Acetylcholine synthesis and neuron differentiation

Development of the nervous system is dependent on the co-operation between cell determination events and the action of epigenetic factors; in addition to well known factors, e.g. growth factors, neurotransmitters have been assigned a role as "morphogens" and modulators of neuronal differentiation in an early developmental phase. The possible role of acetylcholine as a modulator of neuronal differentiation has been considered in two experimental systems. A neuroblastoma cell line, which does not synthesise any neurotransmitter, has been transfected with a choline acetyltransferase construct; activation of acetylcholine synthesis, thus achieved, is followed by a higher expression of neuronal specific traits. The presence in these cells of muscarinic receptors is consistent with the existence of an autocrine loop, which may be responsible for the more advanced differentiation state observed in the transfected cells. Expression of cholinergic markers appears as a common feature of DRG sensory neurons, independently of the neurotransmitter used. Choline acetyltransferase can be detected in DRG at early developmental stages. The distribution of muscarinic receptors in DRG has suggested that activation of acetylcholine synthesis may be related in an early developmental phase to the interaction between neurons and nonneuronal cells and to modulation of cell differentiation. Both systems suggest that acetylcholine may have a role as a modulator of neuronal differentiation.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

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.     

Neuronal phosphoproteins

This article summarizes some of our knowledge concerning intracellular protein phosphorylation pathways in nerve cells. It also summarizes, very briefly, recent direct experimental evidence involving intracellular injection of protein kinases, protein kinase inhibitors, and substrates, indicating that protein phosphorylation mediates the actions of a variety of neurotransmitters on their target cells. Finally, it summarizes in somewhat greater detail the results of studies of three different types of substrate proteins that appear to regulate different types of biological responses in nerve cells: synapsin I, a substrate protein present in virtually all nerve terminals, which appears to regulate neurotransmitter release from those nerve terminals; the acetylcholine receptor, the phosphorylation of which regulates its rate of desensitization in the presence of acetylcholine; and DARPP-32, the phosphorylation of which converts it into a very potent phosphoprotein phosphatase inhibitor that may be involved in the regulation by the neuromodulator dopamine of the effects of the neurotransmitter glutamate. The identification and characterization of additional neuronal phosphoproteins can be expected to lead to the clarification of numerous additional molecular mechanisms by which signal transduction is carried out in nerve cells.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

"In vivo" intraneuronal trafficking of G protein coupled receptors in the striatum: regulation by dopaminergic and cholinergic environment

We have studied "in vivo" neurochemically identified striatal neurons to analyze the localisation and the trafficking of dopamine and acetylcholine G protein coupled receptors (GPCR) (D1R, D2R, m2R and m4R) under the influence of neurotransmitter environment. We have identified receptors in tissue sections through immunohistochemical detection at the light and electron microscopic level. We have identified receptors in normal animals and after acute and chronic stimulations. We have quantified receptors through image analysis at the electron microscopic level in relation to various subcellular compartments. Our results demonstrate that, in normal conditions, GPCRs are mostly associated with plasma membrane of the striatal neurons, mostly at extra-synaptic sites. In certain instances (m4R; D2R), receptors have prominent localisation inside the rough endoplasmic reticulum. Our results also show that two distinct receptors for a same neurotransmitter may have distinct subcellular localisation in a same neuronal population (m2R versus m4R) and that the same neurotransmitter receptor (m4R) can have distinct localisation in distinct neuronal populations (cytoplasm versus cell surface). After acute stimulation, cell surface receptors undergo dramatic subcellular changes that involve plasma membrane depletion, internalisation in endosomes and in multivesicular bodies. Such changes are reversible after the end of the stimulation and are blocked by antagonist action. Chronic stimulation also provokes changes in subcellular localisation with specific pattern: plasma membrane depletion, and exaggerated storage of receptors in rough endoplasmic reticulum and eventually Golgi complex (D1R; m2R and m4R). Decreasing chronic receptor stimulation reverses such changes. These results demonstrate that, "in vivo", in the striatum, GPCRs undergo complex intraneuronal trafficking under the influence of neurochemical environment in conditions that dramatically modulate the number of cell surface receptors available for interaction with neurotransmitters or drugs. This confirms that "in vivo", the trafficking and the subcellular compartmentalization of GPCRs may contribute to regulate neuronal sensitivity and neuronal interactions in physiological, experimental and pathological conditions, including in therapeutic conditions.     

