Search PubMed⌕ Search

Biomedical subjects

Harold L Atwood

Publications and source records attributed to Harold L Atwood.

16 recordsLinked to original sources
PubMed2007

Morphological and functional effects of altered cysteine string protein at the Drosophila larval neuromuscular junction.

The synaptic vesicle-associated cysteine string protein (CSP) is critical for neurotransmitter release at the neuromuscular junction (NMJ) of Drosophila, where the approximately 4% of mutant flies lacking CSP that survive to adulthood exhibit spastic jumping and shaking, temperature-sensitive paralysis, and premature death. Previously, it has been shown that CSP is also required for nerve terminal growth and the prevention of neurodegeneration in Drosophila and mice. At larval csp null mutant NMJs of Drosophila, intracellular recordings from the muscle showed that evoked release is significantly reduced at room temperature. However, it remained unclear whether the reduction in evoked release might be due to a loss of synaptic boutons, loss of synapses, and alterations in trafficking of vesicles to synapses. To resolve these issues, we have examined synaptic structure and function of csp null mutant NMJs at the level of single boutons. csp null mutations proportionally reduce the number of synaptic boutons of both motor neurons (1s and 1b) innervating larval muscles 6 and 7, while the number of synapses per bouton remains normal. However, focal recordings from individual synaptic boutons show that nerve-evoked neurotransmitter release is also impaired in both 1s and 1b boutons. Further, our ultrastructural analyses show that the reduction in evoked release at low stimulation frequencies is not due to a loss of synapses or to alterations in docked vesicles at synapses. Together, these data suggest that CSP promotes synaptic growth and evoked neurotransmitter release by mechanistically independent signaling pathways.

Animals↗
PubMed2006

Modular neuropile organization in the Drosophila larval brain facilitates identification and mapping of central neurons.

Elucidating how neuronal networks process information requires identification of critical individual neurons and their connectivity patterns. For this purpose, we used the third-instar Drosophila larval brain and applied reverse-genetic tools, immunolabeling procedures, and 3D digital reconstruction software. Consistent topological definition of neuropile compartments in the larval brain can be obtained through simple fluorescence-immunolabeling methods. The modular neuropiles can be used as a fiducial framework for mapping the projection patterns of individual neurons labeled with green fluorescent protein (GFP). GFP-labeled neurons often exhibit dendrite-like arbors as well as clustered varicose terminals on neurite branches that innervate identifiable neuropile compartments. We identified candidate cholinergic interneurons in genetic mosaic brains that overlap with the larval optic nerve terminus. By using the neuropile framework, we demonstrate that the candidate visual interneurons are not a subset of the previously identified circadian pacemaker neurons that also contact the larval optic nerve terminus; they may represent parallel pathways in the processing of visual inputs. Thus, in the Drosophila larval brain, modular neuropiles can be used as a framework for systematically identifying, mapping, and classifying interneurons; understanding their roles in behavior can then be pursued further.

Animals↗
PubMed2006

Thermoprotection of synaptic transmission in a Drosophila heat shock factor mutant is accompanied by increased expression of Hsp83 and DnaJ-1.

In Drosophila larvae, acquired synaptic thermotolerance after heat shock has previously been shown to correlate with the induction of heat shock proteins (Hsps) including HSP70. We tested the hypothesis that synaptic thermotolerance would be significantly diminished in a temperature-sensitive strain (Drosophila heat shock factor mutant hsf4), which has been reported not to be able to produce inducible Hsps in response to heat shock. Contrary to our hypothesis, considerable thermoprotection was still observed at hsf4 larval synapses after heat shock. To investigate the cause of this thermoprotection, we conducted DNA microarray experiments to identify heat-induced transcript changes in these organisms. Transcripts of the hsp83, dnaJ-1 (hsp40), and glutathione-S-transferase gstE1 genes were significantly upregulated in hsf4 larvae after heat shock. In addition, increases in the levels of Hsp83 and DnaJ-1 proteins but not in the inducible form of Hsp70 were detected by Western blot analysis. The mode of heat shock administration differentially affected the relative transcript and translational changes for these chaperones. These results indicate that the compensatory upregulation of constitutively expressed Hsps, in the absence of the synthesis of the major inducible Hsp, Hsp70, could still provide substantial thermoprotection to both synapses and the whole organism.

