vesicle trafficking and membrane fusion. Electrophysiological studies at central nervous system synapses of Rab3A null-mutant mice have indicated that nerve stimulation-evoked transmitter release and its short- and long-term modulation are partly dependent on Rab3A, whereas spontaneous uniquantal release is completely independent of it. Here, we studied the acetylcholine (ACh) release at the neuromuscular junction (NMJ) of diaphragm and soleus muscles from Rab3A-deficient mice with intracellular microelectrode methods. Surprisingly, we found 20-40% reduction of spontaneous ACh release but completely intact nerve action potential-evoked release at both high- and low-rate stimulation and during recovery from intense release. The ACh release induced by hypertonic medium was also unchanged, indicating that the pool of vesicles for immediate release is unaltered at the Rab3A-deficient NMJ. These results indicate a selective role of Rab3A in spontaneous transmitter release at the NMJ which cannot or only partly be taken over by the closely related Rab3B, Rab3C, or Rab3D isoforms when Rab3A is deleted. It has been hypothesized that Rab3A mutation underlies human presynaptic myasthenic syndromes, in which severely reduced nerve action potential-evoked ACh release at the NMJ causes paralysis. Our observation that Rab3A deletion does not reduce evoked ACh release at any stimulation rate at the mouse NMJ, argues against this hypothesis.     

Negative cross-talk between presynaptic adenosine and acetylcholine receptors

Functional interactions between presynaptic adenosine and acetylcholine (ACh) autoreceptors were studied at the frog neuromuscular junction by recording miniature end-plate potentials (MEPPs) during bath or local application of agonists. The frequency of MEPPs was reduced by adenosine acting on presynaptic adenosine A1 receptors (EC(50) = 1.1 microm) or by carbachol acting on muscarinic M2 receptors (EC(50) = 1.8 microm). However, carbachol did not produce the depressant effect when it was applied after the action of adenosine had reached its maximum. This phenomenon implied that the negative cross-talk (occlusion) had occurred between A1 and M2 receptors. Moreover, the occlusion was receptor-specific as ATP applied in the presence of adenosine continued to depress MEPP frequency. Muscarinic antagonists [atropine or 1-[[2-[(diethylamino)methyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepine-6-one) (AFDX-116)] had no effect on the inhibitory action of adenosine and adenosine antagonists [8-(p-sulfophenyl)theophylline (8-SPT) or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX)] had no effect on the action of carbachol. These data suggested that membrane-delimited interactions did not occur between A1 and M2 receptors. Both carbachol and adenosine similarly inhibited quantal release triggered by high potassium, ionomycin or sucrose. These results indicated a convergence of intracellular pathways activated by M2 and A1 receptors to a common presynaptic effector located downstream of Ca(2+) influx. We propose that the negative cross-talk between two major autoreceptors could take place during intense synaptic activity and thereby attenuate the presynaptic inhibitory effects of ACh and adenosine.     

ATP but not adenosine inhibits nonquantal acetylcholine release at the mouse neuromuscular junction

The postsynaptic membrane of the neuromuscular synapse treated with antiacetylcholinesterase is depolarized due to nonquantal release of acetylcholine (ACh) from the motor nerve ending. This can be demonstrated by the hyperpolarization produced by the application of curare (H-effect). ATP (1 x 10-5 M) decreased the magnitude of the H-effect from 5 to 1.5 mV. The membrane input resistance and the ACh sensitivity were unchanged, and so changes in these cannot explain the ATP effect. Adenosine alone was without effect on the nonquantal release. On the other hand, both ATP and adenosine depressed the frequency of spontaneous miniature endplate potentials, to 56% and 43% respectively. The protein kinase A inhibitor Rp-cAMP or the guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one did not affect the inhibitory influence of ATP on the H-effect, whereas staurosporine, an inhibitor of protein kinase C, completely abolished the action of ATP. Suramin, an ATP antagonist, enhanced the H-effect to 8.6 mV and, like staurosporine, prevented the inhibitory effect of ATP. ATP thus suppresses the nonquantal release via a direct action on presynaptic metabotropic P2 receptors coupled to protein kinase C, whilst adenosine exerts its action mainly by affecting the mechanisms underlying quantal release. These data, together with earlier evidence, show that nonquantal release of ACh can be modulated by several distinct regulatory pathways, in particular by endogenous substances which may or may not be present in the synaptic cleft at rest or during activity.     

