Biomedical subjects

H L Atwood

Publications and source records attributed to H L Atwood.

At least 19 recordsLinked to original sources

PubMed2006

Presynaptic effectors contributing to cAMP-induced synaptic potentiation in Drosophila.

cAMP analogs and activation of adenylyl cyclase by forskolin strongly potentiate synaptic transmission at the Drosophila neuromuscular junction. These effects are generally attributed to activation of cAMP-dependent protein kinase. Recent reports on crustacean and mammalian synapses have implicated other cAMP-dependent effectors in synaptic potentiation. Drosophila neuromuscular junctions were tested for effects of two known cAMP-dependent effectors: hyperpolarization-activated, cyclic nucleotide-regulated channels (HCNCs) and guanine nucleotide exchange protein activated by cAMP (Epac). Forskolin-induced enhancement of synaptic transmission was drastically reduced by a blocker of HCNCs, but not completely eliminated. A specific agonist for Epac modestly enhanced synaptic potentials. This agonist also stabilized their amplitudes in the presence of a blocker of HCNCs. The observations implicate HCNCs and Epac in cAMP-dependent potentiation that does not require cAMP-dependent protein kinase, indicating that additional previously unexplored factors contribute to synaptic plasticity in Drosophila. Genetic and molecular techniques available for Drosophila can be used to define the underlying molecular basis for cAMP-dependent synaptic potentiation.

Animals↗

PubMed2006

The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivation.

Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cac(ts2) mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cac(ts2) mutation occurs in the cytoplasmic carboxyl tail of the alpha1-subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+-imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cac(ts2) mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Ca(v)2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 degrees C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Ca(v)2.1 channel inactivation.

Amino Acid Sequence↗

PubMed2004

Turning behavior in Drosophila larvae: a role for the small scribbler transcript.

The Drosophila larva is extensively used for studies of neural development and function, yet the mechanisms underlying the appropriate development of its stereotypic motor behaviors remain largely unknown. We have previously shown that mutations in scribbler (sbb), a gene encoding two transcripts widely expressed in the nervous system, cause abnormally frequent episodes of turning in the third instar larva. Here we report that hypomorphic sbb mutant larvae display aberrant turning from the second instar stage onwards. We focus on the smaller of the two sbb transcripts and show that its pan-neural expression during early larval life, but not in later larval life, restores wild type turning behavior. To identify the classes of neurons in which this sbb transcript is involved, we carried out transgenic rescue experiments. Targeted expression of the small sbb transcript using the cha-GAL4 driver was sufficient to restore wild type turning behavior. In contrast, expression of this sbb transcript in motoneurons, sensory neurons or large numbers of unidentified interneurons was not sufficient. Our data suggest that the expression of the smaller sbb transcript may be needed in a subset of neurons for the maintenance of normal turning behavior in Drosophila larvae.

Age Factors↗

PubMed2003

Single neuron activity in the Drosophila larval CNS detected with calcium indicators.

Although the Drosophila larva has been extensively used for genetic studies of synaptic transmission and locomotion, neurophysiological studies have lagged because it is difficult to investigate circuitry and synaptic function in the larval central nervous system (CNS). Here we introduce an optical technique to monitor neuronal activity in the intact Drosophila larval CNS. We loaded neurons retrogradely through cut axons with dextran-conjugated calcium indicators. Fluorescence responses to changes in the concentration of intracellular calcium are sufficiently fast and large to monitor electrical activity in single neurons. Responses to action potentials were detected in motor neuron cell bodies, axons, neurites, dendrites and sensory neuron afferents identified by genetically targeted green fluorescent protein expression. Our findings provide an experimental procedure for testing synaptic function and connectivity within the intact larval CNS.

Animals↗

PubMed2002

Fast calcium signals in Drosophila motor neuron terminals.

Drosophila is a powerful model for neuroscientists, but physiological techniques have not kept pace with advances in molecular genetics. We introduce a reliable assay for intracellular calcium dynamics in Drosophila larval motor neuron terminals, and a new physiological solution that improves the longevity of the larval preparation. By loading calcium indicators into motor neuron terminals through cut axons, we obtained a high signal-to-noise ratio with confocal microscopy, and good temporal resolution of calcium-dependent fluorescence changes. We provide an estimate for the resting intracellular calcium concentration, the first description of calcium kinetics for a single action potential (AP), and improved resolution of calcium kinetics during AP trains. The very rapid decay of the calcium signal following a single AP (tau ~60 ms) indicates a previously unreported fast calcium extrusion mechanism in Drosophila motor neuron terminals well suited for sustaining physiological processes during the high rates of impulse activity which drive locomotor activity.

