Classical Noncholinergic Neurotransmitters and the Vesicular Transport System for Acetylcholine

Abstract: The acetylcholine transporter exhibits such low affinity and specificity for acetylchoiine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [³H]serotonin, [³H]noradrenaline, or [³H]glutamate occurred. Serotonin, noradrenaline, and N -acetylaspartyl glutamate inhibited active transport of [³H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and γ-aminobutyric acid were shown here not to inhibit active transport of [³H]-acetylcholine. Noradrenaline was competitive with respect to [³H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [³H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.     

脑透析术——测定神经递质和神经肽释放的一种新技术

脑透析术（Ｂｒａｉｎｄｉａｌｙｓｉｓ）,又称微透析术（Ｍｉｃｒｏｄｉａｌｙｓｉｓ）是在推挽灌流基础上发展起来的一种连续灌注并采集清醒自由活动动物特定脑区内灌流液的一种新方法。该技术与高效液相色谱（ＨＰＬＣ）或放射免疫测定（ＲＩＡ）等灵敏的检测系统相结合可测定脑内细胞外液中许多神经递质如乙酰胆碱、去甲肾上腺素、多巴胺、５－羟色胺及它们的代谢产物、游离氨基酸、小的肽类、磷酸乙醇胺、维生素和各种离子等的变化。     

Changes in dialysate concentrations of glutamate and GABA in the brain: an index of volume transmission mediated actions?

Brain microdialysis has become a frequently used method to study the extracellular concentrations of neurotransmitters in specific areas of the brain. For years, and this is still the case today, dialysate concentrations and hence extracellular concentrations of neurotransmitters have been interpreted as a direct index of the neuronal release of these specific neurotransmitter systems. Although this seems to be the case for neurotransmitters such as dopamine, serotonin and acetylcholine, the extracellular concentrations of glutamate and GABA do not provide a reliable index of their synaptic exocytotic release. However, many microdialysis studies show changes in extracellular concentrations of glutamate and GABA under specific pharmacological and behavioural stimuli that could be interpreted as a consequence of the activation of specific neurochemical circuits. Despite this, we still do not know the origin and physiological significance of these changes of glutamate and GABA in the extracellular space. Here we propose that the changes in dialysate concentrations of these two neurotransmitters found under specific treatments could be an expression of the activity of the neurone-astrocyte unit in specific circuits of the brain. It is further proposed that dialysate changes of glutamate and GABA could be used as an index of volume transmission mediated actions of these two neurotransmitters in the brain. This hypothesis is based firstly on the assumption that the activity of neurones is functionally linked to the activity of astrocytes, which can release glutamate and GABA to the extracellular space; secondly, on the existence of extrasynaptic glutamate and GABA receptors with functional properties different from those of GABA receptors located at the synapse; and thirdly, on the experimental evidence reporting specific electrophysiological and neurochemical effects of glutamate and GABA when their levels are increased in the extracellular space. According to this concept, glutamate and GABA, once released into the extracellular compartment, could diffuse and have long-lasting effects modulating glutamatergic and/or GABAergic neurone-astrocytic networks and their interactions with other neurotransmitter neurone networks in the same areas of the brain.     

Functional Significance of the Effects of Neurotransmitters on the Na+,K+-ATPase System

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.     

Comparative quantitative aspects of putative neurotransmitters in the central nervous system of spiders (Arachnida: Araneida)

The amounts of eight putative neurotransmitters or modulators (acetylcholine, dopamine, noradrenaline, adrenaline, GABA, taurine, glutamic acid, glycine) were determined from the CNS of 12 species of five different spider families (Theraphosidae, Agelenidae, Araneidae, Lycosidae, Salticidae). Comparatively high contents of acetylcholine and noradrenaline could be found in the CNS of hunting spiders, higher contents of GABA and taurine were visible in the web-building spider families, while extraordinarily high amounts of glutamic acid were confined to the Theraphosidae. The results obtained are compared with findings from other arthropod groups and the role of putative transmitters or modulators in the spider CNS is discussed in relation to behavioural differences within the families investigated.     

