Single calcium channels on a cholinergic presynaptic nerve terminal.

The calyx-type synapse of the chick ciliary ganglion was used to examine single calcium channels in a vertebrate cholinergic presynaptic nerve terminal by means of the cell-attached, patch-clamp technique. Calcium channels were recorded on the internal, transmitter-release face of the nerve terminal, but were not detected on the external face. These channels were recruited at -30 mV, with maximum activation at about +30 mV, and were sometimes clustered at high densities. Single-channel conductance estimates with voltage-pulse or -ramp techniques gave values of 11-14 pS with 110 mM barium, which is in the intermediate, N-type range for calcium channels on a control neuron. This nerve terminal calcium channel, termed the NPT-type, may link action potentials to transmitter release at many vertebrate fast-transmitting synapses.     

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom- induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.     

Nicotinic acetylcholine receptors on a cholinergic nerve terminal in the cockroach,Periplaneta americana

Summary Intracellular microelectrode recording and ionophoretic application of carbamylcholine (CCh) were used to compare the cholinergic sensitivity of postsynaptic dendrites of an identified neurone with that of an identified presynaptic cholinergic axon.The axon of the lateral filiform hair sensory neurone (LFHSN) in the first-instar cockroachPeriplaneta americana was found to be as sensitive to CCh as the dendritic regions of giant interneurone 3 (GI 3). The CCh response of both neurones was unaffected by replacing Ca²⁺ with Mg²⁺, confirming that the ACh receptors are present on the neurones under test. The CCh response of both neurones was mimicked by ionophoretic application of nicotine. The responses were blocked by 10^–5 M mecamylamine and 10^–6 M d-tubocurarine and were not affected by muscarinic antagonists, suggesting that the ACh receptors present on GI 3 and LFHSN are predominantly nicotinic.The muscarinic agonist oxotremorine and the antagonists atropine and quinuclidinyl benzilate had no modulatory effect on LFHSN-GI 3 synaptic transmission.The latency of the LFHSN response to CCh was consistent with the hypothesis that ACh receptors are situated on the main axon/terminal within the neuropil of the ganglion. It has previously been shown that this region of the axon does not form output synapses (Blagburn et al. 1985a). This indirect evidence indicates that presynaptic or extrasynaptic ACh receptors are present in the membrane of a cholinergic axon.LFHSN was depolarized by synaptically-released ACh after normal or evoked spike bursts, suggesting that the nicotinic ACh receptors act as autoreceptors. However, it was not possible to obtain direct evidence to support the hypothesis that these receptors modulate ACh release.Abbreviations CCh carbamylcholine - GI giant interneurone - FHSN filiform hair sensory neurone - LFHSN lateral filiform hair sensory neurone - R _in input resistance - V depolarization - V _m resting potential     

Genetic mosaic analysis reveals FGF receptor 2 function in terminal end buds during mammary gland branching morphogenesis

FGF signaling is associated with breast cancer and is required for mammary placode formation in the mouse. In this study, we employed a genetic mosaic analysis based on Cre-mediated recombination to investigate FGF receptor 2 (Fgfr2) function in the postnatal mammary gland. Mosaic inactivation of Fgfr2 by the MMTV-Cre transgene enabled us to compare the behavior of Fgfr2 null and Fgfr2 heterozygous cells in the same gland. Fgfr2 null cells were at a competitive disadvantage to their Fgfr2 heterozygous neighbors in the highly proliferative terminal end buds (TEBs) at the invasion front, owing to a negative effect of loss of Fgfr2 function on cell proliferation. However, Fgfr2 null cells were tolerated in mature ducts. In these genetic mosaic mammary glands, the epithelial network is apparently built by TEBs that over time are composed of a progressively larger proportion of Fgfr2-positive cells. However, subsequently, most cells lose Fgfr2 function, presumably due to additional rounds of Cre-mediated recombination. Using an independent strategy to create mosaic mammary glands, which employed an adenovirus-Cre that acts only once, we confirmed that Fgfr2 null cells were out-competed by neighboring Fgfr2 heterozygous cells. Together, our data demonstrate that Fgfr2 functions in the proliferating and invading TEBs, but it is not required in the mature ducts of the pubertal mammary gland.     