Characterization of vinpocetine effects on DA and DOPAC release in striatal isolated nerve endings

The effect of vinpocetine, a nootropic drug with anti-ischemic potential, on the release of DA and its main metabolite, DOPAC, was investigated in striatum isolated nerve endings under resting and depolarized conditions. Vinpocetine does not modify the baseline release of DA or the exocytotic release of DA evoked by high K(+), but inhibits the release of DA evoked by veratridine reversal of the DA transporter. In addition to these results, which confirm the vinpocetine selective blockade of voltage-sensitive presynaptic Na(+) channels (VSSC) previously reported [Neurochem. Res. 24 (1999) 1585], vinpocetine increases DOPAC release either under resting, veratridine or high K(+) depolarized conditions. This latter effect, which does not involve VSSC, was characterized. The parallel determination of the released and retained catecholamine concentrations revealed that vinpocetine increases DOPAC release at the expense of internal DA in a dose-dependent manner (low microM range). In contrast to vinpocetine, the selective MAO-A inhibitor, clorgyline, increases DA and decreases DOPAC formation. The combined action of vinpocetine and clorgyline does not indicate, however, that the activation of MAO is the mechanism responsible for the increase in DOPAC caused by vinpocetine. Reserpine, although more potent and efficient than vinpocetine, qualitatively exerts the same pattern of changes on DA and DOPAC concentrations. It is concluded that, in addition to the inhibition of presynaptic VSSC permeability, which selectively inhibits the transporter-mediated release of all neurotransmitters, vinpocetine increases DOPAC by impairing the vesicular storage of DA. Our results indicate that the cytoplasm extravesicular DA is metabolized by MAO to DOPAC. Most of the DOPAC formed is exported to the extracellular medium.     

Differential effect of serum from bipolar versus schizophrenic patients on spontaneous acetylcholine release at mammalian neuromuscular junction

OBJECTIVE: The diagnosis of bipolar disease frequently requires a long time since the age of onset, especially because the disease is misdiagnosed with schizophrenia. The aim of the present work was to investigate whether sera from bipolar patients have an active substance that allows making a fast identification of the disease. METHODS: Sera from healthy volunteers, euthymic and non-stabilized bipolar patients, and schizophrenic patients were passively transferred into CF1 mice and after 2 day injections, MEPP frequency from diaphragm muscles was recorded. The same procedure was performed with sera fraction of high and low MW (cut-off 3000). RESULTS: Sera from non-stabilized bipolar patients induced a decreased MEPP frequency and occluded the presynaptic inhibitory effect of the specific adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) in the recipient mice, while in the euthymic bipolar group spontaneous secretion reached control values although the action of CCPA was still prevented. Similar results were obtained with low MW sera fraction from euthymic and non-stabilized bipolar patients. The addition of adenosine deaminase to the sera fraction prevented the modification of spontaneous ACh release. In mice injected with sera from schizophrenic patients, MEPP frequency was within control values and CCPA induced its typical inhibitory action. CONCLUSIONS: These results indicate that bipolar patients contain in their blood an active substance compatible with adenosine, which was able to modify spontaneous ACh release in the recipient mice. This effect was not observed with sera from healthy volunteers and schizophrenic patients. The increase of adenosine concentration may result from synaptic hyperactivity that presumably plays a role in the symptoms of bipolar disorder and/or may derive from peripheral cells through a more general mechanism. SIGNIFICANCE: The different results obtained with bipolar and schizophrenic sera raise the possibility that the passive transfer model could be used as a diagnostic test in the future.     