A method for concurrent measurement of picomole quantities of acetylcholine, choline, dopamine, norepinephrine, serotonin, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, tryptophan, tyrosine, glycine, aspartate, glutamate, alanine, and gamma-aminobutyric acid in single tissue samples from different areas of rat central nervous system.

A rapid and sensitive method for separation and concurrent assay of 14 compounds at the picomole level in individual rat brain parts is described. The putative amino acid neurotransmitters (aspartate, γ-aminobutyric acid, glutamate, and glycine) are extracted from a 20–30 mg portion of the tissue with 5% TCA and assayed as their respective DNP-amino acid methyl ester derivatives by glc. Four other putative neurotransmitters (acetylcholine, dopamine, norepinephrine, and serotonin) and some of their precursors and metabolites (choline, tryptophan, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, and tyrosine) are extracted in 1n formic acid-acetone (v/v:15/85) from the remaining tissue. The lipids are removed with a heptane-chloroform (v/v:8/1) wash and the aqueous phase is dried at 37°C under N₂. The dried extract is dissolved in water (pH 4). With one portion of this solution, acetylcholine and choline are assayed using a radioenzymatic method whereas with the rest, dopamine, norepinephrine, serotonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophan, tryptophan, and tyrosine are separated with three ion exchange resins arranged in tandem. These compounds are assayed fluorometrically with modified microadaptations of previously reported methods.     

Transporter mediated GABA release in the retina: role of excitatory amino acids and dopamine

In general, the release of neurotransmitters in the central nervous system is accomplished by a calcium-dependent process which constitutes a common feature of exocytosis, a conserved mechanism for transmitter release in all species. However, neurotransmitters can also be released by the reversal of their transporters. In the retina, a large portion of GABA is released by this mechanism, which is under the control of neuroactive agents, such as excitatory amino acids and dopamine. In this review, we will focus on the transporter mediated GABA release and the role played by excitatory amino acids and dopamine in this process. First, we will discuss the works that used radiolabeled GABA to study the outflow of the neurotransmitter and then the works that took into consideration the endogenous pool of GABA and the topography of GABAergic circuits influenced by excitatory amino acids and dopamine.     

Transporter mediated GABA release in the retina: Role of excitatory amino acids and dopamine

In general, the release of neurotransmitters in the central nervous system is accomplished by a calcium-dependent process which constitutes a common feature of exocytosis, a conserved mechanism for transmitter release in all species. However, neurotransmitters can also be released by the reversal of their transporters. In the retina, a large portion of GABA is released by this mechanism, which is under the control of neuroactive agents, such as excitatory amino acids and dopamine. In this review, we will focus on the transporter mediated GABA release and the role played by excitatory amino acids and dopamine in this process. First, we will discuss the works that used radiolabeled GABA to study the outflow of the neurotransmitter and then the works that took into consideration the endogenous pool of GABA and the topography of GABAergic circuits influenced by excitatory amino acids and dopamine.     

Colocalization of ChAT, DβH and NADPH-d in the pancreatic neurons of the newborn guinea pig

Choline acetyltransferase (ChAT) as a rate-limiting enzyme in the biosynthetic pathway of acetylcholine is thought to be present in all cholinergic neurons. However, its immunoreactivity has not been successfully applied to the study of cholinergic neurons in the pancreas. In a previous study in the pancreas of newborn guinea pig we reported the colocalization of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS) with various neuropeptides as well as dopamine-β-hydroxylase (DβH), the enzyme responsible for converting dopamine to noradrenaline. Whether NADPH-d is colocalized with ChAT in the pancreatic neurons is not known. Also it would be interesting to find out whether noradrenaline and acetylcholine could be colocalized in the same pancreatic neurons. In the present study, a method for triple labelling of ChAT, DβH and NADPH-d was used to answer the above questions. Colocalization of ChAT, DβH and NADPH-d was constantly demonstrated in the same neurons in the same sections. It is concluded that some of the pancreatic neurons may utilize more than one neurotransmitter such as nitric oxide (NO), acetylcholine and noradrenaline to achieve their function. The possible cotransmission of acetylcholine and noradrenaline was extremely intriguing, and its mechanism and significance needs to be further investigated.     