Renin-angiotensin system inhibition on noradrenergic nerve terminal function in pacing-induced heart failure

Chronic angiotensin-converting enzyme (ACE) inhibition has been shown to improve cardiac sympathetic nerve terminal function in heart failure. To determine whether similar effects could be produced by angiotensin II AT(1) receptor blockade, we administered the ACE inhibitor quinapril, angiotensin II AT(1) receptor blocker losartan, or both agents together, to rabbits with pacing-induced heart failure. Chronic rapid pacing produced left ventricular dilation and decline of fractional shortening, increased plasma norepinephrine (NE), and caused reductions of myocardial NE uptake activity, NE histofluorescence profile, and tyrosine hydroxylase immunostained profile. Administration of quinapril or losartan retarded the progression of left ventricular dysfunction and attenuated cardiac sympathetic nerve terminal abnormalities in heart failure. Quinapril and losartan together produced greater effects than either agent alone. The effect of renin-angiotensin system inhibition on improvement of left ventricular function and remodeling, however, was not sustained. Our results suggest that the effects of ACE inhibitors are mediated via the reduction of angiotensin II and that angiotensin II plays a pivotal role in modulating cardiac sympathetic nerve terminal function during development of heart failure. The combined effect of ACE inhibition and angiotensin II AT(1) receptor blockade on cardiac sympathetic nerve terminal dysfunction may contribute to the beneficial effects on cardiac function in heart failure.     

Disruption of vagal efferent axon and nerve terminal function in the postischemic myocardium

Despite the importance of vagal control over the ventricle, little is known regarding vagal efferent conduction and nerve terminal function in the postischemic myocardium. To elucidate postischemic changes in the cardiac vagal efferent neuronal function, we measured myocardial interstitial acetylcholine (ACh) levels by using in vivo cardiac microdialysis and examined the ACh responses to electrical stimulation of the vagi or local administration of ouabain in anesthetized cats. Sixty-minute occlusions of the left anterior descending coronary artery (LAD) followed by 60-min reperfusion abolished electrical stimulation-induced ACh release (20.4 +/- 3.9 vs. 0.9 +/- 0.4 nmol/l; means +/- SE, P < 0.01). In different groups of animals, 60-min LAD occlusion followed by 60-min reperfusion decreased but did not completely abolish ouabain-induced release of ACh (9.2 +/- 1.8 vs. 3.9 +/- 0.7 nmol/l; P < 0.05). These results indicate that function of the vagal efferent axon was completely interrupted, whereas the local ACh release was partially suppressed in the postischemic myocardium. The postischemic disruption of vagal efferent neuronal function might exert deleterious effects on cardiac regulation.     

Silicotungstic acid for cytochemical localization of water soluble substance(s) of cholinergic motor nerve terminal

Summary Silicotungstic acid (STA), an electron dense substance and a powerful precipitating agent of quaternary ammonium salts such as choline and acetylcholine, was employed on the frog motor end-plate in order to prove that STA reacts with diffusible substance(s) in nerve terminals. Thus, STA treatment and osmium tetroxide (OsO₄) fixation were performed in three different ways. No reaction was detectable when STA treatment followed osmification, while simultaneous treatment with STA and OsO₄ darkened both presynaptic and synaptic vesicle membranes. When STA was employed directly on fresh tissues which were subsequently fixed by OsO₄, small black precipitates were observed in the synaptic vesicles and none on other synaptic structures. The possible reaction of STA with acetylcholine is discussed.     