Distinct receptors and different transduction mechanisms for ATP and adenosine at the frog motor nerve endings

Corelease of ATP with ACh from motor endings suggests a physiological role for ATP in synaptic transmission. We previously showed that, on skeletal muscle, ATP directly inhibited ACh release via presynaptic P2 receptors. The receptor identification (P2X or P2Y) and its transduction mechanism remained, however, unknown. In the present study using the voltage-clamp technique we analyzed the properties of presynaptic ATP receptors and subsequent effector mechanisms. ATP or adenosine presynaptically depressed multiquantal end-plate currents, with longer latency for ATP action. ATPgammaS, agonist at P2X receptors, or Bz-ATP, agonist at P2X7 receptors, were ineffective. The action of ATP was prevented by suramin and unchanged by PPADS or TNP-ATP, antagonists of P2X receptors, or RB-2, a blocker of certain P2Y receptors. The depressant action of ATP was reproduced by UTP, metabotropic P2Y receptor agonist. Pertussis toxin (PTX), antagonist of Gi/o-proteins, and inhibitors of phosphatidylcholine specific PLC (D609) and PKC (staurosporine or chelerythrine) prevented the effect of ATP while blockers of PLA2 (OBAA) and COX (aspirin or indomethacin) attenuated it. Inhibitors of phosphatidylinositide-specific PLC (U73122), guanylylcyclase (ODQ), PKA (Rp-cAMPS) or PLD (1-butanol) did not affect the action of ATP. No inhibitor of second messengers (except PTX) changed the action of adenosine. Our data indicate, for motor nerve endings, the existence of inhibitory P2Y receptors coupled to multiple intracellular cascades including phosphatidylinositide-specific PLC/PKC/PLA2/COX. This divergent presynaptic P2 signalling (unlike the single effector mechanism for P1 receptors) could provide feedback inhibition of transmitter release and perhaps be involved in presynaptic plasticity.     

Evidence that cholinergic axon terminals are equipped with both muscarinic and adenosine receptors

The release of 3H-acetylcholine (ACh) from longitudinal muscle strips of guinea pig ileum, which were previously incubated with 3H-choline, was measured by scintillation spectrometry. The release of ACh evoked by electrical field stimulation was inhibited in the following ways: stimulating muscarinic receptors directly with oxotremorine or indirectly with eserine by increasing ACh concentration in the surrounding axon terminals or stimulating adenosine receptors by increasing the biophase concentration of adenosine with dipyridamole. The muscarinic antagonist atropine and the adenosine receptor antagonist theophylline enhanced ACh release. Atropine prevented the effect of eserine and oxotremorine on ACh release and theophylline counteracted the effect of dipyridamole. When the release of ACh was under the inhibitory effect of muscarinic receptor stimulation theophylline did not increase ACh release. Under these conditions atropine caused an extremely high increase in the release of ACh, which was not further enhanced by theophylline. When the extracellular level of adenosine was increased by dipyridamole, eserine, atropine or eserine and atropine together, they were unable to change the release of ACh, while theophylline increased release of ACh. Therefore, it is concluded that the muscarinic receptor mediated inhibition of ACh release is not due to previously released adenosine. Thus, adenosine and muscarinic feedback systems seem to be independent and each cholinergic nerve ending contains both adenosine and muscarinic receptors.     

Possible mechanisms of the effect of physostigmine on the facilitation of acetylcholine release in the guinea pig myenteric plexus

The automodulation of acetylcholine (ACh) release in the guinea pig myenteric plexus-longitudinal muscle preparation was investigated by studying the electric stimulation-evoked release of radiolabeled ACh. When the release associated with neuronal activity was challenged by the muscarinic antagonist atropine, the release was not significantly enhanced. When the acetylcholinesterase (AChE) blocker physostigmine was present, the well-established muscarinic receptor-mediated autoinhibition was operative i.e., the release was significantly reduced. However, when both drugs were added together, the release was much higher than under control conditions. Therefore, it seems likely that there is also a facilitatory system. We made an effort to clear up the mechanism of this facilitation by blocking possible nicotinic presynaptic receptors, by excluding the α₂-adrenoceptor-mediated masking effect of noradrenergic heteromodulation, by preventing a possible ATP-mediated mechanism, and by attempting to prevent the direct effect of physostigmine. None of these manipulations resulted in a decrease of the surplus release. It is concluded, that when the negative feedback modulation of ACh is inhibited and AChE activity is reduced, high levels of ACh facilitates additional release of ACh from the nerve terminals, possibly through a not yet verified class of receptors.     