Changes in levels of monoamines and their metabolites in incompletely ischemic brains of spontaneously hypertensive rats

In order to investigate changes in levels of monoamines and their related substances together with those of other neurotransmitters (acetylcholine and GABA), choline and substances related to energy metabolism (ATP, lactate and glucose) accompanying incomplete cerebral ischemia, a bilateral common carotid artery occlusion model of spontaneously hypertensive rats (SHR) was utilized. Animals were subjected to 1 or 2 h ischemia. Then the concentrations of substances were measured in the cerebral cortex, hippocampus and striatum and compared with control values. Due to the incomplete ischemia, ATP showed a moderate decrease, while lactate and choline increased remarkably, and GABA underwent a moderate increase. With regard to monoamines, both noradrenaline and serotonin levels were reduced in the cerebral cortex and hippocampus, whereas dopamine levels increased in the hippocampus. All monoamine metabolites, i.e. metabolites by monoamine oxidase (MAO), metabolites by catechol-O-methyltransferase (COMT), and metabolites by both MAO and COMT, underwent increases. The 3-methoxytyramine level in particular showed marked increases. Furthermore levels of precursor amino acids as well as 5-hydroxytryptophan rose. Acetylcholine decreased moderately only in the cerebral cortex. Among these changes, sustained increases in all the monoamine metabolites were characteristic in the incompletely ischemic brain, suggesting that both COMT and MAO retain their activities in the incompletely ischemic brain.     

Neurotransmitters and neuromodulators controlling the anterior byssus retractor muscle of Mytilus edulis.

1. The anterior byssus retractor muscle (ABRM) of Mytilus edulis is innervated by at least two kinds of nerves, excitatory and relaxing nerves. The principal neurotransmitters released from these nerves have been shown to be acetylcholine and serotonin, respectively. 2. Some other monoamines, such as dopamine and octopamine, and various peptides, such as FMRFamide-related peptides, Mytilus inhibitory peptides, SCP-related peptides and a catch-relaxing peptide, may also be involved in the regulation of the muscle as neurotransmitters or neuromodulators. 3. The ABRM seems to be typical of invertebrate muscles controlled by multiple neurotransmitters and neuromodulators.     

Effect of Ouabain Applied by Intrastriatal Microdialysis on the In Vivo Release of Dopamine, Acetylcholine, and Amino Acids in the Brain of Conscious Rats

In the present study we have applied a brain microdialysis technique to investigate the effects of ouabain infusion on the release of dopamine, acetylcholine, and amino acids from striatal neurons in freely moving rats. Ouabain caused an increase in the dialysate levels of dopamine; its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC); and the amino acids glutamate, aspartate, taurine, glycine, alanine, serine, asparagine, and threonine. The ouabain-induced increase in dopamine was dose dependent and explosive (100-fold at an infusion concentration of 1 mmol/L) and contrasted strongly with the small effect of the glycoside on the output of DOPAC. We investigated the nature of ouabain-induced transmitter release by determining its sensitivity to coinfusion with tetrodotoxin or the calcium antagonist Mg2+. In the case of dopamine two mechanisms of ouabain-induced release could be established. At lower infusion concentrations ouabain induced an exocytotic type of release whereas at higher concentrations the release was probably carrier mediated. In the case of amino acids we noticed a calcium-independent release which was nerve impulse flow dependent in the case of glutamate and aspartate and impulse flow independent in the case of alanine, serine, glycine, threonine, and asparagine. Ouabain induced a decrease in the release of acetylcholine and glutamine.     

The SLC6 orphans are forming a family of amino acid transporters

Transporters in the human genome are grouped in solute carrier families (SLC). The SLC6 family is one of the biggest transporter families in the human genome comprising 20 members. It is usually referred to as the neurotransmitter transporter family because its founding members encode transporters for the neurotransmitters GABA, noradrenaline, serotonin and dopamine. The family also includes a number of 'orphan' transporters, the function of which has remained elusive until recently. Identification of the broadly specific neutral amino acid transporter SLC6A19 (also called B(0)AT1) suggested that all orphan transporters may in fact be amino acid transporters. This was subsequently confirmed by the identification of SLC6A20 as the long-sought IMINO system, a proline transporter found in kidney, intestine and brain. Very recently, SLC6A15 was identified as the neutral amino acid transporter B(0)AT2. All amino acid transporters appear to cotransport only 1Na(+) together with the amino acid substrate. Both, B(0)AT1 and B(0)AT2 are chloride independent, whereas IMINO is chloride dependent. The amino acid transporters of the SLC6 family are functionally and sequence related to the recently crystallized leucine transporter from Aquifex aeolicus. The structure elegantly explains many of the mechanistic features of the SLC6 amino acid transporters.     

