M. Knipper

Positive feedback between acetylcholine and the neurotrophins nerve growth factor and brain-derived neurotrophic factor in the rat hippocampus.

In the rat hippocampus, nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) are synthesized by neurons in an activity‐dependent manner. Glutamate receptor activation increases whereas GABAergic stimulation decreases NGF and BDNF mRNA levels. Here we demonstrate that NGF and BDNF mRNA and NGF protein are up‐regulated in the rat hippocampus by the activation of muscarinic receptors. Conversely, NGF and BDNF enhance the release of acetylcholine (ACh) from rat hippocampal synaptosomes containing the nerve endings of the septal cholinergic neurons. NGF also rapidly increases the high‐affinity choline transport into synaptosomes. The reciprocal regulation of ACh, NGF and BDNF in the hippocampus suggests a novel molecular framework by which the neurotrophins might influence synaptic plasticity.

Characterization of acetylcholine release from insect synaptosomes

Synaptosomes prepared from ganglia of Locusta migratoria, are able to accumulate [3H]choline and convert most of it to acetylcholine. Exposure of the labelled synaptosomes to media containing elevated K+ concentrations evoked a large increase in the efflux of tritiated acetylcholine. Some characteristics of acetylcholine release from insect nerve terminals were studied by continously perfusing synaptosomes with various solutions. Depolarization of the nerve endings with veratridine or K+ induced a release which was dependent on extracellular calcium, whereas Mg2+ inhibited the release. Pretreatment with the Ca2+-ionophore, A 23187, allowed a calcium-induced release under non-depolarizing conditions. The calcium-dependent efflux is thought to reflect stimulus-secretion coupling processes. In the presence of eserine and carbamylcholine the release was inhibited. Analysis of various cholinergic drugs revealed that the evoked efflux was susceptible to muscarinic ligands, it was enhanced by atropine and reduced by oxotremorine. The results suggest a feed-back regulation of acetylcholine release via muscarinic autoreceptors.

Neurotrophin induced second messenger responses in rat brain synaptosomes.

Neurotrophins (NGF, BDNF) elicit significant elevation of cAMP as well as IP3-concentrations in either membrane preparations or intact isolated nerve endings from rat brain hippocampus. The induced second messenger responses in membrane preparations were detectable in the subsecond time range. The IP3-level reached a maximum after a few hundred ms; whereas the cAMP-level continued to rise even after several seconds. The IP3-response but not the cAMP-signal was inhibited in the presence of the tyrosine kinase blocker K252a, suggesting that the two second messenger cascades were triggered via different mechanisms. The results suggest that neurotrophins may induce short-term effects in nerve terminals via second messenger pathways.

Monoclonal antibodies against the high affinity choline transport system.

Monoclonal antibodies have been isolated that specifically block the high affinity, Na(+)-dependent transport of choline in insect synaptosomes and synaptosomal ghosts. Antibodies were derived after immunization of mice with synaptosomal membranes from locust. Antibody VIB6F5, an IgG isotype, significantly inhibited the high affinity translocation of choline, the effect exhibited saturation at increased antibody concentrations. Antibodies recognized a 80 kDa antigen identified by Western blot analysis of synaptosomal membranes. In immunocytochemical approaches VIB6F5 specifically stained distinct areas in the neuropil of head and thoracic ganglia.

Isolation and reconstitution of the high-affinity choline carrier

Monoclonal antibodies, which block the high‐affinity uptake of choline in synaptosomal ghosts, have been used to purify a membrane polypeptide (80 kDa) from insect synaptosomal membranes. This isolated protein was found to catalyse the sodium‐dependent, hemicholinium‐sensitive accumulation of choline after reconstitution into liposomes, thus, apparently represents the high‐affinity choline transporter.

Purification and reconstitution of the high affinity choline transporter.

The high-affinity choline transporter has been solubilized from synaptosomal membranes by various detergents. The solubilized carrier protein has been incorporated into liposomes after removal of the detergent by dialysis. Using the reconstitution of choline transport activity as an assay, the components catalyzing choline translocation were purified from the detergent extract by ion-exchange chromatography on a Mono-Q column followed by immunoaffinity chromatography. Monitoring the active fractions by sodium dodecylsulfate polyacrylamide gel electrophoresis and isoelectrofocussing gave one major protein with an apparent molecular weight of about 90,000 and an isoelectric point of pH 4.7. The isolated protein appeared to be heavily glycosylated as shown by lectin binding; upon treatment with endoglycosidase F the polypeptide was degraded to an apparent molecular weight of about 65,000. Accumulation of choline into liposomes reconstituted with the purified protein was driven by artificially imposed sodium gradients and inhibited by hemicholinium-3.

Muscarinic receptors modulating acetylcholine release from insect synaptosomes

1. Cholinergic synapses in the central nervous system of insects contain inhibitory muscarinic receptors whose stimulation by agonists leads to a diminished output of acetylcholine; antagonists, like atropine, facilitate acetylcholine release. 2. The receptors involved appear to be of the M2-subtype. Upon activation of presynaptic receptors a significant reduction of the intrasynaptosomal cyclic AMP level as well as a significantly increased membrane potential was observed. 3. The observed membrane hyperpolarization is apparently not a consequence of a lower cyclic AMP level, thus both effects may offer alternative or synergistical mechanisms for modulating transmitter release.

Choline fluxes in synaptosomal membrane vesicles

Summary1.Synaptic plasma membrane vesicles isolated from the highly cholinergic nervous tissue of insects were used to study the translocation of choline across the membrane via a high-affinity carrier-mediated mechanism energized by ion gradients as the sole driving force.2.The uphill movement of choline, energized mainly by the Na+ gradient, attained levels of choline severalfold the final equilibrium value at the peak of the overshoot.3.Efflux of choline required the presence of internal sodium ions and was promoted by external choline if Na+ was present. External choline inhibited choline efflux in the absence of sodium.4.It is concluded that the efflux of choline is in many aspects symmetrical with its uptake.

Cloning, sequencing and expression of locust tropomyosin

Abstract Several different clones which contain sequences complementary to the mRNA encoding tropomyosin were isolated from cDNA libraries prepared from locust RNA. Based on the sequence analysis of available clones, the complete primary structure of the locust tropomyosin was explored. The deduced protein sequence showed a repeating pattern of amino acid residues characteristic of a coiled-coil structure. The amino acid sequence of locust tropomyosin contains domains of complete homology but also regions of pronounced variability when compared with tropomyosins of other species. Northern blot as well as Western blot analysis revealed that different forms of tropomyosins are expressed in locust muscles.