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A Maelicke

Publications and source records attributed to A Maelicke.

At least 55 records · Page 3Linked to original sources
PubMed1996

Ligand-gated ion channels in acutely dissociated rat hippocampal neurons with long dendrites.

A technique for dissociation of hippocampi of 3-25-day-old rats is described by which pyramidal and bipolar neurons with many long (up to 200 microns) dendrites can be obtained. Dissociation of CA1 neurons was achieved by mechanical means, in the absence of Ca2+, and without the use of proteolytic enzymes. The functional properties of the dissociated neurons were assessed using the whole-cell patch-clamp technique. Whole-cell currents were elicited by U-tube application of the agonists N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA), and acetylcholine (ACh), and spontaneous miniature currents were also observed in these neurons. ACh-elicited currents were blocked by methyllycaconitine (MLA, 1 nM) and Pb2+ (0.1-10 microM). These results establish acutely dissociated neurons as a simple and reliable preparation for the study of the pharmacology, kinetics and subcellular distribution of ligand-gated ion channels.

Acetylcholine↗
PubMed1996

Activation and blockade of mouse muscle nicotinic channels by antibodies directed against the binding site of the acetylcholine receptor.

1. Using the patch-clamp technique, we have found that mouse muscle nicotinic acetylcholine receptor (nAChR) channels can be activated by low concentrations of a monoclonal antibody (MoAb), referred to as WF6, which is directed against the acetylcholine (ACh) binding site. Similar effects were seen using IgG or F(ab)2 fragments from the sera of patients with myasthenia gravis (MG), which contain polyclonal anti-nAChR antibodies. 2. The mean open times of MoAb and the slope conductance of single WF6-activated single channels were similar to those of ACh-activated channels under the same experimental conditions. 3. On outside-out patches, single channel activity was elicited by MoAb WF6 and MG F(ab)2 fragments, and was blocked by (+)-tubocurarine. We therefore concluded that MoAb WF6 and the MG F(ab)2 fragments activate the nAChR. 4. MoAb WF6 and MG F(ab)2 fragments blocked the current activated by pulsed application of 10(-4) M ACh to a significant extent. The block was partly reversible. The rate constants for the binding and dissociation of MoAb WF6 from the receptor were determined quantitatively.

Animals↗
PubMed1996

Alpha4-1 subunit mRNA of the nicotinic acetylcholine receptor in the rat olfactory bulb: cellular expression in adult, pre- and postnatal stages.

In addition to their role in signal transduction, nicotinic acetylcholine receptors have been shown in vi-tro to be involved in neuronal growth cone regulation during development. This idea is supported by recent histochemical findings showing that iso- and archicortical nicotinic alpha4-1 receptor mRNA expression precedes cholinergic fiber ingrowth. To test whether this also holds true for rhinencephalic parts of the telencephalon, we have studied the olfactory bulb by digoxigenin-mediated in situ hybridization, using an alpha4-1 isoform-specific riboprobe and an alkaline-phosphatase-based detection system. Development is characterized by early intense alpha4-1 mRNA expression (embryonic day 14), reaching a peak around postnatal day 2 when all olfactory bulb layers are invested with numerous alpha4-1 transcript-bearing neurons. Subsequently, the density of labeled neurons decreases to reach adult levels (postnatal day 120), where strongly labeled neurons remain in the mitral cell layer, outer external plexiform layer, and glomerular layer. The unifying pattern of iso-, archicortical, and rhinencephalic alpha4-1 mRNA expression is its early onset, i.e. preceding cholinergic innervation. This points to a possible role of nicotinic receptors regarding neuronal migration in all three regions.

Animals↗
PubMed1996

Agonist responses of neuronal nicotinic acetylcholine receptors are potentiated by a novel class of allosterically acting ligands.

