Effects of various experimental manipulations on neostriatal acetylcholine and dopamine release

The release of endogenous acetylcholine and dopamine and the appearance of their metabolites, choline and dihydroxyphenylacetic acid (DOPAC), from neostriatal slices prepared from Fischer 344 rats was examined under various experimental conditions. There was a dose-dependent increase in the amount of neurotransmitter or metabolite as the medium potassium concentration was increased from 5 to 50 mM. Over an eight minute period in Krebs Ringer bicarbonate buffer containing 25 mM potassium, the rate of release of acetylcholine was 6 to 13 times greater than that of dopamine. The dopamine endogenous to the slice preparation appeared to have little effect on the release of endogenous acetylcholine since manipulations that significantly altered dopamine release (depletion with 6-hydroxydopamine or uptake inhibition with nomifensine) had minimal effects on the cholinergic neurons. In contrast, increasing the endogenous acetylcholine in the preparation by inhibiting acetylcholinesterase resulted in a 1.2 to 12 fold increase in dopamine release depending upon the incubation time and the potassium concentration. These studies indicate that within the neostriatal slices there is minimal influence of the endogenous dopamine on the cholinergic neurons, whereas the extracellular acetylcholine can influence dopamine release when its concentration is increased by inhibition of acetylcholinesterase.     

Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development.     

Action of a beta-bungarotoxin on autonomic ganglia and adrenergic neurotransmission.

A beta-bungarotoxin was isolated from the venom of Bungarus multicinctus by column chromatography on Sephadex G-50 and SP-Sephadex. The toxin produced presynaptic effects on neuromuscular transmission with characteristics similar to those described by others. In a sympathetic ganglion, the toxin increased spontaneous acetylcholine (ACh) release and decreased ACh release evoked by preganglionic nerve stimulation. The toxin did not block the response of isolated ileum to cholinergic nerve stimulation, did not block the release of noradrenaline from the adrenergic nerve terminals of a nictitating membrane preparation, and did not alter the responses of smooth and cardiac muscle preparations to noradrenaline. It is suggested that the specificity of beta-bungarotoxin for certain nerve terminals is related either to selective binding of the toxin or to the selective presence of a necessary substrate for its action. An attempt to show selective binding of 125I-toxin to cholinergic nerve terminals in skeletal muscle was not successful.     

The resting release of acetylcholine by a retinal neuron

The cholinergic amacrine cells of the rabbit retina secrete acetylcholine by two mechanisms. One is activated by stimulation of the retina by light or depolarization of the amacrine cells by K+ ions. It requires the presence of extracellular Ca2+. The second is independent of extracellular Ca2+ and is unaffected by large depolarizations of the cells. It bears some similarity to the acetylcholine 'leakage' described at the neuromuscular junction. Although the Ca2+-independent mechanism accounts for about two thirds of the total acetylcholine release in the dark, the amount of acetylcholine released in this way is small compared with the release of acetylcholine triggered by stimulation of the retina with light. Its biological significance is unclear.     

Hexamethonium sensitivity of the swim musculature of the pteropod mollusc, <Emphasis Type="Italic">Clione limacina</Emphasis>

Swimming in reduced electrophysiological preparations of the pteropod mollusc, Clione limacina, was blocked by bath application of hexamethonium even though pattern generator activity continued with this treatment. Neuromuscular recordings indicated that hexamethonium blocked synaptic input from Pd-3 and Pd-4 motoneurons to slow-twitch muscle cells, while connections from Pd-1A and Pd-2A motoneurons to fast-twitch muscle cells were variable in their response to hexamethonium—synaptic inputs were suppressed in most cases and occasionally blocked, but the latter only with high concentrations and long incubations. Acutely dissociated wing muscle cells showed a concentration-dependency in the percentage of contracted cells with bath application of acetylcholine, and this contractile activity was blocked in preparations that were first bathed in hexamethonium. Intracellular recordings from dissociated slow-twitch muscle cells showed conductance-increase depolarizations of approximately 20 mV following 1 s pressure ejections of 10⁻⁴ M acetylcholine from micropipettes placed immediately adjacent to the muscle cells. These responses were blocked when hexamethonium was bath applied prior to the pressure-applied acetylcholine. The results suggest the Pd-3/Pd-4 motoneuron to slow-twitch muscle cell junctions are cholinergic with nicotinic-like receptors, while the Pd-1A/Pd-2A to fast-twitch muscle cell connections are likely cholinergic, but with a different receptor type.     