Inhibition of synaptically evoked cortical acetylcholine release by intracortical glutamate: involvement of GABAergic neurons

Cortical acetylcholine (ACh) has been shown to regulate diverse cognitive processes and its release can be regulated by neuromodulators that act presynaptically at cholinergic terminals. The neocortex receives dense glutamatergic input from thalamocortical and other fibres. The present study used in vivo microdialysis to examine, and pharmacologically characterize, the effect of glutamate on cortical ACh release evoked by electrical stimulation of the pedunculopontine tegmental nucleus in urethane-anaesthetized rats. All drugs were administered locally within the cortex by reverse dialysis. Application of glutamate had no detectable effect on spontaneous ACh release but reduced evoked cortical ACh efflux in a concentration-dependent manner. This effect was mimicked by the glutamate transporter blocker L-trans-pyrrolidine-2,4-dicarboxylic acid, as well as by the ionotropic glutamate receptor agonists N-methyl-D-aspartic acid and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, and was blocked by the ionotropic glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and (+/-)-3-(2-carboxypiperazin-4yl)-propyl-1-phosphonic acid. Glutamate application also increased extracellular adenosine levels but the simultaneous delivery of the broad-spectrum adenosine receptor antagonist caffeine failed to affect the inhibitory action of glutamate on evoked ACh release. However, the effect of glutamate was fully blocked by simultaneous delivery of the GABAA receptor antagonist bicuculline and partially blocked by the GABAB receptor antagonist phaclofen. These results suggest that ionotropic glutamate receptor activation by glutamate inhibits evoked cortical ACh release via an indirect pathway involving GABAergic neurons in the cortex.     

Adenosine released by astrocytes contributes to hypoxia-induced modulation of synaptic transmission

Astrocytes play a critical role in brain homeostasis controlling the local environment in normal as well as in pathological conditions, such as during hypoxic/ischemic insult. Since astrocytes have recently been identified as a source for a wide variety of gliotransmitters that modulate synaptic activity, we investigated whether the hypoxia-induced excitatory synaptic depression might be mediated by adenosine release from astrocytes. We used electrophysiological and Ca2+ imaging techniques in hippocampal slices and transgenic mice, in which ATP released from astrocytes is specifically impaired, as well as chemiluminescent and fluorescence photometric Ca2+ techniques in purified cultured astrocytes. In hippocampal slices, hypoxia induced a transient depression of excitatory synaptic transmission mediated by activation of presynaptic A1 adenosine receptors. The glia-specific metabolic inhibitor fluorocitrate (FC) was as effective as the A1 adenosine receptor antagonist CPT in preventing the hypoxia-induced excitatory synaptic transmission reduction. Furthermore, FC abolished the extracellular adenosine concentration increase during hypoxia in astrocyte cultures. Several lines of evidence suggest that the increase of extracellular adenosine levels during hypoxia does not result from extracellular ATP or cAMP catabolism, and that astrocytes directly release adenosine in response to hypoxia. Adenosine release is negatively modulated by external or internal Ca2+ concentrations. Moreover, adenosine transport inhibitors did not modify the hypoxia-induced effects, suggesting that adenosine was not released by facilitated transport. We conclude that during hypoxia, astrocytes contribute to regulate the excitatory synaptic transmission through the release of adenosine, which acting on A1 adenosine receptors reduces presynaptic transmitter release. Therefore, adenosine release from astrocytes serves as a protective mechanism by down regulating the synaptic activity level during demanding conditions such as transient hypoxia.     

Effect of endogenous opioid peptides on acetylcholine release from the cat superior cervical ganglion: selective effect of a heptapeptide

The present experiments show the presence of both metenkephalin-like and met-enkephalin-Arg6-Phe7-like immunoreactivity in the superior cervical ganglion of the cat; this was determined by radioimmunoassay after high-pressure liquid chromatography separation of tissue extracts. There was measurable efflux of both peptides, as determined by radioimmunoassay of ganglionic perfusates; this measure was increased by thiorphan, an enkephalinase inhibitor. The effect of the 2 peptides on ACh release was determined: The stable analog of methionine-enkephalin, D-Ala2-methionine-enkephalinamide, did not affect ACh release from the ganglion; in contrast, methionine-enkephalin-Arg6-Phe7 significantly depressed evoked ACh release. The effect of met-enkephalin-Arg6-Phe7 to decrease ACh release was antagonized, although only partially, by the opioid antagonist naloxone. Thus, it appears that methionine-enkephalin-Arg6-Phe7 alters ACh release from the superior cervical ganglion by acting, at least in part, on a presynaptic opioid receptor. The results suggest that in the cat superior cervical ganglion, the heptapeptide enkephalin might have a significant role in the regulation of synaptic transmission, which is unrelated to its potential function as a precursor for methionine-enkephalin.     

