Developmental regulation of tyrosine phosphorylation of the nicotinic acetylcholine receptor in Torpedo electrocyte

Tyrosine phosphorylation is thought to play a critical role in the clustering of acetylcholine receptors (AChR) at the developing neuromuscular junction. Yet, in vitro approaches have led to conflicting conclusions regarding the function of tyrosine phosphorylation of AChR beta subunit in AChR clustering. In this work, we followed in situ the time course of tyrosine phosphorylation of AChR in developing Torpedo electrocyte. We observed that tyrosine phosphorylation of the AChR beta and delta subunits occurs at a late stage of embryonic development after the accumulation of AChRs and rapsyn in the membrane and the onset of innervation. Interestingly, in the mature postsynaptic membrane, we observed two populations of AChR differing both in their phosphotyrosine content and distribution. Our data are consistent with the notion that tyrosine phosphorylation of the AChR is related to downstream events in the pathway regulating AChR accumulation rather than to initial clustering events.     

Roles of rapsyn and agrin in interaction of postsynaptic proteins with acetylcholine receptors.

At the neuromuscular junction, aggregates of acetylcholine receptors (AChRs) are anchored in the muscle membrane by association with rapsyn and other postsynaptic proteins. We have investigated the interactions between the AChR and these proteins in cultured C2 myotubes before and after treatment with agrin, a nerve-derived protein that induces AChRs to cluster. When AChRs were isolated from detergent extracts of untreated C2 myotubes, they were associated with rapsyn and, to a lesser degree, with utrophin, beta-dystroglycan, MuSK, and src-related kinases, but not with syntrophin. Treatment with agrin increased the association of AChRs with MuSK, a receptor tyrosine kinase that forms part of the agrin receptor complex, without affecting other interactions. Analysis of rapsyn-deficient myotubes, which do not form protein clusters in response to agrin, revealed that rapsyn is required for association of the AChR with utrophin and beta-dystroglycan, and for the agrin-induced increase in association with MuSK, but not for constitutive interactions with MuSK and src-related kinases. In rapsyn -/- myotubes, agrin caused normal tyrosine phosphorylation of AChR-associated and total MuSK, whereas phosphorylation of the AChR beta subunit, both constitutive and agrin-induced, was strongly reduced. These results show first that aneural myotubes contain preassembled AChR protein complexes that may function in the assembly of the postsynaptic apparatus, and second that rapsyn, in addition to its role in AChR phosphorylation, mediates selected protein interactions with the AChR and serves as a link between the AChR and the dystrophin/utrophin glycoprotein complex.     

Developmental mRNA expression of the alpha10 nicotinic acetylcholine receptor subunit in the rat cochlea

A recently discovered alpha10 subunit of the nicotinic acetylcholine receptor (nAChR) family is believed to form a heteromeric receptor with the alpha9 nAChR subunit in auditory hair cells. In the present study, the alpha10 nAChR subunit expression in the developing and adult rat inner ear was analyzed by PCR and localized using isotopic in situ hybridization. Unlike the alpha9 subunit, the alpha10 subunit was not detected at embryonic day 18 (E18). From E21 through postnatal day 15 (P15), the alpha10 subunit was localized over both inner hair cell (IHC) and outer hair cell (OHC) regions, but in the mature cochlea detectable levels of alpha10 mRNA were found only over the OHC region. From E21 through adult ages, there was also a small but consistent basal to apical gradient of alpha10 expression; that is, higher levels in basal regions and lower levels in apical regions. Previously, we detected the alpha9 nAChR subunit over IHCs as early as E18 and throughout adult ages with a clear basal-apical gradient of expression. Our studies raise the question of whether the alpha9 and alpha10 subunits are differentially regulated during embryonic and postnatal development.     

Targeted in vivo expression of nicotinic acetylcholine receptors in mouse brain using lentiviral expression vectors

Nicotinic acetylcholine receptors (nAChRs) in the brain exhibit diverse functional properties and ubiquitous distribution. Yet, except for providing a receptor for the exogenously applied nicotine of tobacco products, their role in the normal functioning of the brain has remained elusive. We have used a lentiviral expression vector to re-express the beta2 subunit specifically in the ventral tegmental area (VTA) of beta2-/- mice. The viral vector efficiently expresses beta2- subunit protein leading to new nAChR-binding sites. VTA neurons transduced by the lentiviral vector are responsive to intravenous nicotine when analyzed using in vivo electrophysiology. Nicotine-induced dopamine release from the nucleus accumbens (NuAcc) was also restored in re-expressing beta2-/- mice. Intra-VTA injection of nicotine was found to be reinforcing in both wild-type and beta2-subunit re-expressing beta2-/- mice, but not in beta2-/- mice. Furthermore, in the absence of applied nicotine, the spontaneous slow exploratory behavior of the mice was restored, whereas fast navigation did not change. This latter behavioral analysis suggests a role for beta2* nAChR, specifically expressed in the VTA, in mammalian cognitive function.     