Neurotranmission systems as targets for toxicants: a review

Neurotransmitters are chemicals that transmit impulses from one nerve to another or from nerves to effector organs. Numerous neurotransmitters have been described in mammals, amongst them acetylcholine, amino acids, amines, peptides and gases. Toxicants may interact with various parts of neurotransmission systems, including synthetic and degradative enzymes, presynaptic vesicles and the specialized receptors that characterize neurotransmission systems. Important toxicants acting on the cholinergic system include the anticholinesterases (organophosphates and carbamates) and substances that act on receptors such as nicotine and the neonicotinoid insecticides, including imidacloprid. An important substance acting on the glutamatergic system is domoic acid, responsible for amnesic shellfish poisoning. 4-Aminobutyric acid (GABA) and glycine are inhibitory neurotransmitters and their antagonists, fipronil (an insecticide) and strychnine respectively, are excitatory. Abnormalities of dopamine neurotransmission occur in Parkinson’s disease, and a number of substances that interfere with this system produce Parkinsonian symptoms and clinical signs, including notably 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is the precursor of 1-methyl-4-phenylpyridinium. Fewer substances are known that interfere with adrenergic, histaminergic or seroninergic neurotransmission, but there are some examples. Among peptide neurotransmission systems, agonists of opioids are the only well-known toxic compounds.     

An analysis of free amino acids,neurotransmitters and enzymes in the nervous system of Solpugida (Arachnida)

1. The composition of amino acids, biogenic amines and related enzymes in the brain and other regions of the nervous system was analyzed for the solpugids, Eremobates palpisetulosus and Eremorhax magnus. The most abundant amino acids were Glu, Asp, Pro, Ala and Gly.2. GABA, dopamine, noradrenaline, acetylcholine and acetylcholinesterase were present in the brain. AChE activity was highest in the protocerebrum and cheliceral nerves; no activity was found in the optic or genital nerves.3. Concentrations of amines and ACh were found to increase in the brain during postembryonic development.     

Dysfunction of Cholinergic and Dopaminergic Neuronal Systems in β-Amyloid Protein-Infused Rats

Abstract: Accumulations of β-amyloid protein are characteristic and diagnostic features of the brain of Alzheimer's disease patients; however, the physiological role of this protein in CNS is unknown. We have previously reported that continuous infusion of β-amyloid protein into rat cerebral ventricle impairs learning ability and decreases choline acetyltransferase activity, a marker enzyme of cholinergic neuron. In this study, the effects of β-amyloid protein infusion on the release of neurotransmitters in cholinergic and dopaminergic neuronal systems were investigated by using an in vivo brain microdialysis method. Nicotine-stimulated release of acetylcholine and dopamine in these animals was significantly lower than that in vehicle-infused rats. Further, dopamine release induced by high-K stimulation was decreased in β-amyloid protein-infused rats compared with vehicle-infused rats. These results suggest that the release of the two transmitters, acetylcholine and dopamine, was decreased by β-amyloid protein and that learning deficits observed in the β-amyloid protein-infused rats are partly due to the impairment of neurotransmitter release. Furthermore, continuous infusion of β-amyloid protein may be a useful method to produce the animal model of Alzheimer's disease.     