Acclimatization↗
PubMed2005

The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses.

We have identified EMS-induced mutations in Drosophila Miro (dMiro), an atypical mitochondrial GTPase that is orthologous to human Miro (hMiro). Mutant dmiro animals exhibit defects in locomotion and die prematurely. Mitochondria in dmiro mutant muscles and neurons are abnormally distributed. Instead of being transported into axons and dendrites, mitochondria accumulate in parallel rows in neuronal somata. Mutant neuromuscular junctions (NMJs) lack presynaptic mitochondria, but neurotransmitter release and acute Ca2+ buffering is only impaired during prolonged stimulation. Neuronal, but not muscular, expression of dMiro in dmiro mutants restored viability, transport of mitochondria to NMJs, the structure of synaptic boutons, the organization of presynaptic microtubules, and the size of postsynaptic muscles. In addition, gain of dMiro function causes an abnormal accumulation of mitochondria in distal synaptic boutons of NMJs. Together, our findings suggest that dMiro is required for controlling anterograde transport of mitochondria and their proper distribution within nerve terminals.

Animals↗
PubMed2005

AP180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca2+-triggered exocytosis.

AP180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been implicated in retrieving SV proteins. In Drosophila, deletion of its homologue, Like-AP180 (LAP), has been shown to increase the size of SVs but decrease the number of SVs and transmitter release. However, it remains elusive whether a reduction in the total vesicle pool directly affects transmitter release. Further, it is unknown whether the lap mutation also affects vesicle protein retrieval and synaptic protein localization and, if so, how it might affect exocytosis. Using a combination of electrophysiology, optical imaging, electron microscopy, and immunocytochemistry, we have further characterized the lap mutant and hereby show that LAP plays additional roles in maintaining both normal synaptic transmission and protein distribution at synapses. While increasing the rate of spontaneous vesicle fusion, the lap mutation dramatically reduces impulse-evoked transmitter release at steps downstream of calcium entry and vesicle docking. Notably, lap mutations disrupt calcium coupling to exocytosis and reduce calcium cooperativity. These results suggest a primary defect in calcium sensors on the vesicles or on the release machinery. Consistent with this hypothesis, three vesicle proteins critical for calcium-mediated exocytosis, synaptotagmin I, cysteine-string protein, and neuronal synaptobrevin, are all mislocalized to the extrasynaptic axonal regions along with Dap160, an active zone marker (nc82), and glutamate receptors in the mutant. These results suggest that AP180 is required for either recycling vesicle proteins and/or maintaining the distribution of both vesicle and synaptic proteins in the nerve terminal.

Animals↗
PubMed2005

Calcium sensitivity of neurotransmitter release differs at phasic and tonic synapses.

The efficacy of synaptic transmission varies greatly among synaptic contacts. We have explored the origins of differences between phasic and tonic crustacean neuromuscular junctions. Synaptic boutons of a phasic motor neuron release three orders of magnitude more quanta to a single action potential and show strong depression to a train, whereas tonic synapses are nearly unresponsive to single action potentials and display an immense facilitation. Phasic and tonic synapses display a similar nonlinear dependence on extracellular [Ca2+]. We imposed similar spatially uniform intracellular [Ca2+] ([Ca2+]i) steps in phasic and tonic synapses by photolysis of presynaptic caged calcium. [Ca2+]i was measured fluorometrically while transmitter release was monitored electrophysiologically from single boutons in which the [Ca2+]i was elevated. Phasic synapses released the readily releasable pool (RRP) of vesicles at a much higher rate and with a shorter delay than did tonic synapses. Comparison of several kinetic models of molecular events showed that a difference in Ca2+-sensitive priming of vesicles in the RRP combined with a revision of the kinetic Ca2+-binding sequence to the secretory trigger produced the best fit to the markedly different responses to Ca2+ steps and action potentials and of the characteristic features of synaptic plasticity in phasic and tonic synapses. The results reveal processes underlying one aspect of synaptic diversity that may also regulate changes in synaptic strength during development and learning and memory formation.