The non-neuronal cholinergic system in humans: expression,function and pathophysiology

Acetylcholine, a prime example of a neurotransmitter, has been detected in bacteria, algae, protozoa, and primitive plants, indicating an extremely early appearance in the evolutionary process (about 3 billion years). In humans, acetylcholine and/or the synthesizing enzyme, choline acetyltransferase (ChAT), have been found in epithelial cells (airways, alimentary tract, urogenital tract, epidermis), mesothelial (pleura, pericardium), endothelial, muscle and immune cells (mononuclear cells, granulocytes, alveolar macrophages, mast cells). The widespread expression of non-neuronal acetylcholine is accompanied by the ubiquitous presence of cholinesterase and receptors (nicotinic, muscarinic). Thus, the non-neuronal cholinergic system and non-neuronal acetylcholine, acting as a local cellular signaling molecule, has to be discriminated from the neuronal cholinergic system and neuronal acetylcholine, acting as neurotransmitter. In the human placenta anti-ChAT immunoreactivity is found in multiple subcellular compartments like the cell membrane (microvilli, coated pits), endosomes, cytoskeleton, mitochondria and in the cell nucleus. These locations correspond with the results of experiments where possible functions of non-neuronal acetylcholine have been identified (proliferation, differentiation, organization of the cytoskeleton and the cell-cell contact, locomotion, migration, ciliary activity, immune functions). In the human placenta acetylcholine release is mediated by organic cation transporters. Thus, structural and functional differences are evident between the non-neuronal and neuronal cholinergic system. Enhanced levels of acetylcholine are detected in inflammatory diseases. In conclusion, it is time to revise the role of acetylcholine in humans. Its biological and pathobiological roles have to be elucidated in more detail and possibly, new therapeutical targets may become available.     

Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris

Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.Key words: invertebrate, cephalopod, choline acetyltransferase, neuron, immunohistochemistry.     

Pharmacological action of the heptapeptide GFSKLYFamide in the muscle of the sea cucumber Holothuria glaberrima (Echinodermata)

The holothurian neuropeptide GFSKLYFamide (GlyPheSerLysLeuTyrPheNH₂), GFSKLYFa, was characterized recently and shown to be present in nerve fibers that apparently innervate various muscle systems. We have studied the potential neurotransmitter role of this peptide by assaying its effects on the contractility of visceral and somatic muscles. GFSKLYFa in nanomolar concentrations induces a relaxation of the muscle tension in the intestine. A similar effect is observed on the longitudinal muscle bands of the body wall of the sea cucumber. The relaxing action of GFSKLYFa is dose dependent suggesting that its action is mediated by receptors present in the muscle cells. In addition, GFSKLYFa induces the relaxation of the acetylcholine contracted intestine. Our investigation provides additional evidence indicating that GFSKLYFa might be a neurotransmitter acting at the neuromuscular junctions of the sea cucumber Holothuria glaberrima.     

Effects of cholinergic drugs on muscle contraction in Moniliformis moniliformis (Acanthocephala)

In whole Moniliformis moniliformis spontaneous muscle contractions were rhythmic; longitudinal contractions were measured with a force transducer. The cholinergic agonists levamisole and nicotine significantly increased muscle tension in whole worms; these contractions were tonic and were antagonised by the ganglionic blocker pentolinium and by piperazine. In addition, levamisole-induced contractions were inhibited by gallamine, hexamethonium, and norepinephrine. In worm segments, where drugs in solution were injected through the worms, acetylcholine (ACh) and nicotinic agonists were effective in causing contractions, whereas muscarinic agonists in concentrations up to 1 mM had no effect. Although muscle contraction in M. moniliformis was induced by nicotinic agonists, these contractions were effectively antagonised by a range of chemicals that block ganglionic, skeletal, and muscarinic sites in vertebrates. The presence of ACh in M. moniliformis and the effects of nicotinic agonists on muscle contraction suggest that ACh is a putative excitatory neurotransmitter.     

Nematode acetylcholinesterases: molecular forms and their potential role in nematode behavior

Nematode movement is reliant upon the somatic musculature that runs longitudinally along the body wall. Neuromuscular synapses occur in the ventral and dorsal cords and employ the excitatory neurotransmitter, acetylcholine (ACh), for modulation of muscle activity. Acetylcholine activity is terminated by hydrolysis by acetylcholinesterase (AChE). Here, Charles Opperman and Stella Chang discuss the molecular forms and potential role of this enzyme.     

Altered Cholinergic Metabolism in Rat CNS Following Aluminum Exposure: Implications on Learning Performance

Abstract: The effects of Al on the central cholinergic system have been studied. Al, at a dose of 10 mg/kg of body weight/day for 4 weeks, had a deleterious effect on the activities of biosynthetic (choline acetyltransferase) and hydrolytic (acetylcholinesterase) enzymes of the neurotransmitter acetylcholine. The levels of acetylcholine were also significantly lowered in different brain regions at the end of the dose regimen. There was a significant decrease in high-affinity choline uptake following Al exposure. Muscarinic acetylcholine receptor binding studies revealed a decreased number of binding sites ( B _max), with the maximum effects being manifested in the hippocampus. Exogenous addition of 10 µ M desferrioxamine restored the muscarinic receptor binding completely. The impaired cholinergic functioning had severe effects on cognitive functions. Neurobehavioral deficits were manifested in terms of decreased active (52%) and passive (73.30%) avoidance tests. The results suggest that Al exerts its toxic effects by altering cholinergic transmission, which is ultimately reflected in neurobehavioral deficits.     