Acetylcholin als non‐neuronales Signalmolekül

The non‐neuronal cholinergic system Lately there was increasing evidence that acetylcholine plays an important role as a signalling molecule in non‐neuronal cells, besides its well known function as a neurotransmitter. The so‐called non‐neuronal cholinergic system (NNCS) was detected in many organs such as lung, skin and placenta, where it is involved in the regulation of diverse physiological processes. Additionally there is increasing evidence that the NNCS may be involved in the pathogenesis of diseases such as cystic fibrosis. Therefore detailed investigation of the NNCS is essential for a better understanding of human physiology and for developing improved therapies.     

Hierarchical neuronal modeling of cognitive functions: from synaptic transmission to the Tower of London

Recent progress in the molecular biology of synaptic transmission, in particular of neurotransmitter receptors, offers novel information relevant to ‘realistic’ modeling of neural processes at the single cell and network level. Sophisticated computer analyses of 2D crystals by high resolution electron microscopy yield images of single neurotransmitter receptor molecules with tentative identifications of ligand binding sites and of conformational transitions. The dynamics of conformational changes can be accounted for by a ‘multistate allosteric network’ model. Allosteric receptors also possess the structural and functional properties required to serve as coincidence detectors between pre- and post-synaptic signals and, therefore, can be used as building blocks for a chemical Hebb synapse. These properties were introduced into networks of formal neurons capable of producing and detecting temporal sequences. In more elaborate models of pre-frontal cortex functions, allosteric receptors control the selection of transient ‘pre-representations’ and their stabilization by external or internal reward signals. We apply this scheme to Shallice's Tower of London test, and we show how a hierarchical neuronal architecture can implement executive or planning functions associated with frontal areas.     

Structural organization of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.     

Spectrophotometric detection of monovalent cation flux in cells: fluorescence microscope measurement of acetylcholine receptor-mediated ion flux in PC-12 cells

A new and convenient spectroscopic method for measuring monovalent cation flux in cells is described. The technique is based on fluorescence quenching of an entrapped fluorophore (anthracene-1,5-dicarboxylic acid) by Cs+. A conventional fluorescence microscope can be used to measure the Cs+ flux. The usefulness of the technique is illustrated by measurement of acetylcholine receptor-mediated Cs+ flux in PC-12 cells, a sympathetic neuronal cell line. The results are the same as those obtained when radioactive tracer ions were used. The technique is applicable to any transmembrane process in which Cs+ can substitute for either Na+ or K+. The method has been developed to identify the different neurotransmitter receptors that control the translocation of monovalent cations and to locate the cells in central nervous system cell preparations that contain these receptors. The advantage of an optical method over tracer ion methods for biochemical and pharmacological studies of transmembrane processes in cells is described.     

How fast does an acetylcholine receptor channel open? Laser-pulse photolysis of an inactive precursor of carbamoylcholine in the microsecond time region with BC3H1 cells.

The integrated function of the nervous system depends on specific and rapid transmission of signals between its constituent cells. The nicotinic acetylcholine receptor is the best known of a group of membrane-bound proteins responsible for such transmission; for this process to occur, a specific neurotransmitter, in this case acetylcholine, must bind to the receptor, which then forms transmembrane channels through which cations pass. The resulting change in transmembrane voltage determines whether or not a signal is transmitted. The question of how fast this process takes place in any neurotransmitter receptor has remained one of the interesting and most challenging in the field. To answer it, many attempts have been made to evaluate the rate constant for the opening of the acetylcholine receptor channel, but in almost all these studies the rate was measured after the receptor-mediated reaction, which involves the open channel and many intermediate states, had reached a quasi equilibrium. This resulted in a plethora of reported values for the rate constant that differ by a factor of up to 50-fold, even when the measurements were made with the same type of cell. The new approach described here involves the use of single cells of a mammalian cell line (BC3H1), containing muscle-type acetylcholine receptors, and the rapid introduction of neurotransmitter to the cell surface. The rapid delivery was achieved by converting a previously synthesized photolabile precursor of carbamoylcholine to carbamoylcholine, a stable amino-group-containing analogue of acetylcholine, with a single laser pulse and an observed photolysis rate of 7300 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential localization of glutamate receptors opposite sites of high presynaptic release