Animals↗
PubMed2005

The multiple functions of cysteine-string protein analyzed at Drosophila nerve terminals.

The synaptic vesicle-associated cysteine-string protein (CSP) is important for synaptic transmission. Previous studies revealed multiple defects at neuromuscular junctions (NMJs) of csp null-mutant Drosophila, but whether these defects are independent of each other or mechanistically linked through J domain mediated-interactions with heat-shock cognate protein 70 (Hsc70) has not been established. To resolve this issue, we genetically dissected the individual functions of CSP by an in vivo structure/function analysis. Expression of mutant CSP lacking the J domain at csp null-mutant NMJs fully restored normal thermo-tolerance of evoked transmitter release but did not completely restore evoked release at room temperature and failed to reverse the abnormal intraterminal Ca2+ levels. This suggests that J domain-mediated functions are essential for the regulation of intraterminal Ca2+ levels but only partially required for regulating evoked release and not required for protecting evoked release against thermal stress. Hence, CSP can also act as an Hsc70-independent chaperone protecting evoked release from thermal stress. Expression of mutant CSP lacking the L domain restored neurotransmission and partially reversed the abnormal intraterminal Ca2+ levels, suggesting that the L domain is important, although not essential, for the role of CSP in regulating intraterminal Ca2+ levels. We detected no effects of csp mutations on individual presynaptic Ca2+ signals triggered by action potentials, suggesting that presynaptic Ca2+ entry is not primarily impaired. Both the J and L domains were also required for the role of CSP in synaptic growth. Together, these results suggest that CSP has several independent synaptic functions, affecting synaptic growth, evoked release, thermal protection of evoked release, and intraterminal Ca2+ levels at rest and during stimulation.

Analysis of Variance↗
PubMed2004

Synaptic vesicles: test for a role in presynaptic calcium regulation.

Membrane-bound organelles such as mitochondria and the endoplasmic reticulum play an important role in neuronal Ca(2+) homeostasis. Synaptic vesicles (SVs), the organelles responsible for exocytosis of neurotransmitters, occupy more of the volume of presynaptic nerve terminals than any other organelle and, under some conditions, can accumulate Ca(2+). They are also closely associated with voltage-gated Ca(2+) channels (VGCCs) that trigger transmitter release by admitting Ca(2+) into the nerve terminal in response to action potentials (APs). We tested the hypothesis that SVs can modulate Ca(2+) signals in the presynaptic terminal. This has been a difficult question to address because neither pharmacological nor genetic approaches to block Ca(2+) permeation of the SV membrane have been available. To investigate the possible role of SVs in Ca(2+) regulation, we used imaging techniques to compare Ca(2+) dynamics in motor nerve terminals before and after depletion of SVs. We used the temperature-sensitive Drosophila dynamin mutant shibire, in which SVs can be eliminated by stimulation. There was no difference in the amplitude or time course of Ca(2+) responses during high-frequency trains of APs, or single APs, in individual presynaptic boutons before and after depletion of SVs. SVs have a limited role, if any, in the rapid sequestration of Ca(2+) within the neuronal cytosol or the synaptic microdomain. We also conclude that SVs are not important for regulation of synaptic VGCCs.

Action Potentials↗
PubMed2002

Quantal size and variation determined by vesicle size in normal and mutant Drosophila glutamatergic synapses.

Quantal size and variation at chemical synapses could be determined presynaptically by the amount of neurotransmitter released from synaptic vesicles or postsynaptically by the number of receptors available for activation. We investigated these possibilities at Drosophila glutamatergic neuromuscular synapses formed by two separate motor neurons innervating the same muscle cell. At wild-type synapses of the two neurons we found a difference in quantal size corresponding to a difference in mean synaptic vesicle volume. The same finding applied to two mutants (dlg and lap) in which synaptic vesicle size was altered. Quantal variances at wild-type and mutant synapses were similar and could be accounted for by variation in vesicular volume. The linear relationship between quantal size and vesicular volume for several different genotypes indicates that glutamate is regulated homeostatically to the same intravesicular concentration in all cases. Thus functional differences in synaptic strength among glutamatergic neurons of Drosophila result in part from intrinsic differences in vesicle size.

