Calcium-stores mediate adaptation in axon terminals of olfactory receptor neurons in Drosophila.

Background: In vertebrates and invertebrates, sensory neurons adapt to variable ambient conditions, such as the duration or repetition of a stimulus, a physiological mechanism considered as a simple form of non-associative learning and neuronal plasticity. Although various signaling pathways, as cAMP, cGMP, and the inositol 1,4,5-triphosphate receptor (InsP3R) play a role in adaptation, their precise mechanisms of action at the cellular level remain incompletely understood. Recently, in Drosophila, we reported that odor-induced Ca2+-response in axon terminals of olfactory receptor neurons (ORNs) is related to odor duration.

Presynaptic Ca2+ stores contribute to odor-induced responses in Drosophila olfactory receptor neurons.

In both vertebrates and invertebrates, olfactory receptor neurons (ORNs) respond to several odors. They also adapt to stimulus variations, and this is considered to be a simple form of non-associative learning and neuronal plasticity. Different mechanisms have been described to support neuronal and/or synaptic plasticity.

Non-invasive in vivo imaging of calcium signaling in mice.

Rapid and transient elevations of Ca(2+) within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca(2+) concentration ([Ca(2+)]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca(2+)-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms.

Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters.

Measurements of local Ca2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto-fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution.

Trachynilysin, a neurosecretory protein isolated from stonefish (Synanceia trachynis) venom, forms nonselective pores in the membrane of NG108-15 cells.

Trachynilysin, a protein toxin isolated from the venom of the stonefish Synanceia trachynis, has been reported to elicit massive acetylcholine release from motor nerve endings of isolated neuromuscular preparations and to increase both cytosolic Ca2+ and catecholamine release from chromaffin cells. In the present study, we used the patch clamp technique to investigate the effect of trachynilysin on the cytoplasmic membrane of differentiated NG108-15 cells in culture. Trachynilysin increased membrane conductance the most when the negativity of the cell holding membrane potential was reduced.

Effect of brefeldin A on acetylcholine release from glioma C6BU-1 cells.

The glial C6BU-1 cell line, loaded with acetylcholine can release this neurotransmitter. This study was aimed at determining whether disruption of the Golgi-vesicular traffic by brefeldin A would change the acetylcholine release from these cells and affect proteins involved in transmitter release like the 15 kDa proteolipid, common to V-ATPase and mediatophore. Cells were treated for 24 or 36 h with brefeldin A (35.

Developmental and adult expression of rat calcium-sensing receptor transcripts in neurons and oligodendrocytes.

The calcium-sensing receptor (CaSR) is a member of a growing family of heptahelical receptors with an unusually large extracellular domain. To further delineate its functions in neurons and glia, we have investigated the expression pattern of CaSR transcripts in the postnatal and adult rat brain, spinal cord and dorsal root ganglia by in situ hybridization. CaSR-expressing cells were spatially and temporally regulated in myelinated structures with a caudo-rostral pattern that paralleled that of myelin basic protein, a marker of myelination, with a downregulation observed in the adult.

Evoked acetylcholine release by immortalized brain endothelial cells genetically modified to express choline acetyltransferase and/or the vesicular acetylcholine transporter.

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT.

Cyclic ADP-ribose and calcium-induced calcium release regulate neurotransmitter release at a cholinergic synapse of Aplysia.

1. Presynaptic injection of cyclic ADP-ribose (cADPR), a modulator of the ryanodine receptor, increased the postsynaptic response evoked by a presynaptic spike at an identified cholinergic synapse in the buccal ganglion of Aplysia californica. 2.

Enhancement of quantal transmitter release and mediatophore expression by cyclic AMP in fibroblasts loaded with acetylcholine.

Neuronal properties such as neurotransmitter uptake and release can be expressed in non-neuronal cells. We show here that fibroblasts-mouse cell line L-M(TK-)-are able to take up acetylcholine from the external medium and to release it in response to a calcium influx. Release was assessed biochemically by a luminescence method, but it was also elicited from individual fibroblasts and recorded in real-time using a Xenopus myocyte as an acetylcholine detector.

Thrombin-induced membrane currents in native Xenopus follicles.

The presence and properties of thrombin receptors have been investigated in Xenopus Laevis follicles. In follicles, voltage-clamped at -60 mV or -80 mV, sub-micromolar concentrations of thrombin (Thr) induce smooth inward currents carried by chloride ions, which desensitize over tens of minutes. This desensitization is prevented by PKC inhibitors (staurosporine and H7).

A syntaxin-related protein controls acetylcholine release by different mechanisms in Aplysia.

Polyclonal antibodies raised against rat syntaxin-1B and an affinity-purified fraction have been used to study the functional role of this protein in transmitter release from Aplysia neurons. In a ganglionic protein extract, this fraction recognized a 37,000 molecular weight protein which therefore might be the Aplysia homologue of rat brain syntaxin-1B. Immunoglobulins were injected in the presynaptic cell of an identified cholinergic synapse of the buccal ganglion of Aplysia californica.

Characterization of a presynaptic glutamate receptor.