BACKGROUND: The localization of glutamate receptors is essential for the formation and plasticity of excitatory synapses. These receptors cluster opposite neurotransmitter release sites of glutamatergic neurons, but these release sites have heterogeneous structural and functional properties. At the Drosophila neuromuscular junction, receptors expressed in a single postsynaptic cell are confronted with an array of hundreds of apposed active zones. Hence, this is an ideal preparation for the investigation of whether receptor clustering is sensitive to the morphological and physiological properties of the apposed active zones. RESULTS: To investigate the relationship between the localization of glutamate receptors and the properties of the apposed active zones, we investigated receptor localization in mutants in which receptors are limited. We find that receptors are not uniformly distributed opposite the full array of active zones but that some active zones have a disproportionately large share of receptors as assayed by receptor levels and response to transmitter. The active zones at which receptors preferentially cluster are larger and have a higher neurotransmitter release probability than the average active zone. We find a similar relationship between glutamate receptor clusters and active-zone size at wild-type synapses. CONCLUSIONS: When confronted with an array of active zones, glutamate receptors preferentially cluster opposite the largest and most physiologically active sites. These results suggest an activity-dependent matching of pre- and postsynaptic function at the level of a single active zone.     

Heterogeneity of receptor immunoreactivity at synapses of glycine-utilizing neurons.

Neurons often contain, and probably release, more than one neuroactive substance that may have diverse or opposite actions on the postsynaptic cell. It remains unexplained how these neurons utilize their multiple neuroactive substances while maintaining appropriate resolution of neurotransmitter functions. Here, we have examined the ultrastructural localization of glycine receptors by using a monoclonal antibody directed to the intracellular domain of the strychnine-sensitive glycine receptor. We have found that glycine receptors are only localized to 56% of the synapses made by presumed 'glycinergic' (more accurately, glycine-utilizing) amacrine cells in the turtle retina. The remaining synapses made by these same boutons show no evidence of glycine receptors. As there is no evidence to suggest the presence of a second type of glycine receptor, these data indicate that only a portion of the postsynaptic sites contacted by the glycine-utilizing neurons can respond to glycine. They also suggest that a neuron containing multiple neuroactive substances can selectively affect postsynaptic elements by means of heterogeneous receptor localization.     

Shaping cellular form and function by autophagy

In addition to its familiar role in non-selective bulk degradation of cellular material, autophagy can also bring about specific changes in the structure and function of cells. Autophagy has been proposed to operate in a substrate-selective mode to carry out this function, although evidence to demonstrate selectivity has been lacking. A recent study of synapse formation in the nervous system of the nematode Caenorhabditis elegans now provides experimental evidence for substrate-selective autophagy. Synapses form when presynaptic cells contact their postsynaptic partners during development. This contact induces the assembly of synaptically-localized protein complexes in the postsynaptic cell that contain scaffolding proteins and neurotransmitter receptors. When presynaptic contact was blocked, autophagy in the postsynaptic cell was induced. Substrate selectivity was evident in this system: the gamma-aminobutyric acid type A receptor (GABA(A) receptor), an integral-membrane neurotransmitter receptor, trafficked from the cell surface to autophagosomes. By contrast, the acetylcholine receptor, a structurally-similar neurotransmitter receptor, remained on the cell surface. This result provides experimental support for the idea that autophagy can bring about changes in cell structure and behavior by degrading specific cellular proteins, particularly cell surface receptors that are often important for regulating cell growth, differentiation and function.     