Similar to the gamma-aminobutyric acidA receptor and the N-methyl-D-aspartate subtype of glutamate receptor, neuronal nicotinic acetylcholine receptors are subject to positive modulatory control by allosterically acting ligands. Exogenous ligands such as galanthamine and the neurotransmitter 5-hydroxytryptamine, when applied in submicromolar concentrations with nicotinic agonists, significantly increase the frequency of opening of nicotinic receptor channels and potentiate agonist-activated currents. Because these effects have been shown to be blocked by the monoclonal antibody FK1, they are mediated by binding sites that are located on alpha subunits of nicotinic receptors and distinct from those for acetylcholine and acetylcholine-competitive ligands. At higher concentrations, the potentiating effect of these ligands decreases and is eventually overcome by an inhibition of the agonist-induced response. The sensitizing actions of galanthamine, 5-hydroxytryptamine, and related compounds, at submicromolar concentrations, may reflect the existence of cross-talk between adjacent neuroreceptors and synapses in the central nervous system and thus suggests the formation of transiently active chemical networks in the vertebrate brain.

Allosteric Site↗
PubMed1996

Paraoxon: cholinesterase-independent stimulation of transmitter release and selective block of ligand-gated ion channels in cultured hippocampal neurons.

Paraoxon (O,O-diethyl O-p-nitrophenyl phosphate) is the neurotoxic metabolite of the insecticide parathion (O,O-diethyl O-p-nitrophenyl phosphorothioate). The effects of organophosphorus compounds on peripheral synapses have been attributed to inhibition of cholinesterase and to direct actions on muscarinic and nicotinic receptors, but less is known about the actions of organophosphorus compounds, including paraoxon, in the central nervous system. We investigated initially the effects of paraoxon on spontaneous transmitter release by recording miniature postsynaptic currents (MPSCs) from cultured rat hippocampal neurons using the whole-cell mode of the patch-clamp technique. Paraoxon (0.3 microM) in the presence of tetrodotoxin (0.3 microM) and atropine (1 microM) caused a significant increase in the frequency of gamma-aminobutyric acid- and glutamate-mediated MPSCs, but did not change the peak amplitudes or decay-time constants of these MPSCs. In contrast, application of nicotinic agonists or antagonists did not change the MPSC frequency. The presynaptic effect of paraoxon shown here was not mediated by actions on muscarinic or nicotinic receptors, or by elevated acetylcholine levels secondary to inhibition of cholinesterase. In addition, agonists were applied to assess the postsynaptic effects of paraoxon on excitatory and inhibitory amino acid receptors. Paraoxon (30 microM-1 mM) blocked the ion channels of glycine, gamma-aminobutyric acidA, N-methyl-D-aspartic acid and nicotinic acetylcholine receptors, but not the ion channels of kalnate- and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. The combined effects of paraoxon on spontaneous transmitter release and on the functions of several ligand-gated receptors may constitute mechanisms relevant to the neurotoxicity of paraoxon.

Animals↗
PubMed1996

Diversity of nicotinic acetylcholine receptors in rat brain. V. alpha-Bungarotoxin-sensitive nicotinic receptors in olfactory bulb neurons and presynaptic modulation of glutamate release.