Widespread decrease of nicotinic acetylcholine receptors in Parkinson's disease

OBJECTIVE: Nicotinic acetylcholine receptors have close interactions with the dopaminergic system and play critical roles in cognitive function. The purpose of this study was to compare these receptors between living PD patients and healthy subjects. METHODS: Nicotinic acetylcholine receptors were imaged in 10 nondemented Parkinson's disease patients and 15 age-matched healthy subjects using a single-photon emission computed tomography ligand [(123)I]5-iodo-3-[2(S)-2-azetidinylmethoxy]pyridine. Using an arterial input function, we measured the total distribution volume (V; specific plus nondisplaceable), as well as the delivery (K(1)). RESULTS: Parkinson's disease showed a widespread significant decrease (approximately 10%) of V in both cortical and subcortical regions without a significant change in K(1). INTERPRETATION: These results indicate the importance of extending the study to demented patients.     

The role of nicotinic acetylcholine receptors in lymphocyte development

The sizes of lymphocyte populations in lymphoid organs of nicotinic acetylcholine receptor knockout and chimera (knockout/wild-type) mice were studied by flow cytometry. The absence of beta2 subunit decreased, while nicotine treatment increased B lymphocyte numbers in the bone marrow. In chimera mice, either beta2 or alpha7 subunits influenced lymphocyte populations in primary lymphoid organs, while in the spleen, only alpha7 receptors were critical. More annexin V-positive B cells were found in the bone marrow of knockout than wild-type animals. We conclude that nicotinic receptors are involved in regulating lymphocyte development and control the B lymphocyte survival.     

The role of nicotinic acetylcholine receptors in the mechanisms of anesthesia

Nicotinic acetylcholine receptors are members of the ligand-gated ion channel superfamily, that includes also gamma-amino-butiric-acid(A), glycine, and 5-hydroxytryptamine(3) receptors. Functional nicotinic acetylcholine receptors result from the association of five subunits each contributing to the pore lining. The major neuronal nicotinic acetylcholine receptors are heterologous pentamers of alpha4beta2 subunits (brain), or alpha3beta4 subunits (autonomic ganglia). Another class of neuronal receptors that are found both in the central and peripheral nervous system is the homomeric alpha7 receptor. The muscle receptor subtypes comprise of alphabetadeltagamma (embryonal) or alphabetadeltaepsilon (adult) subunits. Although nicotinic acetylcholine receptors are not directly involved in the hypnotic component of anesthesia, it is possible that modulation of central nicotinic transmission by volatile agents contributes to analgesia. The main effect of anesthetic agents on nicotinic acetylcholine receptors is inhibitory. Volatile anesthetics and ketamine are the most potent inhibitors both at alpha4beta2 and alpha3beta4 receptors with clinically relevant IC(50) values. Neuronal nicotinic acetylcholine receptors are more sensitive to anesthetics than their muscle counterparts, with the exception of the alpha7 receptor. Several intravenous anesthetics such as barbiturates, etomidate, and propofol exert also an inhibitory effect on the nicotinic acetylcholine receptors, but only at concentrations higher than those necessary for anesthesia. Usual clinical concentrations of curare cause competitive inhibition of muscle nicotinic acetylcholine receptors while higher concentrations may induce open channel blockade. Neuronal nAChRs like alpha4beta2 and alpha3beta4 are inhibited by atracurium, a curare derivative, but at low concentrations the alpha4beta2 receptor is activated. Inhibition of sympathetic transmission by clinically relevant concentrations of some anesthetic agents is probably one of the factors involved in arterial hypotension during anesthesia.     

Modulation of acetylcholine release by nicotinic receptors in the rat brain

Since physostigmine (Phy) is presently used in the experimental treatment of Alzheimer disease (AD) patients by means of intracerebral ventricular (i.c.v.) administration, we designed a study to determine the effect of the drug administered by the same route on the cholinergic system of the rat brain. Particularly, we studied the involvement of nicotinic cholinergic function. The specific conditions required in this experiment were achieved by a series of short-lasting periods of acetylcholinesterase (AChE) inhibition leading to short-lasting increases of acetylcholine (ACh). These were produced by periodic i.c.v. injections of Phy. At 7 days of Phy administration, a small effect on 3H-nicotine binding was seen only in the striatum of the injected side. In rats treated for 13 days, we observed a 120% increase in the stimulated release of 3H-ACh in hippocampal slices of the injected side of the brain. There also was a significant 88% increase in 3H-nicotinic binding in the hippocampus of the same side while muscarinic binding was unchanged. These results suggest a process of upregulation of presynaptic nicotinic autoreceptors in the hippocampus modulating ACh release but no effect on the muscarinic receptors. Our results also suggest that pulses of ACh in analogy to nicotinic stimulation can cause protracted desensitization and eventually inactivation of the receptor leading to its up-regulation. These results are consistent with findings on the release of ACh from cortical biopsies and of a sustained ACh release in the CSF of AD patients following the same treatment.     