Animals↗

PubMed2001

Drosophila Hsc70-4 is critical for neurotransmitter exocytosis in vivo.

Previous in vitro studies of cysteine-string protein (CSP) imply a potential role for the clathrin-uncoating ATPase Hsc70 in exocytosis. We show that hypomorphic mutations in Drosophila Hsc70-4 (Hsc4) impair nerve-evoked neurotransmitter release, but not synaptic vesicle recycling in vivo. The loss of release can be restored by increasing external or internal Ca(2+) and is caused by a reduced Ca(2+) sensitivity of exocytosis downstream of Ca(2+) entry. Hsc4 and CSP are likely to act in common pathways, as indicated by their in vitro protein interaction, the similar loss of evoked release in individual and double mutants, and genetic interactions causing a loss of release in trans-heterozygous hsc4-csp double mutants. We suggest that Hsc4 and CSP cooperatively augment the probability of release by increasing the Ca(2+) sensitivity of vesicle fusion.

Abdomen↗

PubMed2000

Statistical methods for assessing the dimensions of synaptic vesicles in nerve terminals.

Chemical transmission between neurons occurs by the release of neurotransmitter packaged within vesicles of the presynaptic neuron onto a postsynaptic target. The amount of transmitter contained within a vesicle is in part regulated by the size of the vesicle. Thus, it is of general interest to quantify the dimension of vesicles in understanding the basic principles of chemical synaptic transmission. These vesicles can only be measured by electron microscopic techniques. Obtaining the true dimensions of synaptic structures is therefore complicated by stereological considerations. In this study, we suggest improved methods for determining the distributions (and mean sizes) for populations of vesicle diameters by mathematical processes involving (1) an implicit inversion of the empirical data distribution, (2) an explicit inversion approach, and (3) an approach based on substituting the empirical distribution into the inversion formula and then isotonizing using an iterated convex minorant algorithm. These procedures provide distributions that better represent the true population distributions (and means) for comparisons with other data sets of vesicle diameter measures.

Animals↗

PubMed2000

Assessing accurate sizes of synaptic vesicles in nerve terminals.

Chemical synaptic transmission occurs when vesicles within a presynaptic neuron fuse to the membrane and release their neurotransmitter content into the synaptic cleft, eliciting a response in the postsynaptic cell. If concentration of neurotransmitter is the same in all synaptic vesicles, the volume of the vesicle determines how much transmitter is released. Thus, variation in vesicular volume may contribute to observed variance of synaptic quantal unit size. The present study provides an approach to more fully and accurately characterize the dimensions of synaptic vesicles within a population containing varied sizes of vesicles. The methodology can be applied in a wide range of stereological problems. The approach characterizes the distribution of vesicle sizes within a population and provides a means to assess effects of experimental manipulations on vesicle dimensions. The mathematical treatments to obtain the true distribution of vesicle sizes involve extraction of the observed distribution from an enlarged population containing smaller vesicle diameters produced by sectioning of the specimens. A FORTRAN program is provided.

Algorithms↗

PubMed2000

Cysteine-string protein increases the calcium sensitivity of neurotransmitter exocytosis in Drosophila.

Previous studies suggest that the vesicular cysteine-string protein (CSP) may modulate presynaptic Ca(2+) channel activity in fast neurotransmitter release. To test this hypothesis, we analyzed the dynamics of presynaptic Ca(2+) ion influx with the Ca(2+) indicator fluo-4 AM at csp mutant neuromuscular junctions of Drosophila. From 24 to 30 degrees C, stimulus-evoked, relative presynaptic Ca(2+) signals were increasingly larger in csp mutant boutons than in controls. Above 30 degrees C, Ca(2+) signals declined and were similar to controls at 34 degrees C. A prolonged decay of Ca(2+) signals in mutant boutons at high temperatures indicated abnormally slow Ca(2+) clearance. Cytosolic Ca(2+) at rest was determined with the ratiometric Ca(2+) indicator fura-2 AM and was similar in mutant and control boutons at 24 degrees C but higher in mutant boutons at 34 degrees C. Despite larger Ca(2+) signals in mutant boutons, evoked neurotransmitter release was always reduced in csp mutants and exhibited pronounced facilitation. Thus, a lack of Ca(2+) entry cannot explain the reduction of neurotransmitter release in csp mutants. At all temperatures tested, raising extracellular Ca(2+) increased transmitter release elicited by single stimuli in csp mutants. Collectively, these data suggest multiple functions for CSP at synaptic terminals. Increased Ca(2+) signals coupled with reduced release suggest a direct function of CSP in exocytosis downstream from Ca(2+) entry. Because the reduction of evoked release in csp mutants is counteracted by increased Ca(2+) levels, we suggest that CSP primarily increases the Ca(2+) sensitivity of the exocytotic machinery.