Innervation of holothurian body wall muscle: inhibitory effects and localization of 5-HT

We investigated innervation to body wall muscles as well as distribution of 5-HT (serotonin) and its effects on longitudinal muscles of body wall (LMBW) of the sea cucumber Apostichopus japonicus. With serial sections we found neural branches and fibers extending from hyponeural part of radial nerve towards LMBW and circular muscles of body wall. With the aqueous aldehyde (Faglu) method yellow fluorescence indicating indolamines was observed in LMBW and in the mesentery connecting LMBW to the body wall. With indirect immunohistochemistry 5-HT-like immunoreactivity was observed in LMBW and in mesentery. These results strongly suggested that both LMBW and mesentery contained 5-HT. The effects of monoamine neurotransmitters were studied in LMBW. Putative neurotransmitters tested were 5-HT, adrenaline, noradrenaline, dopamine, and DOPA at the concentration of 10(-6) M. The application of 5-HT caused no contraction or relaxation, but it inhibited the contraction induced by 10(-6)-10(-5) M acetylcholine (ACh). Catecholamines were ineffective by themselves and had no effects on the contraction induced by ACh. The present histological, histochemical, and pharmacological studies strongly suggested that holothurian LMBW was innervated by inhibitory serotonergic neurons of the hyponeural nervous system.     

Pharmacological heterogeneity of release-regulating presynaptic AMPA/kainate receptors in the rat brain: study with receptor antagonists

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors mediating hippocampal [(3)H]noradrenaline or [(3)H]serotonin release, striatal [(3)H]dopamine release and cortical [(3)H]acetylcholine release were pharmacologically characterized using several AMPA/kainate receptor antagonists. The releases of the four transmitters elicited by exposing synaptosomes to AMPA were antagonized by NBQX, indicating that they reflect AMPA/kainate receptor activation. GYKI52466 did not inhibit the AMPA-induced release of [(3)H]noradrenaline, [(3)H]dopamine or [(3)H]serotonin, while it weakly affected the AMPA-mediated release of [(3)H]acetylcholine. On the contrary, LY300164 and LY303070 were potent antagonists able to discriminate among AMPA/kainate receptor subtypes. Both compounds blocked the AMPA receptors mediating [(3)H]dopamine and [(3)H]acetylcholine release. However, LY303070, but not LY300164, inhibited the AMPA-induced release of [(3)H]noradrenaline, while the AMPA-mediated [(3)H]serotonin release was sensitive to LY300164 but not to LY303070. SYM2206 mimicked LY300164 and prevented the AMPA-induced release of [(3)H]dopamine, [(3)H]acetylcholine and [(3)H]serotonin, but not that of [(3)H]noradrenaline. NS102 failed to antagonize the AMPA-induced release of all four transmitters. LY293558 prevented the AMPA-mediated release of [(3)H]noradrenaline, [(3)H]dopamine, [(3)H]acetylcholine or [(3)H]serotonin. Differently, LY377770 did not inhibit the AMPA-mediated release of [(3)H]noradrenaline and [(3)H]acetylcholine, but it potently blocked the AMPA-induced release of [(3)H]serotonin and, less so, of [(3)H]dopamine. AMOA inhibited the AMPA-induced release of [(3)H]serotonin or [(3)H]acetylcholine, but not that of [(3)H]noradrenaline or [(3)H]dopamine. GAMS prevented the AMPA-mediated release of [(3)H]acetylcholine and, more weakly, that of [(3)H]dopamine, but it failed to inhibit the release of [(3)H]noradrenaline or [(3)H]serotonin elicited by AMPA. gamma-DGG did not affect the AMPA-mediated release of any of the four transmitters studied. In conclusion, based on the antagonist profiles obtained, the four receptors here analyzed all belong to the AMPA-preferring subclass of glutamate receptors; however, they appear to differ from each other, probably due to differences in subunit composition. The compounds LY300164, LY303070, LY377770, AMOA and GAMS may be useful to discriminate among AMPA-preferring receptor subtypes.     

Noradrenaline, dopamine, 5-hydroxytryptamine and tryptophan concentrations in the brains of four cohabiting species of fish

1. The concentrations of the neurotransmitters noradrenaline, dopamine and 5-hydroxy-tryptamine and the amino acid tryptophan were determined in the telencephalon, optic lobes and rest of the brains of four species of fish collected from a stream in central Saskatchewan. 2. The species investigated were white sucker (Catostomus commersoni), longnose sucker (Catostomus catostomus), longnosed dace (Rhinichthys cataractae) and northern pike (Esox lucius). 3. Significant differences were found in the concentrations of amines in different regions of the brain within species and within the same brain region between species. 4. These results may be related to the phylogenetic differences or to patterns in brain development evolved by fish species to adapt to particular lifestyles.