Higher expression of alpha7 nicotinic acetylcholine receptors in human fetal compared to adult brain

Neuronal nicotinic acetylcholine receptors are thought to be involved in regulation of several processes during neurogenesis of the brain. In this study the expression of the alpha7 nicotinic acetylcholine receptor subtype was investigated in human fetal (9-11 weeks of gestation), middle-aged (28-51 years) and aged (68-94 years) medulla oblongata, pons, frontal cortex, and cerebellum. The specific binding of the alpha7 receptor antagonist [(125)I]alpha-bungarotoxin was significantly higher in fetal than in both middle-aged and aged medulla oblongata and aged pons. No significant decrease in [(125)I]alpha-bungarotoxin binding sites was observed from fetal to adult cortex and cerebellum. The alpha7 mRNA expression was significantly higher in all fetal brain regions investigated, except for aged cortex, than in corresponding middle-aged and aged tissue. The high expression of alpha7 nicotinic acetylcholine receptors in fetal compared to adult brain supports the view that these receptors play an important role during brain development.     

The possible contribution of neuronal nicotinic acetylcholine receptors in depression

Although tobacco use and smoking were introduced long ago, it was only recently that the nicotine contained in the tobacco leaves was recognized as an addictive substance acting on the central nervous system (CNS). However, even prior to this recognition, several studies have reported an association between smoking and psychiatric disorders. One of the many observations was that smoking cessation is accompanied by a marked increase in the probability of major depression. In parallel with the discovery of the neuronal nicotinic acetylcholine receptors and their extensive expression in the CNS, this association sheds new light on the influence of cholinergic transmission in depression. In this article, we examine the various modes of action of nicotine in the CNS and discuss the mechanisms by which this alkaloid can prevent or precipitate mood disorders, and the possibility of discovering new therapeutic avenues for the treatment of depression.     

The calsyntenins--a family of postsynaptic membrane proteins with distinct neuronal expression patterns

We have identified two novel postsynaptic membrane proteins that are highly similar to calsyntenin-1 in their extracellular parts but vary considerably in their cytoplasmic segment. Calsyntenin-1 has recently been identified in our lab as a postsynaptic membrane protein with a highly acidic cytoplasmic segment with putative Ca(2+)-binding capacity (Vogt et al., 2001, Mol. Cell. Neurosci. 17: 151-166). Based on their structural similarity to calsyntenin-1, we have called the novel proteins calsyntenin-2 and -3, respectively. By immunoelectron microscopy, the calsyntenin protein family was localized in the postsynaptic membrane of excitatory central nervous system (CNS) synapses. In situ hybridization analysis revealed that calsyntenin-1 was abundant in most neurons of the CNS with relatively little variation in its expression level. Calsyntenin-2 and -3 expressions varied much more with highest levels in GABAergic neurons. Based on their distinct expression patterns and the differences in their cytoplasmic segments, we suggest a cell-type-specific functional role for the three calsyntenins in excitatory synaptic transmission.     

Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture

The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer＇s disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which influence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to persistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in persistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer＇s disease.     

Developmental expression patterns of bone morphogenetic proteins, receptors, and binding proteins in the chick retina

Bone morphogenetic proteins (BMPs), a large subfamily of the transforming growth factor-beta (TGF-beta) superfamily of growth factors, have been implicated in patterning of the central nervous system, but their role in the retina is much less well known. As an initial step in addressing this issue, we have investigated by in situ hybridization the expression patterns of BMP-2, -4, -5, -6, and -7, BMP receptor kinases (BRKs) -1, -2, and -3, and BMP binding proteins noggin and chordin, in the chick embryonic eye at embryonic day 3 (E3), and in isolated retinas at E6, E8, and E18. Strikingly, all mRNAs examined had spatially restricted patterns of expression in the early eye, with the receptors found primarily in the ventral portion of the retina and in the optic stalk, and the ligands and binding proteins localized to other regions of the retina and/or retinal pigment epithelium. Dorso-ventrally restricted patterns of expression persisted at E8, but were no longer apparent at E18, whereas layer-specific patterns of expression were detectable at both E8 and E18. This distribution of BMP family members, receptors, and binding proteins within the retina appears consistent with a possible role in patterning and/or differentiation of this tissue.     