Animals↗

PubMed2000

Role of cAMP cascade in synaptic stability and plasticity: ultrastructural and physiological analyses of individual synaptic boutons in Drosophila memory mutants.

Mutations of the genes rutabaga (rut) and dunce (dnc) affect the synthesis and degradation of cAMP, respectively, and disrupt learning in Drosophila. Combined ultrastructural analysis and focal electrophysiological recording in the larval neuromuscular junction revealed a loss of stability and fine tuning of synaptic structure and function in both mutants. Increased ratios of docked/undocked vesicles and poorly defined synaptic specializations characterized dnc synapses. In contrast, rut boutons possessed fewer, although larger, synapses with lower proportions of docked vesicles. At reduced Ca(2+) levels, decreased quantal content coupled with an increase in failure rate was seen in rut boutons and reduced pair-pulse facilitation were found in both rut and dnc mutants. At physiological Ca(2+) levels, strong enhancement, instead of depression, in evoked release was observed in some dnc and rut boutons during 10 Hz tetanus. Furthermore, increased variability of synaptic transmission, including fluctuation and asynchronicity of evoked release, paralleled an increase in synapse size variation in both dnc and rut boutons, which might impose problems for effective signal processing in the nervous system. Pharmacological and genetic studies indicated broader ranges of physiological alteration by dnc and rut mutations than either the acute effects of cAMP analogs or the available mutations that affect cAMP-dependent protein kinase (PKA) activity. This is consistent with previous reports of more severe learning defects in dnc and rut mutations than these PKA mutants and allows identification of the phenotypes involving long-term developmental regulation and those conferred by PKA.

Alleles↗

PubMed2000

Synaptic ultrastructure in nerve terminals of Drosophila larvae overexpressing the learning gene dunce.

We investigated synaptic ultrastructure of individual nerve ending varicosities at the Drosophila larval neuromuscular junction in transgenic larvae overexpressing the learning gene dunce (dnc) in the nervous system. It was previously shown that cAMP is reduced to one-third normal in these larvae and that they have fewer nerve terminal varicosities and smaller junction potentials, although transmitter release from individual nerve ending varicosities is not significantly altered. We tested the hypothesis that synaptic ultrastructure is modified to compensate for possible reduced efficacy of synaptic transmission resulting from lower than normal cAMP. Synaptic size and number of presynaptic dense bodies (active zone structures) per synapse are modestly enhanced in transgenic larvae overexpressing the dnc gene product and in rutabaga (rut(1)) mutant larvae, which have reduced adenylyl cyclase activity and reduced neural cAMP. The incidence of complex synapses (possessing 2 or more presynaptic dense bodies) was not consistently different in experimental larvae compared to controls. The observations suggest that chronic reduction of cAMP levels in the nervous system of Drosophila larvae, although leading to a modest compensatory change in synaptic structure, does not markedly alter several synaptic ultrastructural parameters which are thought to influence the strength of transmitter release; thus, homeostatic mechanisms do not act to maintain normal-sized junction potentials by altering synaptic structure.

Animals↗

PubMed2000

Neurotransmitter levels and synaptic strength at the Drosophila larval neuromuscular junction are not altered by mutation in the sluggish-A gene, which encodes proline oxidase and affects adult locomotion.