The presence of functional nicotinic acetylcholine receptors (nAChRs) on cultured neurons of the rat olfactory bulb was evaluated using the whole-cell patch-clamp technique. Application of acetylcholine (ACh) to 78% of the tested olfactory bulb neurons evoked whole-cell currents (referred to as direct response), which are very similar in characteristics to type IA currents. Their peak amplitude increased, while their rise-time and decay-time constants decreased with increasing agonist concentration. In 12% of the neurons, ACh evoked single or multiple miniature postsynaptic currents (referred to as indirect response) for which amplitude, rise time, and decay-time constants were not dependent upon the ACh concentration. Methyllycaconitine (1 nM), a selective competitive antagonist at the alpha-bungarotoxin-sensitive neuronal nAChR, reversibly blocked both responses, whereas 6-cyano-7-nitroquinoxaline-2,3-di-one (10 microM) reversibly blocked only the indirect responses. Whereas tetrodotoxin (0.2-2 microM) failed to affect the indirect response, Ca(+2)-free, Mg(+2)-containing external solution decreased reversibly and significantly the frequency of ACh-evoked miniature postsynaptic currents. The pharmacology and kinetics of the two types of responses are consistent with the existence in the olfactory bulb neurons of alpha-bungarotoxin-sensitive nAChRs at both postsynaptic and presynaptic sites, the presynaptic receptors being located on glutamatergic synapses where they modulate the release of the transmitter. The dimensions of the soma and dendrites of the neurons suggest that the direct response is obtained from periglomerular and/or granular neurons, and the indirect response from short-axon and/or external tufted cells. The present results suggest that 1) nicotinic synaptic transmission could play an important role in modulating the bulbar output at the glomerular level, and 2) a presynaptic modulatory effect is one of the functions for the alpha-bungarotoxin-sensitive nAChRs in the mammalian central nervous system.

Acetylcholine↗
PubMed1995

Cellular expression of alpha 4 subunit mRNA of the nicotinic acetylcholine receptor in the developing rat telencephalon.

By contrast to adult brain, little is known on the development of nicotinic acetylcholine receptor (nAChR) expression. Using a digoxigenin-labeled riboprobe for in situ hybridization, alpha 4 nAChR subunit mRNA expression was studied in embryonic and postnatal rat neocortex and hippocampus where it was transiently increased in neuronal subpopulations and preceded cholinergic fiber ingrowth. alpha 4 expression was increased in neocortical layer VIb between E20 and P2 and, about birth, in dentate gyrus granule cells subsequently decreasing to adult levels. nAChR mRNA expression is increased at the developing neuromuscular endplate preceding cholinergic innervation which triggers changes in non-alpha nAChR isoform expression. It has to be elucidated whether similar changes may occur in the telencephalon.

Animals↗
PubMed1995

Nicotinic receptor function in the mammalian central nervous system.

The diversity of neuronal nicotinic receptors (nAChRs) in addition to their possible involvement in such pathological conditions as Alzheimer's disease have directed our research towards the characterization of these receptors in various mammalian brain areas. Our studies have relied on electrophysiological, biochemical, and immunofluorescent techniques applied to cultured and acutely dissociated hippocampal neurons, and have been aimed at identifying the various subtypes of nAChRs expressed in the mammalian central nervous system (CNS), at defining the mechanisms by which CNS nAChR activity is modulated, and at determining the ion permeability of CNS nAChR channels. Our findings can be summarized as follows: (1) hippocampal neurons express at least three subtypes of CNS nAChRs--an alpha 7-subunit-bearing nAChR that subserves fast-inactivating, alpha-BGT-sensitive currents, which are referred to as type IA, and alpha 4 beta 2 nAChR that subserves slowly inactivating, dihydro-beta-erythroidine-sensitive currents, which are referred to as type II, and an alpha 3 beta 4 nAChR that subserves slowly inactivating, mecamylamine-sensitive currents, which are referred to as type III; (2) nicotinic agonists can activate a single type of nicotinic current in olfactory bulb neurons, that is, type IA currents; (3) alpha 7-subunit-bearing nAChR channels in the hippocampus have a brief lifetime, a high conductance, and a high Ca2+ permeability; (4) the peak amplitude of type IA currents tends to rundown with time, and this rundown can be prevented by the presence of ATP-regenerating compounds (particularly phosphocreatine) in the internal solution; (5) rectification of type IA currents is dependent on the presence of Mg2+ in the internal solution; and (6) there is an ACh-insensitive site on neuronal and nonneuronal nAChRs through which the receptor channel can be activated. These findings lay the groundwork for a better understanding of the physiological role of these receptors in synaptic transmission in the CNS.

Acetylcholine↗
PubMed1995

Gene expression of the nicotinic acetylcholine receptor alpha 4 subunit in the frontal cortex in Parkinson's disease patients.