Effects of nociceptin on cardiac norepinephrine and acetylcholine release evoked by ouabain

We investigated whether the novel peptide, nociceptin, modulates neuronal transmission at autonomic nerve endings. Using a cardiac dialysis technique, the effects of locally applied nociceptin on cardiac acetylcholine (ACh) and norepinephrine (NE) release were examined in anesthetized cats. Dialysis probes were implanted in the left ventricular wall, with the concentration of dialysate NE or ACh serving as an indicator of NE or ACh output at cardiac sympathetic or parasympathetic nerve endings. Locally applied ouabain evoked increases in NE and ACh output. Nociceptin suppressed the ouabain induced ACh increment. The ouabain induced NE release was not altered by nociceptin. However, in the presence of desipramine (a NE uptake inhibitor), nociceptin suppressed the ouabain-induced NE release. Inhibition by nociceptin of ouabain-induced release of NE or ACh was blocked by pretreatment with nocistatin (a nociceptin action blocking peptide). Nociceptin-induced inhibition of ACh or NE release is attributable to pre-synaptic modulation rather than a reversal of the ouabain effect. These findings demonstrate that nociceptin inhibits cardiac autonomic neurotransmission via a presynaptic opioid receptor-like1(ORL1) receptor.     

A2A adenosine receptors are located on presynaptic motor nerve terminals in the mouse

Extracellular adenosine is present at the mammalian neuromuscular junction (NMJ) by virtue of its release from activated nerve terminals and muscle fibers, and as a metabolite of adenosine tri-phosphate, which is coreleased with acetylcholine. Two activities for adenosine have been described: an inhibitory effect presumed to be modulated by the A1 receptor subtype, and a facilitatory effect mediated by the A2A receptor subtype. To date, only pharmacological evidence is available for these actions. We have used an antibody against the A2A receptor subtype, and demonstrated that A2A receptors are present on presynaptic motor nerve terminals at NMJs but not on associated glial or muscle cells, in the mouse. These results therefore provide additional evidence that there are multiple adenosine receptors present at the NMJ, and that stimulation of quantal and nonquantal release of acetylcholine (ACh) could be mediated by A2A receptors.     

Modulation of calcium-dependent and-independent acetylcholine release from motor nerve endings

Inhibition of acetylcholine (ACh) release by adenosine is an important mechanism by which the secretory apparatus is regulated at both mammalian (Ginsborg and Hirst, 1972; Hirsh et al., 2002; Silinsky, 2004) and amphibian (Silinsky, 1980; Silinsky and Solsona, 1992; Redman and Silinsky, 1993, 1994; Robitaille et al., 1999) neuromuscular junctions (NMJs). ACh is known to be costored with ATP in cholinergic vesicles (Zimmermann, 1994), and it has been demonstrated that at amphibian NMJs, adenosine derived from neurally released ATPis the mediator of neuromuscular depression exhibited at low frequencies of nerve stimulation (Redman and Silinsky, 1994) (Fig. 1). At the mouse motor nerve ending the inhibitory actions of adenosine on transmitter release are linked to a reduction in the nerve-terminal calcium current associated with neurotransmitter release (Silinsky, 2004). In contrast, at the frog motor nerve, inhibition of ACh release by adenosine occurs in the absence of any effect on nerve-terminal calcium currents (Silinsky and Solsona, 1992; Redman and Silinsky, 1994; Robitaille et al., 1999). That is, at the frog NMJ adenosine inhibits ACh release through an effect on a process that takes place downstream from calcium entry. Although the exact site at which adenosine inhibits transmitter release is unknown, both the speed (50-100 ms; E. M. Silinsky, unpublished observations) and the stimulation-independent nature of inhibition suggest that this process must occur through an action on vesicles that are already primed and ready for release. Thus, the likely sites for mediating the action of adenosine are those core components of the neurotransmitter release process, the three SNARES (SNAP-25, syntaxin, and synaptobrevin), and synaptotagmin. However, there are difficulties in addressing which of these individual elements of the secretory apparatus might be involved in the actions of adenosine. We could use fractions of botulinum toxin to eliminate individual components of the secretory apparatus. However, each of these core components of the release machinery is individually essential for the neurotransmitter release process. Therefore, we decided to approach this problem by alternative means.     