The sluggish-A (slgA) gene of Drosophila melanogaster has been shown to encode for the enzyme proline oxidase, a mitochondrial enzyme which catalyzes the first step in the conversion of L-proline to L-glutamate. The slgA transcript is expressed in both larval and adult Drosophila melanogaster. Mutations in this gene lead to reduced proline oxidase activity and an elevation of free proline levels. Adult mutant flies show a striking reduction of motor activity. Since proline oxidase may contribute to the supply of the neurotransmitter glutamate in the nervous system, a reduction in proline oxidase activity could reduce neural glutamate pools and affect synaptic transmission in neurons utilizing glutamate as a transmitter, including peripheral motor neurons. We tested the hypothesis that glutamate, and synaptic transmission mediated by glutamate, are reduced at synapses of glutamatergic motor neurons in slgA mutants. Levels of glutamate and proline in different cell compartments, and functional properties of synaptic transmission were compared in slgA and control specimens. Proline is elevated in muscle cells of slgA mutants, indicating that the slgA gene regulates tissue proline levels. In nerve terminal varicosities, proline levels were low in both mutants and controls. Glutamate levels in nerve terminal varicosities of slgA mutants and controls were similar. In addition, we found that glutamatergic synaptic transmission at individual nerve endings and at the whole-cell level was similar in slgA mutants and controls. Thus, proline oxidase does not play a major role in generating neuronal glutamate pools at the Drosophila larval neuromuscular junction, and larval neuromuscular performance is not altered significantly in slgA mutants. Metabolic pathways other than that involving proline oxidase are able to sustain glutamatergic synaptic function in Drosophila larvae.

Animals↗

PubMed1999

Crustacean frequenins: molecular cloning and differential localization at neuromuscular junctions.

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium-binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq-like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin- and dynamin-like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq-like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium-binding regions (EF hands) are conserved. The widespread occurrence of frq-like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons.

Animals↗

PubMed1999

Calcium entry related to active zones and differences in transmitter release at phasic and tonic synapses.

Synaptic functional differentiation of crayfish phasic and tonic motor neurons is large. For one impulse, quantal release of neurotransmitter is typically 100-1000 times higher for phasic synapses. We tested the hypothesis that differences in synaptic strength are determined by differences in synaptic calcium entry. Calcium signals were measured with the injected calcium indicator dyes Calcium Green-1 and fura-2. Estimated Ca(2+) entry increased almost linearly with frequency for both axons and was two to three times larger in phasic terminals. Tonic terminal Ca(2+) at 10 Hz exceeded phasic terminal Ca(2+) at 1 Hz, yet transmitter release was much higher for phasic terminals at these frequencies. Freeze-fracture images of synapses revealed on average similar numbers of prominent presynaptic active zone particles (putative ion channels) for both neurons and a two- to fourfold phasic/tonic ratio of active zones per terminal volume. This can account for the larger calcium signals seen in phasic terminals. Thus, differences in synaptic strength are less closely linked to differences in synaptic channel properties and calcium entry than to differences in calcium sensitivity of transmitter release.

Animals↗

PubMed1999

Neuroprotection at Drosophila synapses conferred by prior heat shock.

Synapses are critical sites of information transfer in the nervous system, and it is important that their functionality be maintained under stressful conditions to prevent communication breakdown. Here we show that synaptic transmission at the Drosophila larval neuromuscular junction is protected by prior exposure to heat shock that strongly induces expression of heat shock proteins, in particular hsp70. Using a macropatch electrode to record synaptic activity at individual, visualized boutons, we found that prior heat shock sustains synaptic performance at high test temperatures through pre- and postsynaptic alterations. After heat shock, nerve impulses release more quantal units at high temperatures and exhibit fewer failures of release (presynaptic modification), whereas the amplitude of quantal currents remains more constant than does that in nonheat-shocked controls (postsynaptic modification). The time course of these physiological changes is similar to that of elevated hsp70. Thus, stress-induced neuroprotective mechanisms maintain function at synapses by modifying their properties.

Analysis of Variance↗

PubMed1999

Drosophila larval neuromuscular junction's responses to reduction of cAMP in the nervous system.