Glutamate receptors mediate excitatory neurotransmission in the brain and are important in the formation of memory and in some neurodegenerative disorders. A complementary DNA clone that encoded a 33-kilodalton protein (GR33) was obtained by screening a library with an antibody generated against glutamate binding proteins. The sequence of GR33 is identical to that of the recently reported presynaptic protein syntaxin.

In the GluR1 glutamate receptor subunit a glutamine to histidine point mutation suppresses inward rectification but not calcium permeability.

Recent papers have described glutamine to arginine point mutations of the cloned AMPA/Kainate receptor subunits that alter current-voltage relationship and suppress Ca2+ permeability, thus linking these two characteristics. We describe a glutamine to histidine mutation at the same position, which alters current-voltage relationship but retains Ca2+ permeability, thus dissociating the two properties.

Cloning and functional characterization of a novel mas-related gene, modulating intracellular angiotensin II actions.

The mas oncogene codes for a GTP binding protein-coupled receptor that determines a physiological response to angiotensin when expressed in Xenopus laevis oocytes or in the neuronal cell line NG115-401L. However, another gene, rat thoracic aorta gene, structurally related to mas, is devoid of any functional similarity with the angiotensin receptor(s). The relationships between the mas-related proteins and the angiotensin receptors were investigated by identifying and characterizing new members of the mas gene family.

Electrophysiological and pharmacological properties of GluR1, a subunit of a glutamate receptor-channel expressed in Xenopus oocytes.

A cDNA clone encoding an excitatory amino acid receptor was isolated from a rat brain cDNA library by Hollmann et al. (Nature, 342 (1989) 643-648). In Xenopus oocytes, this clone, GluR1, expressed a functional receptor-channel activated by kainate (KA), domoate (D), glutamate and quisqualate (QA).

Effect of low-density lipoproteins on Ca(2+)-dependent K+ channels in the membrane of smooth muscle cells isolated from human fetal aorta.

The effect of low-density lipoproteins (LDL) on the kinetics of Ca(2+)-dependent K+ channels was investigated in patch-clamp experiments with fetal smooth muscle cells (SMC) from human aorta cultured in normal medium (control SMC), on cells cultured in LDL-free medium for 24-48 h (LDL-SMC), and on cells from the preceding group that had been cultured for a further 24-48 h in a medium containing 50 micrograms ml-1 of LDL (LDL + SMC). Under identical conditions (at 50 mV, in 5 x 10(-7) M [Ca2+]i), the channel kinetics of control SMC and of LDL-SMC were the same, but for LDL + SMC the average time the channels were open (To) was either shorter or longer than in the control cells: To was about 30 ms in control SMC and around either 10 or 50 ms in LDL + SMC. For each group, a plot of log(To) versus membrane potential gave parallel lines, indicating that the voltage dependence remained unchanged.

Comparison of Ca2+-dependent K+ channels in the membrane of smooth muscle cells isolated from adult and foetal human aorta.

Ca2+-activated K+ ionic currents in the membrane of cultured smooth muscle cells isolated from foetal and adult human aorta were studied using whole cell and single-channel patch-clamp techniques. Whole cell currents in adult smooth muscle cells were 3-8 times larger than in foetal cells of similar sizes. The elementary conductance and ionic selectivity of single Ca2+-activated K+ were identical for both types of cells.

Arsenazo III transients and calcium current in a normally non-spiking neuronal soma of crayfish.

Arsenazo III was used to investigate Ca2+ transients in the normally non-excitable soma of the motor giant neurones of the crayfish Procambarus clarkii. Two kinds of regenerative potentials could be obtained depending on membrane potential conditioning: a fast spike after a pre-hyperpolarization to -90 mV and a slow action potential after a pre-depolarization to -50 mV. Only the second of these was accompanied by an Arsenazo III transient.

Cyclic adenosine monophosphate, calcium, acetylcholine and the current induced by adenosine in the Xenopus oocyte.

The K+ current response to bath-applied adenosine has been studied on follicle-enclosed full grown oocytes from Xenopus laevis, using the two electrodes voltage-clamp technique. The response to adenosine was mimicked by forskolin, an activator of adenylate cyclase. Forskolin applied at low concentration potentiated the response to adenosine.

Post-synaptic calcium influx at the giant synapse of the squid during activation by glutamate.

Changes in free calcium were monitored in the post-synaptic axon of the giant synapse of the squid, using the calcium indicators aequorin and Arsenazo III. The peak size of the calcium-dependent optical signals recorded from aequorin and Arsenazo III both showed a linear relation with the amount of calcium injected ionophoretically into the axon, but the Arsenazo signal had a slower time course than the aequorin. Ionophoretic application of glutamate to the post-synaptic axon depolarized the axon and caused a rise in intracellular free calcium.

beta-Adrenergic induced K+ current in Xenopus oocytes: role of cAMP, inhibition by muscarinic agents.

The K+ current induced by isoprenaline acting on beta-adrenergic receptors in Xenopus laevis has been studied in oocytes still surrounded by their follicular cells and inner ovarian epithelium. Forskolin, an adenylate cyclase activator, induced a similar K+ current and when used at subliminal concentration it potentiated the current induced by isoprenaline. Inhibition of phosphodiesterase by methylisobutylxanthine also enhanced the response to isoprenaline.