Elevation of intracellular calcium levels in neurons by nicotinic acetylcholine receptors

The recognition that intracellular free calcium serves as a ubiquitous intracellular signal has motivated efforts to elucidate mechanisms by which cells regulate calcium influx. One route of entry that may offer both spatial and temporal fine resolution for altering calcium levels is that provided by cation-permeable, ligand-gated ion channels. Biophysical measurements as well as calcium imaging techniques demonstrate that neuronal nicotinic acetylcholine receptors as a class have a high relative permeability to calcium; some subtypes equal or exceed all other known receptors in this respect. Activation of nicotinic receptors on neurons can produce substantial increases in intracellular calcium levels by direct passage of calcium through the receptor channel. When multiple classes of nicotinic receptors are expressed by the same neuron, each appears capable of increasing calcium in the cell but may differ with respect to location, temporal response, agonist sensitivity, or regulation in achieving it. As a result, nicotinic receptors must be considered strong candidates for signaling molecules through which neurons regulate a diverse array of cellular events.     

Molecular mechanisms of regulation of neuronal muscarinic receptor sensitivity

Like other neurotransmitter receptors, muscarinic acetylcholine receptors are subject to regulation by the state of receptor activation. Prolonged increases in the concentration of muscarinic agonists result in a decrease in receptor density and loss of receptor sensitivity, both in vivo and in vitro. On the other hand, when the receptor is deprived of acetylcholine for a long duration in vivo, the receptor becomes more sensitive in responding to muscarinic agonists. However, it has been more difficult to demonstrate increases in receptor concentration that accompany this supersensitive state. The purpose of this review is to provide current information related to the characteristics of muscarinic receptor regulation and the molecular mechanisms underlying this phenomenon, regarding both the density of receptors and their transduction mechanisms. Furthermore, possible feedback regulatory roles of different second messenger signals are discussed. Particular emphasis is dedicated to molecular mechanisms of regulation of neuronal muscarinic receptors.     

Polarized signaling: basolateral receptor localization in epithelial cells by PDZ-containing proteins

Extracellular signals are normally presented to one surface of epithelial cells and to one end of neurons, and so neuronal and epithelial cell signaling is inherently polarized. Another aspect of signaling polarity is that receptors are often asymmetrically distributed on the surfaces of polarized cells. Recent evidence from studies of Caenorhabditis elegans shows that signaling polarity plays an important role in development. The underlying mesoderm induces the overlying ectoderm to form the vulva, and asymmetric distribution of the signal receptor on the basolateral surface of the epithelium is crucial for this signaling. In neurons, the localization of neurotransmitter receptors and ion channels at synapses allows neurons to be exquisitely sensitive to synaptic inputs. Exciting recent reports suggest that receptor localization to neuronal synapses and the basolateral membrane domains of epithelia may involve a common molecular mechanism involving localization by PDZ-containing proteins.     

Neurotransmitter receptor expression and activity during neuronal differentiation of embryonal carcinoma and stem cells: from basic research towards clinical applications

Abstract.  Embryonal carcinoma and embryonic stem cells have served as models to understand basic aspects of neuronal differentiation and are promising candidates for regenerative medicine. Besides being well characterized regarding the capability of embryonal carcinoma and embryonic stem cells to be precursors of different tissues, the molecular mechanisms controlling neuronal differentiation are hardly understood. Neuropeptide and neurotransmitter receptors are expressed at early stages of differentiation prior to synaptogenesis, triggering transient changes in calcium concentration and inducing neurone-specific gene expression. In vitro neuronal differentiation of embryonal carcinoma and embryonic stem cells closely resembles early neuronal development in vivo . Murine P19 EC cells are a well-characterized model for in vitro differentiation, which upon treatment with retinoic acid differentiate into neurones. Expression and activity of various receptor proteins is regulated during their differentiation. Stimulation of kinin-B2, endothelin-B, muscarinic acetylcholine, and N-methyl-D-aspartate receptors results in transient increases of intracellular free calcium concentration [Ca²⁺]_i in P19 cells undergoing neuronal differentiation, whereas embryonal cells do not respond or show a smaller change in [Ca²⁺]_i than differentiating cells. Receptor inhibition, as studied with the example of the kinin-B2 receptor, aborts neuronal maturation of P19 cells, demonstrating the crucial importance of B2 receptors during the differentiation process. Future success in obtaining desired neuronal phenotypes from pluripotent cells in vitro may offer new therapeutic perspectives for curing genetic and acquired dysfunctions of the developing and adult nervous system.