Silicotungstic acid for cytochemical localization of water soluble substance(s) of cholinergic motor nerve terminal

Silicotungstic acid (STA), an electron dense substance and a powerful precipitating agent of quaternary ammonium salts such as choline and acetylcholine, was employed on the frog motor end-plate in order to prove that STA reacts with diffusible substance(s) in nerve terminals. Thus, STA treatment and osmium tetroxide (OsO4) fixation were performed in three different ways. No reaction was detectable when STA treatment followed osmification, while simultaneous treatment with STA and OsO4 darkened both presynaptic and synaptic vesicle membranes. When STA was employed directly on fresh tissues which were subsequently fixed by OsO4, small black precipitates were observed in the synaptic vesicles and none on other synaptic structures. The possible reaction of STA with acetylcholine is discussed.     

Genetic analysis of clathrin function in yeast

The use of yeast mutants to study the function and dynamics of clathrin-coated membranes has offered new insights into clathrin's role in the secretory pathway and has raised additional questions. Most strains of yeast can incur a disruption of clathrin heavy or light chain genes and remain viable. However, in rare cases, alleles of genes other than clathrin affect the viability of clathrin-deficient cells. The relationship of the products of these genes to clathrin awaits clarification. Phenotypic characterization of clathrin-deficient yeast mutants suggests that clathrin is not essential for the generation of secretory pathway transport vesicles at the ER or the Golgi complex but is required for the intracellular retention of a Golgi membrane protein, Kex2p. With this genetic evidence for clathrin's function in vivo, biochemical and genetic experiments can be designed to address the mechanism by which clathrin effects retention of Kex2p. Clathrin-deficient yeast carry out protein secretion, receptor-mediated endocytosis of mating pheromone, and efficient targeting of newly synthesized vacuolar proteins. These observations challenge aspects of clathrin's proposed involvement in protein transport through the secretory pathway and to lysosomes in mammalian cells. However, the differences are beginning to recede in the face of additional experiments; the formation of clathrin coated vesicles is no longer commonly thought to be obligately coupled to transport through the secretory pathway in mammalian cells (Rothman 1986; Brodsky, 1988), and the role of clathrin in retaining a Golgi membrane protein in yeast may have its precedents in receptor-mediated endocytosis by mammalian cells or in secretory granule formation in endocrine cells. A unified theory of clathrin function is emerging (Brodsky, 1988) which suggests that the clathrin coat assemblage (clathrin heavy and light chains and the associated proteins) acts as a facilitator of intracellular protein transport by sorting and concentrating cargo molecules. The results from studies of clathrin-deficient yeast support this theory. Future experiments will determine whether clathrin provides its functions at different transport stages in different organisms or whether all eukaryotic cells employ clathrin at the same stages of intracellular protein transport.     

Choline mustard: an irreversible ligand for use in studies of choline transport mechanisms at the cholinergic nerve terminal

It has been shown in our laboratory that choline mustard aziridinium ion is a potent and irreversible inhibitor of choline transport into rat brain synaptosomes; this compound showed selectivity for the sodium-dependent, high affinity carrier in that it was 30 times more potent as an inhibitor when compared with the effect on sodium-independent, low affinity choline uptake. In the present study, this mustard analogue did not inhibit synaptosomal uptake of 5-hydroxytryptamine, noradrenaline, or gamma-aminobutyric acid, thereby confirming further the specificity of this compound for the choline carrier. Studies of the effect of depolarization of the nerve terminals on the inactivation of choline carriers by choline mustard were performed. It was determined that alkylation of the carrier was significantly increased in nerve endings previously depolarized. The enhancing effect of depolarization on choline transport velocity and on the alkylation of choline carriers by choline mustard was dependent upon the presence of sodium in the external medium. Possible mechanisms for the enhanced inactivation of choline carriers by choline mustard aziridinium ion are proposed, and kinetic interactions of choline mustard with the high affinity choline carrier and with choline acetyltransferase are reviewed and discussed.     