Immunohistochemical localization of nicotinic acetylcholine receptors in the guinea pig bowel and pancreas

Although nicotinic acetylcholine receptors (nAChRs) are known to be present on neural elements in both the bowel and the pancreas, the precise location of these receptors has not previously been determined. Immunocytochemistry, by using a rat monoclonal antibody (mAb35), which recognizes α-bungarotoxin (α-Bgt)-insensitive nAChRs, and a polyclonal antibody raised against the α-Bgt-sensitive receptor subunit, α7, was used to locate receptor protein in guinea pig gut and pancreas. mAb35-receptor (mAb35-R) immunoreactivity was abundant in both enteric plexuses, enterochromaffin cells, and pancreatic ganglia. Immunostaining was associated with the cell membrane, and clusters of mAb35-R were observed on cell somas and dendrites. Receptor immunoreactivity was also observed on terminals and axons, suggesting that a subset of nAChRs is presynaptic. Internal sites of mAb35-R were observed in permeabilized ganglia. Cells expressing the receptors were closely associated with ChAT-immunoreactive nerve fibers. In addition, the majority of ChAT-positive neurons expressed both cell surface and internal stores of mAb35-R. In the bowel, clusters of mAb35-R were present on the soma and dendrites of Dogiel type I motorneurons and secretomotor neurons. Receptors were detected at the plasma membrane of calbindin-immunoreactive myenteric neurons. In contrast, calbindin-immunoreactive submucosal neurons did not express cell surface mAb35-R, supporting the idea that they are sensory neurons. A subset of enteric neurons expressed both mAb35-R and glutamate receptor (GluR1) immunoreactivity. In the pancreas, mAb35-R immunoreactivity was only observed in ganglia. α7-immunoreactivity was found on both enteric cell bodies and nerve fibers. Based on these results, it appears that visceral nAChRs are composed of at least four subunits and that both pre- and postsynaptic nAChRs are present in the gut and pancreas. J. Comp. Neurol. 390:497–514, 1998. © 1998 Wiley-Liss, Inc.     

Role of presynaptic nicotinic acetylcholine receptors in the regulation of gastrointestinal motility

Presynaptic nicotinic acetylcholine receptors (nAChRs) located on cholinergic terminals facilitate the release of acetylcholine (ACh), thereby constituting a fail-safe mechanism at strategic locations, such as the neuromuscular junction, where reliable transmission is vital. Accumulating data indicate that myenteric neurons in the enteric nervous system possess not only somatodendritic nAChRs, which mediate cholinergic transmission between neurons, but also presynaptic nAChRs. Functional evidence shows that these receptors mediate a positive feedback with respect to ACh release from myenteric motoneurons, and might therefore play an important role in the regulation of gastrointestinal motility. These presynaptic nAChRs were found to be more sensitive to nicotinic ligands than somatodendritic nAChRs and could therefore be primary targets of exogenous compounds, such as nicotine. This interaction might provide a neurochemical basis for the effect of smoking on gastrointestinal motility. Another important human pharmacological implication is based on our recent observation that monoamine uptake inhibitor-type antidepressant drugs are able to inhibit presynaptic nAChRs in the enteric nervous system. The disruption of the nAChR-mediated positive feedback modulation by antidepressants might explain the frequent occurrence of constipation, a common side effect, attributed to these drugs. Clarification of the role of presynaptic nAChRs in feedback mechanisms in the enteric nervous system might be instrumental in the development of new drugs affecting gastrointestinal motility.     

Pr-lynx1, a modulator of nicotinic acetylcholine receptors in the insect

Insect nicotinic acetylcholine receptors (nAChRs) are targets for insecticides. Despite the importance of the nAChR as a major target for insecticide action, modulators of nAChRs in insects remain unidentified. Here we describe the cloning and identification of a nAChR modulator gene in an insect. This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library, and the gene itself encodes a protein 120 amino acids in length, named Pr-lynx1. Pr-lynx1 shares all the features, including a cysteine-rich consensus motif and common gene structure, of the Ly-6/neurotoxin superfamily. The recombinant Pr-lynx1, which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells, is normally present at the cell surface as a GPI-anchored protein. Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various tissues, such as the ganglion, brain, mandibular muscle, proventriculus, leg muscle, and epidermis. This expression pattern is similar to the distribution of nAChRs as assayed by alpha3 nAChR immunoreactivity. Co-expression of Pr-lynx1 in Xenopus oocytes expressing alpha3beta4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents, indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs. This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species.