We investigated the effects of chronically lowered cyclic adenosine monophosphate (cAMP) on the morphology and physiology of the Drosophila larval neuromuscular junction, using two fly lines in which cAMP was significantly lower than normal in the nervous system: (a) transgenic flies in which the dunce (dnc) gene product was overexpressed in the nervous system, and (b) flies mutant for the rutabaga gene (rut1) which have reduced adenylyl cyclase activity. In comparison with controls, larvae with reduced cAMP exhibited a smaller number of synaptic varicosities. This effect was more pronounced in transgenic larvae, in which the reduction of neural cAMP was more pronounced. Synaptic transmission was also reduced in both cases, as evidenced by smaller excitatory junctional potentials (EJPs). Synaptic currents recorded from individual synaptic varicosities of the neuromuscular junction indicated almost normal transmitter release properties in transgenic larvae and a modest impairment in rut1 larvae. Thus, reduction in EJP amplitude in transgenic larvae is primarily due to reduced innervation, while in rut1 larvae it is attributable to the combined effects of reduced innervation and a mild impairment of transmitter release. We conclude that the major effect of chronically lowered cAMP is reduction of innervation rather than impairment of transmitter release properties.

Adenylyl Cyclases↗

PubMed1999

Visible evidence for differences in synaptic effectiveness with activity-dependent vesicular uptake and release of FM1-43.

Activity-dependent uptake and release of the fluorescent probe FM1-43 were used to compare synaptic performance (rates of transmitter release and synaptic vesicle turnover) at different frequencies in phasic and tonic motor neurons innervating the crayfish leg extensor muscle and in the tonic motor neuron of the opener muscle. The phasic extensor motor neuron, which has a high quantal content of transmitter release, accumulated and released FM1-43 more rapidly than the tonic motor neuron, especially at low frequencies of stimulation. Individual bright spots appeared on the varicosities of the junctional terminals during stimulation in FM1-43; these spots corresponded to zones of immunostaining for the synaptic vesicle associated protein synaptotagmin, but they were larger and less numerous than synapses identified by electron microscopy and appear to represent one to several synapses with their associated clusters of synaptic vesicles. The number of bright spots observed on varicosities of the tonic terminal after stimulation at >/=20 Hz is generally similar to values for responding units (n) calculated from binomial distributions derived from quantal analysis. At frequencies of </=10 Hz, bright spots did not usually appear on tonic extensor varicosities, and the quantal release patterns were best fitted with Poisson distributions. Another tonic motor neuron, the excitor of the opener muscle, showed individual bright spots at lower frequencies of stimulation, consistent with its higher quantal output at these frequencies and corresponding with the binomial fits for quantal release distributions. In this axon, the number of distinctive bright spots increased with frequency in the 2- to 20-Hz range, indicating increased participation of synapses during frequency facilitation. In the tonic extensor neuron terminals, the brightness and the size of the individual spots increased with frequency, and new foci of dye uptake appeared at the edges of preexisting spots. Relative intensity change varied considerably among individual spots during dye loading at different frequencies. Similarly, individual spots on a single tonic terminal destained at different rates when stimulated after previous loading with FM1-43. These results suggest differential performance of individual synapses or small groups of synapses, some being more effective in transmitter release than others, as inferred from previous ultrastructural and quantal analysis studies. The large overall differences between phasic and tonic synapses suggest differential regulation of transmitter release at individual synapses in the two neurons.

Animals↗

PubMed1999

Quantal unit populations at the Drosophila larval neuromuscular junction.

Focal extracellular recording at visualized boutons of the Drosophila larval neuromuscular junction was used to determine frequency and time course of the spontaneously occurring quantal events. When simultaneous intracellular recordings from the innervated muscle cell were made, more than one class of quantal event occurred at some of the individual boutons. "True" signals (arising at the bouton within the focal macropatch electrode) were often contaminated by additional signals generated outside the lumen of the focal electrode. Inclusion of these contaminating signals gave spuriously low values for relative amplitude, and spuriously high values for spontaneous quantal emission, for the synapses within the focal electrode. The contaminating signals, which appeared to be conducted along the subsynaptic reticulum surrounding the nerve terminals, generally were characterized by relatively small extracellular signals associated with normal intracellular events in the muscle fiber. From plots of simultaneous extracellular and intracellular recordings, the individual data points were classified according to the angles they subtended with the x axis (extracellular signal axis). Statistical procedures were developed to separate the true signals and contaminants with a high level of confidence. Populations of quantal events were found to be well described by Gaussian mixtures of two or three components, one of which could be characterized as the true signal population. Separation of signals from contaminants provides a basis for improving the estimates of quantal size and spontaneous frequency for the synapses sampled by the focal extracellular electrode.

Animals↗