Mechanisms of ATP action on motor nerve terminals at the frog neuromuscular junction

We have shown previously that ATP inhibits transmitter release at the neuromuscular junction through the action on metabotropic P2Y receptors coupled to specific second messenger cascades. In the present study we recorded K(+) or Ca(2+) currents in motor nerve endings or blocked K(+) or Ca(2+) channels in order to explore the nature of downstream presynaptic effectors. Endplate currents were presynaptically depressed by ATP. Blockers of Ca(2+)-activated K(+)-channels, such as iberiotoxin, apamin or tetraethylammonium, did not change the depressant action of ATP. By contrast, K(+) channel blocker 4-aminopyridine (4-AP) and raised extracellular Ca(2+) attenuated the effect of ATP. However, these effects of 4-AP and high Ca(2+) were reversed by Mg(2+), suggesting Ca(2+)-dependence of the ATP action. Ba(2+) promoted the depressant action of ATP as did glibenclamide, a blocker of ATP-sensitive K(+) channels, or mild depolarization produced by 7.5 mm K(+). None of the K(+) channel blockers affected the depressant action of adenosine. Focal recording revealed that neither ATP nor adenosine affected the fast K(+) currents of the motor nerve endings. However, unlike adenosine, ATP or UTP, an agonist of P2Y receptors, reversibly reduced the presynaptic Ca(2+)-current. This effect was abolished by suramin, an antagonist of P2 receptors. Depressant effect of ATP on the endplate and Ca(2+)-currents was mimicked by arachidonate, which precluded the action of ATP. ATP reduced acetylcholine release triggered by ionomycin or sucrose, suggesting inhibition of release machinery. Thus, the presynaptic depressant action of ATP is mediated by inhibition of Ca(2+) channels and by mechanism acting downstream of Ca(2+) entry.     

Adenosine A1 receptors inhibit Ca2+ channels coupled to the release of ACh, but not of GABA, in cultured retina cells

We investigated the effect of adenosine A1 receptors on the release of acetylcholine (ACh) and GABA, and on the intracellular calcium concentration ([Ca2+]i) response in cultured chick amacrine-like neurons, stimulated by KCl depolarization. The KCl-induced release of [3H]ACh, but not the release of [14C]GABA, was potentiated when adenosine A1 receptor activation was prevented by perfusing the cells with adenosine deaminase (ADA) or with 1,3-dipropyl-8-cycloentylxanthine (DPCPX). The changes in the [Ca2+]i induced by KCl depolarization, measured in neurite segments of single cultured cells, were also modulated by endogenous adenosine, acting on adenosine A1 receptors. Our results show that adenosine A1 receptors inhibit Ca2+ entry coupled to ACh release, but not to the release of GABA, suggesting that the synaptic vesicles containing each neurotransmitter are located in different zones of the neurites, containing different VSCC and/or different densities of adenosine A1 receptors.     

Presynaptic membrane receptors in acetylcholine release modulation in the neuromuscular synapse

Over the past few years, we have studied, in the mammalian neuromuscular junction (NMJ), the local involvement in transmitter release of the presynaptic muscarinic ACh autoreceptors (mAChRs), purinergic adenosine autoreceptors (P1Rs), and trophic factor receptors (TFRs; for neurotrophins and trophic cytokines) during development and in the adult. At any given moment, the way in which a synapse works is largely the logical outcome of the confluence of these (and other) metabotropic signalling pathways on intracellular kinases, which phosphorylate protein targets and materialize adaptive changes. We propose an integrated interpretation of the complementary function of these receptors in the adult NMJ. The activity of a given receptor group can modulate a given combination of spontaneous, evoked, and activity‐dependent release characteristics. For instance, P1Rs can conserve resources by limiting spontaneous quantal leak of ACh (an A₁R action) and protect synapse function, because stimulation with adenosine reduces the magnitude of depression during repetitive activity. The overall outcome of the mAChRs seems to contribute to upkeep of spontaneous quantal output of ACh, save synapse function by decreasing the extent of evoked release (mainly an M₂ action), and reduce depression. We have also identified several links among P1Rs, mAChRs, and TFRs. We found a close dependence between mAChR and some TFRs and observed that the muscarinic group has to operate correctly if the tropomyosin‐related kinase B receptor (trkB) is also to operate correctly, and vice versa. Likewise, the functional integrity of mAChRs depends on P1Rs operating normally. © 2014 Wiley Periodicals, Inc.     