Habenula "cholinergic" neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes

Acetylcholine is an important neurotransmitter, and the habenulo-interpeduncular projection is a major cholinergic pathway in the brain. To study the physiological properties of cholinergic transmission in the interpeduncular nucleus (IPN), we used a transgenic mouse line in which the light-gated cation channel ChannelRhodopsin-2 is selectively expressed in cholinergic neurons. Cholinergic axonal terminals were activated by light pulses, and postsynaptic responses were recorded from IPN neurons. Surprisingly, brief photostimulation produces fast excitatory postsynaptic currents that are mediated by ionotropic glutamate receptors, suggesting wired transmission of glutamate. By contrast, tetanic photostimulation generates slow inward currents that are largely mediated by nicotinic acetylcholine receptors, suggesting volume transmission of acetylcholine. Finally, vesicular transporters for glutamate and acetylcholine are coexpressed on the same axonal terminals in the IPN. These results strongly suggest that adult brain "cholinergic" neurons can corelease glutamate and acetylcholine, but these two neurotransmitters activate postsynaptic neurons via different transmission modes.     

Modification of electroplax excitability by veratridine

Veratridine influences membrane-potential changes arising both from the action potential and from the application of external cholinergic agonists in the isolated monocellular electroplax preparation. The action potential shows a long depolarizing after-potential in the presence of veratridine. The effects of various pharmacological agents and of external ion changes on this after-potential are similar to those reported for other nerve and muscle fibers and are consistent with the view that veratridine acts chiefly to increase the Na⁺ conductance.Membrane depolarizations by cholinergic agonists are inhibited by veratridine at pH 7 but strikingly amplified at pH 9. The former effect appears to involve interaction with the cholinergic receptor at the surface of the membrane, while the latter potentiation parallels the increase in the spike after-potential at pH 9 and presumably arises from a Na⁺ conductance increase.Veratridine appears to interact with the component involved in the Na⁺ conductance in the interior membrane phase. The possible localization of this component in both the conducting and synaptic membrane is discussed.     

Cell-cell interaction during synaptogenesis

1. Neuromuscular synapse formation was studied using nerve and muscle cells dissociated from Xenopus embryos and kept in culture for 1 to 3 days. Within a few minutes of manipulated contact with isolated cholinergic neurons, miniature endplate potential-like depolarizations (MEPPs) due to spontaneous release of acetylcholine (ACh) from the neurons were detected in the muscle cells. 2. Addition of an antibody to a frog neural cell adhesion molecule (anti-NCAM) into the culture medium of nerve-muscle co-cultured for 1-3 days decreased the percentage of functional nerve-muscle contacts. 3. Acute exposure to anti-NCAM (1 hour) inhibited significantly muscle cell contact-triggered ACh release from initially identified cholinergic neurons. 4. Lysed muscle cells manipulated into contact with neurons induced ACh release, whereas lysed neurons did not, suggesting the presence of specific molecules on the muscle cell membrane capable of triggering ACh release from the cholinergic neuron. 5. Transient appearance of electrical coupling was detected between neuronal soma and muscle cell, suggesting the possibility of exchange of modulators for the formation and maintenance of neuromuscular synapses. 6. Neuromuscular synaptogenesis constitutes a complex process where at least two different types of direct cell-cell interaction seem to occur: a) cell surface molecule contact (and binding) for cell recognition and triggering of ACh release, and b) transient intercytoplasmic communication between the cells for possible passage of modulatory molecules.     