Immunohistochemical localization of cholinergic nerve terminals

Summary Most of the published light-microscopic methods for the localization of cholinergic nerve pathways present various difficulties of interpretation. The production and characterization of an antiserum that binds specifically to cholinergic terminals is described. The antiserum was raised to small synaptosomes prepared from the purely cholinergic electric organ of Torpedo marmorata. It was shown to lyse cholinergic synaptosomes in a mixed population derived from guinea-pig cortex. After partial purification by adsorption onto nonspecific antigens, it was used to label nerve endings in several tissues of Torpedo, rats and guinea pigs using indirect immunofluorescence histochemistry. The antiserum appears to provide a highly specific means of localizing cholinergic nerve endings in these tissues.     

ATP citrate lyase in cholinergic nerve endings

The activity of ATP-citrate lyase in homogenates of five selected rat brain regions varied from 2.93 to 6.90 nmol/min/mg of protein in the following order: cerebellum < hippocampus < parietal cortex < striatum < medulla oblongata and that of the choline acetyltransferase from 0.15 to 2.08 nmol/min/mg of protein in cerebellum < parietal cortex < hippocampus=medulla oblongata < striatum. No substantial differences were found in regional activities of lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase or acetyl-CoA synthase. High values of relative specific activities for both choline acetyltransferase and ATP-citrate lyase were found in synaptosomal and synaptoplasmic fractions from regions with a high content of cholinergic nerve endings. There are significant correlations between these two enzyme activities in general cytocol (S₃), synaptosomal (B) and synaptoplasmic (B_s) fractions from the different regions (r=0.92–0.99). These data indicate that activity of ATP-citrate lyase in cholinergic neurons is several times higher than that present in glial and noncholinergic neuronal cells.     

Genetic analysis of Drosophila larval optic nerve development.

To identify genes necessary for establishing connections in the Drosophila sensory nervous system, we designed a screen for mutations affecting development of the larval visual system. The larval visual system has a simple and stereotypic morphology, can be recognized histologically by a variety of techniques, and is unnecessary for viability. Therefore, it provides an opportunity to identify genes involved in all stages of development of a simple, specific neuronal connection. By direct observation of the larval visual system in mutant embryos, we identified 24 mutations affecting its development; 13 of these are larval visual system-specific. These 13 mutations can be grouped phenotypically into five classes based on their effects on location, path or morphology of the larval visual system nerves and organs. These mutants and phenotypic classifications provide a context for further analysis of neuronal development, pathfinding and target recognition.     

Cellular analysis of appetitive learning in invertebrates

In recent years significant progress has been made in the analysis of the cellular mechanisms underlying appetitive learning in two invertebrate species, the pond snail Lymnaea stagnalis and the honeybee Apis mellifera. In Lymnaea, both chemical (taste) and tactile appetitive conditioning paradigms were used and cellular traces of behavioural classical conditioning were recorded at several specific sites in the nervous system. These sites included sensory pathways, central pattern generator and modulatory interneurones as well as motoneurones of the feeding network. In the honeybee, a chemical (odour) appetitive conditioning paradigm resulted in cellular changes at different sites in the nervous system. In both the pond snail and the honeybee the activation of identified modulatory interneurones could substitute for the use of the chemical unconditioned stimulus, making these paradigms even more amenable to more detailed cellular and molecular analysis.     

Genetic analysis of cadherin function in animal morphogenesis.

Cadherins are a superfamily of Ca(2+)-dependent adhesion molecules found in metazoans. Several classes of cadherins have been defined from which two - classic cadherins and Fat-like cadherins - have been studied by genetic approaches. Recent in vivo studies in Caenorhabditis elegans and Drosophila show that cadherins play an active role in a number of distinct morphogenetic processes. Classic cadherins function in epithelial polarization, epithelial sheet or tube fusion, cell migration, cell sorting, and axonal patterning. Fat-like cadherins are required for epithelial morphogenesis, proliferation control, and epithelial planar polarization.