Cognitive impairment in Parkinson's disease is accompanied by a marked decrease of cerebrocortical nicotinic receptors. To study the putative site of impaired receptor synthesis, frontal cortices of Parkinson patients with cognitive dysfunction have been screened for the expression of the nicotinic receptor alpha 4 subunit gene. Quantitative assessment of alpha 4 mRNA-expressing neurons did not show significant differences between patients and controls. Therefore, decreased nicotinic receptor sites cannot be attributed to alterations at the transcriptional level of the alpha 4 gene. Alternative causes have to be searched for at the translational and/or postranslational level.

Aged↗
PubMed1995

Cellular distribution in the rat telencephalon of mRNAs encoding for the alpha 3 and alpha 4 subunits of the nicotinic acetylcholine receptor.

Pharmacological and electrophysiological studies provide evidence for the involvement of different nicotinic acetylcholine receptor isoforms in rat neocortical and hippocampal signal transduction. Yet, rather little is known on the cellular localization of these isoforms. With the availability of isoform specific nucleic acid probes and sensitive non-isotopic detection systems, nicotinic receptors can be studied on the mRNA level in individual neurons. In this way, we have paradigmatically studied the distribution of the alpha 3 and alpha 4 isoform mRNAs of the nicotinic receptor in the rat telencephalon. In the cerebral cortex, alpha 3 transcripts were mainly located in pyramidal neurons of layers V and VI and in some non-pyramidal cells in layer IV, while alpha 4 mRNA was detected in different types of neurons located in almost all layers. In the hippocampus, local distribution of both transcripts was comparable. Only very few labeled neurons were observed in the dentate gyrus. In the CA region, the specific mRNAs were detected in pyramidal perikarya and individual neurons in the strata oriens and lacunosum-moleculare. Our data show that the applied method is sufficiently sensitive and isoform-selective in order to study the differential expression of nicotinic receptors on the cellular level in the mammalian brain.

Animals↗
PubMed1994

EX-1, a surface antigen of mouse neuronal progenitor cells and mature neurons.

Using membrane fragments of PCC7-Mz1 embryonal carcinoma cells, an established in vitro model of neural differentiation (Lang et al., J. Cell Biol., 109 (1989) 2481-2493), we have raised monoclonal antibodies (mAb) against developmental stage-specific cell surface antigens. As shown by double-immunofluorescence labeling studies, employing differentiating PCC7-Mz1 cells, primary cultures of mouse cerebellum cells and cryosections of mouse brain and other tissues, rat mAb anti-mouse EX-1 recognizes a membrane protein which is exclusively expressed by cells of the neuronal cell lineage. EX-1-expressing neuronal precursor cells were identified by double labeling with antibodies directed against stem cell markers or BrdU, EX-1-expressing postmitotic neurons by labeling with antibodies directed against phenotypic markers. In the developing mouse brain, the EX-1 antigen is expressed in the neuroepithelium already at prenatal day 8, i.e. clearly before the onset of mature neuron-specific marker expression. Increasing co-expression with the latter is observed from embryonic day E10 throughout neuronal maturation, but not with markers of other cell types tested. From these studies, the EX-1 antigen is the earliest marker for the mouse brain neuronal cell lineage so far discovered.

Animals↗
PubMed1994

Monoclonal antibodies FK1 and WF6 define two neighboring ligand binding sites on Torpedo acetylcholine receptor alpha-polypeptide.

Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl residues at positions 192 and 193 of the nicotinic acetylcholine receptor alpha-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonists, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B. M., Wu, X., Tang, F., Bertazzon, T., Schröder, B., Reinhardt-Maelicke, A., and Maelicke, A. (1991) Biochemistry 30, 2575-2584). Recently we have shown that the sequence region flanking lysine alpha 125 contains elements of the binding site for physostigmine and related ligands, including antibody FK1 (Schrattenholz, A., Godovac-Zimmerman, J., Schäfer, H.-J., Albuquerque, E. X., and Maelicke, A. (1993) Eur. J. Biochem. 216, 671-677). Here we report the identification by enzyme-linked immunosorbent assay techniques, employing fragments of the Torpedo nicotinic acetylcholine receptor alpha-subunit N-terminal region and a panel of synthetic peptides matching in sequence preselected portions of this subunit, of the sequence regions alpha 118-145 and alpha 181-216 as contributing to the FK1 epitope. Of the synthetic peptides employed, alpha 118-137 displayed the highest affinity of FK1 binding. Binding of FK1 and WF6 to single residue-substituted analogs of the sequence alpha 181-200 indicated that the two antibodies have different attachment point patterns within this sequence region. These results, and those of ligand competition studies, suggest that the binding sites for FK1 and physostigmine, and those of WF6 and acetylcholine, are within the same general region of the receptor's three-dimensional structure. The sites neighbor each other, with limited overlap in the case of occupation by high molecular weight ligands.

Acetylcholine↗
PubMed1994

Expression of nicotinic acetylcholine receptors in the rat superior cervical ganglion on mRNA and protein level.

The expression of nicotinic acetylcholine receptors (nAChR) in the rat superior cervical ganglion was investigated by Western blotting, immunohistochemistry and non-radioactive in situ hybridization applying probes for the alpha 4-1 and beta 2 subunit mRNA. Immunoblot analysis of homogenized ganglia using the anti-nAChRs antibody WF6 revealed a labeled protein band of apparent molecular weight of 40 kDa which is typical for the alpha subunit of nAChRs. Applying double-labeling immunofluorescence with antibodies against tyrosine hydroxylase, nAChR-like molecules were identified in most postganglionic neurons and in a subpopulation of small intensely fluorescent (SIF) cells. alpha 4-1 and beta 2 subunit mRNAs were detected in all perikarya of postganglionic sympathetic neurons but not in SIF cells. These results suggest that antibodies raised against purified Torpedo AChR bind to nAChR in sympathetic ganglia and indicate that alpha 4-1 and beta 2 subunits are constituents of nAChRs in sympathetic postganglionic neurons but not of SIF cells.

Animals↗
PubMed1994

Cellular distribution of nicotinic acetylcholine receptor subunit mRNAs in the human cerebral cortex as revealed by non-isotopic in situ hybridization.

The pharmacology of telencephalic nicotinic acetylcholine receptors (nAChRs) has become an important issue in recent years. While in the human brain a direct pharmacological assessment is difficult to achieve the visualization of nAChRs has been enabled by histochemical techniques providing an ever increasing and improving resolution. Receptor autoradiography was used to visualize binding sites on the level of cortical layers whereas immunohistochemistry has allowed for the cell type-specific and ultrastructural localization of receptor protein. Further investigations have to elucidate the cellular sites of NAChR biosynthesis by visualizing subunit-specific transcripts. Using autopsy samples of the human precentral cortex (Area 4) as a paradigm we have applied digoxigenin-labeled cRNA probes to localize transcripts for the alpha 3- and alpha 4-1-subunits of the nAChR. In accordance with findings in the monkey cortex, the alpha 3-subunit seems to be expressed mainly in pyramidal neurons of layers III-VI of the human cerebral cortex. Transcripts for the alpha 4-1-subunit, by contrast, appear to be present in a large number of neurons throughout all layers of the cerebral cortex, consonant with its ubiquitous distribution in the rodent brain. The present findings show that also in human autopsy brains the cell type-specific detection of nAChR transcripts is possible. For the future, this technique will enable to investigate the expression of receptor transcripts in diseased human brains as compared to controls.

Adult↗
PubMed1994

Identification of purine binding sites on Torpedo acetylcholine receptor.

Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[3H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the beta-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of local anesthetics.

Adenosine Triphosphate↗