Thromboxane effects on canine trachealis neuromuscular function

The objective of this study is to determine which inflammatory mediators had direct effects on canine trachealis muscle neuromuscular control to identify candidate mediators of the hyperresponsiveness observed in vitro after O3 exposure. Studies were carried out in the sucrose gap at 29 degrees C and in the muscle bath at 37 degrees C. Leukotriene (LT) B4, LTD4, and prostaglandin (PG) D2 had neither direct nor significant effects on the excitatory junction potentials (EJP's), the secondary membrane potential oscillations, or the associated contractions that followed field stimulation of cholinergic nerves. U 46619, a stable analogue of thromboxane (Tx) A2, enhanced (10(-10)-10(-7) M) the duration and the amplitude of secondary oscillations and associated contractions without affecting the EJP's. In the muscle bath, U 46619 enhanced field-stimulated contractions; this was antagonized competitively by SQ 29548. In both the sucrose gap and the muscle bath, higher concentrations (10(-9) M and higher) caused direct effects, small depolarizations, and contractions. These effects of U 46619 were unaffected by indomethacin or guanethidine but were abolished by SQ 29548, an antagonist selective at TxA2-PGH2 receptors. U 46619 at 10(-9) M did not affect electrical or mechanical responses to acetylcholine and at 10(-9) M did not increase the sensitivity to acetylcholine. Platelet-activating factor (PAF) was inactive in all muscle-bath and most sucrose-gap experiments. In 7 of 20 of the latter, it caused effects qualitatively like those of U 46619, but whether it acted through release of TxA2 could not be tested because of the rapid tachyphylaxis to PAF. We conclude that TxA2 may mediate the hyperresponsiveness found in vitro after O3 treatment.     

The dystrophin-associated protein complex maintains muscle excitability by regulating Ca(2+)-dependent K(+) (BK) channel localization

The dystrophin-associated protein complex (DAPC) consists of several transmembrane and intracellular scaffolding elements that have been implicated in maintaining the structure and morphology of the vertebrate neuromuscular junction (NMJ). Genetic linkage analysis has identified loss-of-function mutations in DAPC genes that give rise to degenerative muscular dystrophies. Although much is known about the involvement of the DAPC in maintaining muscle integrity, less is known about the precise contribution of the DAPC in cell signaling events. To better characterize the functional role of the DAPC at the NMJ, we used electrophysiology, immunohistochemistry, and fluorescent labeling to directly assess cholinergic synaptic transmission, ion channel localization, and muscle excitability in loss-of-function (lf) mutants of Caenorhabditis elegans DAPC homologues. We found that all DAPC mutants consistently display mislocalization of the Ca(2+)-gated K(+) channel, SLO-1, in muscle cells, while ionotropic acetylcholine receptor (AChR) expression and localization at the NMJ remained unaltered. Synaptic cholinergic signaling was also not significantly impacted across DAPC(lf) mutants. Consistent with these findings and the postsynaptic mislocalization of SLO-1, we observed an increase in muscle excitability downstream of cholinergic signaling. Based on our results, we conclude that the DAPC is not involved in regulating AChR architecture at the NMJ, but rather functions to control muscle excitability, in an activity-dependent manner, through the proper localization of SLO-1 channels.     

The comparative roles of acetylcholine and noradrenaline in regulation of spontaneous spike activity of cortical neurons

The effects of acetylcholine and noradrenaline applications on neuronal sponta-neous activity were investigated in slices of guinea-pig parietal cortex. Iontophoretic ejections of both neurotransmitters to the cortical neurons evoked the same-type slowly-developing and long-lasting increase in the rate of spike activity. The different temperature sensitivity of cholinergic and noradrenergic reactions were revealed. During the temperature shift from 32-34 degrees C to 35-36 degrees C the cholinergic effect on neuronal spike activity became extremely strong, that is why even silent at t = 32-32 degrees C neurons became to acetylcholine responsive. Temperature-dependent changes in spike reaction to acetylcholine were accompanied by stable increase in spontaneous spike activity. The noradrenergic reactions did not change with temperature in limits from 32-34 to 35-36 degrees C. In this temperature range spike reactions to glutamate, the main excitation transmitter in the cortex, remained constant. The results obtained suggest that acetylcholine is the main neurotransmitter regulating spontaneous spike activity in cortical neurons.     

Some molecular properties of an isolated acetylcholine receptor ion-translocation protein

An improved procedure for isolation and purification of acetylcholine receptor from Torpedo californica electroplax membranes is described. The purified material contains the neurotransmitter recognition site and a second binding subsite which complexes inorganic cations and bis-quaternary cholinergic analogs. In addition to the transmitter recognition site the isolated macromolecule contains the molecular features necessary for ion-translocation during postsynaptic depolarization, since a chemically excitable membrane can be formed from purified acetylcholine receptor and Torpedo phospholipids.     

Minireview: Cholinergic and monoaminergic substrates of startle habituation

Research is reviewed arising from the proposition that behavioral habituation is mediated by brain mechanisms operated by the neurotransmitter, acetylcholine. Effects of cholinergic drugs on habituation of the startle response in rats fail to support involvement of acetylcholine. Likewise, serotonergic drug effects do not favor the more recent view that startle habituation depends on brain serotonin, nor is there sufficient evidence for an essential role of either dopamine or noradrenalin. Because of persistence of habituation following challenge with a variety of pharmacological agents, the phenomenon probably depends upon a complex interplay between a number of transmitters and behavioral processes. Contrary to earlier belief, no single transmitter should be seen as crucially responsible for startle habituation.