Acetylcholine suppresses the spread of excitation in the visual cortex revealed by optical recording: possible differential effect depending on the source of input

Optical recording with a voltage-sensitive dye was performed in visual cortical slices of the rat to determine the effect of acetylcholine (ACh) on the spread of excitation. In the presence of ACh, the spread of excitation initiated by stimulation at the white matter/layer VI (WM/VI) was greatly suppressed throughout the cortex, with less suppression in the middle layers. By comparing the effect of ACh with that of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the fraction of the synaptic component that was sensitive to ACh was evaluated. ACh suppressed approximately 40-50% (maximum 55.8%, n = 11) of the initial synaptic component in the superficial and deep layers. In the middle, however, the effect was weakest and only approximately 20-30% (minimum 20.9%, n = 11) of the initial synaptic component was suppressed. On the basis of histological analysis, the region with the weakest ACh effect extended from upper V to lower II/III. To identify the site of ACh action in terms of pre- versus postsynaptic localization, exogenous glutamate was applied. Because ACh did not suppress the excitation induced by glutamate, the site of the ACh action was indicated to be presynaptic. When layer II/III was stimulated instead of WM/VI, the suppression was uniform throughout the cortex. A muscarinic receptor antagonist, atropine, blocked the suppression by ACh. In conclusion, our results indicate the following two points. First, ACh strongly suppresses intracortical connectivity through presynaptic muscarinic receptors. Secondly, in contrast to the intracortical connection, some group(s) of fibres, possibly thalamocortical afferents that arise from white matter and terminate in the middle cortical layers are suppressed much less by ACh. While ACh has been reported to have confusingly diverse effects, e.g. direct depolarization and hyperpolarization as well as synaptic facilitation and suppression, its effect on the propagation of excitation is very clear; suppression on intracortical connection, leaving thalamocortical inputs rather intact. We postulate that cholinergic innervation enables the afferent input to have a relatively dominant effect in the cortex.     

G proteins are involved in the regulation of transmitter release at an Aplysia cholinergic synapse

At an identified cholinergic synapse of the Aplysia buccal ganglion, presynaptic injections of guanosine 5'-O-3-thiotriphosphate (GTP-gamma-S) depressed the amplitude of evoked postsynaptic responses. This reduction of acetylcholine (ACh) release by GTP-gamma-S, prevented by pre-injection of guanosine 5'-O-2-thiodiphosphate (GDP-beta-S) in the presynaptic neuron, was due to a reduction of the number of ACh quanta released. The mean amplitude of the evoked miniature postsynaptic current (MPSC) was unchanged. The presynaptic Ca2+ influx was lowered.     

Binding sites for [3H]AF-DX 116 and effect of AF-DX 116 on endogenous acetylcholine release from rat brain slices

The present study shows that the putative M2 ligand, [3H]AF-DX 116, binds to two classes of muscarinic sites in homogenates of rat hippocampus, striatum and cerebral cortex: one with a high affinity (Kd less than 5 nM)/low capacity (Bmax = 30-63 fmol/mg protein), and a second of lower affinity (Kd greater than 65 nM) and higher capacity (Bmax greater than 190 fmol/mg protein). In experiments which tested the effects of the muscarinic antagonists on acetylcholine (ACh) release from brain slices, the non-selective antagonist (-)-quinuclidinyl benzylate and atropine significantly enhanced the potassium (25 mM)-evoked release of ACh. This effect was mimicked by the M2 ligand AF-DX 116, but neither the M1-selective antagonist pirenzepine, nor the putative M3-muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), altered ACh release. Also, the muscarinic agonist, oxotremorine, significantly depressed evoked ACh release from brain slices, an effect that was completely antagonized by atropine or by AF-DX 116, but not by pirenzepine or 4-DAMP. Thus, it appears that presynaptic muscarinic autoreceptors in the rat hippocampus, striatum and cerebral cortex belong to the M2 subtype of muscarinic receptors.