Animals↗
PubMed2002

Inverse relationship between release probability and readily releasable vesicles in depressing and facilitating synapses.

We tested the hypothesis that the probability of vesicular exocytosis at synapses is positively correlated with the pools of readily releasable synaptic vesicles, as shown for mammalian neurons grown in tissue culture. We compared synapses of two identified glutamatergic neurons: phasic (high-output, depressing) and tonic (low-output, facilitating) crustacean motor neurons, which differ 100- to 1000-fold in quantal content. Estimates of vesicles available for exocytosis were made from depletion during forced release and from electron microscopic observation of vesicles docked at synaptic membranes near active zones. Both measurements showed a significantly larger pool of readily releasable vesicles in facilitating synapses, despite their much lower quantal output during stimulation. Thus, the probability for release of docked vesicles is very much lower at facilitating synapses, and the presence of more docked vesicles does not predict higher synaptic release probability in these paired excitatory neurons.

Action Potentials↗
PubMed2002

Presynaptic regulation of neurotransmission in Drosophila by the g protein-coupled receptor methuselah.

Regulation of synaptic strength is essential for neuronal information processing, but the molecular mechanisms that control changes in neuroexocytosis are only partially known. Here we show that the putative G protein-coupled receptor Methuselah (Mth) is required in the presynaptic motor neuron to acutely upregulate neurotransmitter exocytosis at larval Drosophila NMJs. Mutations in the mth gene reduce evoked neurotransmitter release by approximately 50%, and decrease synaptic area and the density of docked and clustered vesicles. Pre- but not postsynaptic expression of normal Mth restored normal release in mth mutants. Conditional expression of Mth restored normal release and normal vesicle docking and clustering but not the reduced size of synaptic sites, suggesting that Mth acutely adjusts vesicle trafficking to synaptic sites.

Action Potentials↗
PubMed2002

Intracellular ionic concentration by calibration from fluorescence indicator emission spectra, its relationship to the K(d), F(min), F(max) formula, and use with Na-Green for presynaptic sodium.

The emission spectra calibration curves for a fluorescence indicator and the F(min), F(max), and K(d) formula were shown to be related. Using the known calibrated fluorescence emitted by Sodium Green (Na-Green) and photo-multiplier-tube quantum efficiency, we calculated the detection signal over a range of sodium concentrations. The calculated calibration curves were compared for optical filters passing a narrow band, medium band or full spectrum. We found that a method based on the full emission spectrum was the most appropriate. Given a known resting concentration of intracellular sodium, calibrated readings can be converted to concentration values. This method is applicable to any fluorescence indicator when curves for emission spectra over a range of concentrations are available. We measured sodium concentration changes during trains of action potentials (APs) at a crayfish motor axon's presynaptic terminals injected with Na-Green. During low frequency AP trains, net sodium increases asymptotically with frequency. Average net Na-flux per AP decreases for increasing terminal size. The terminals of crayfish motor axon have surface area to volume ratio which is 7700 times larger than for squid. Thus, in comparison to squid, crayfish terminals exhibit a larger change in [Na(+)](i) during equivalent AP activity.

Action Potentials↗
PubMed2002

Impairment of central pattern generation in Drosophila cysteine string protein mutants.

The cysteine string proteins are integral synaptic vesicle proteins, critical for fast, Ca2+ -regulated exocytosis. Drosophila larvae with null mutations for the cysteine string protein (csp) gene have temperature-sensitive impairments of neurotransmission and presynaptic calcium removal at the neuromuscular junction. Using the larval Drosophila preparation to examine central pattern generation, we characterized the temperature sensitivity of locomotor patterns in wildtype and csp mutant larvae. Intraburst frequency of motoneuronal activity reached 100 Hz and was sufficiently high to rescue the temperature-sensitive synaptic failure in the mutant. Nevertheless, we show that deletion of the csp gene resulted in a severe deficiency in the generation of coordinated larval motor rhythms. Csp mutants that could generate patterned motor activity had slower, poorly coordinated rhythms with altered temperature sensitivity. We conclude that the temperature-sensitive paralysis characteristic of csp mutants is not a direct result of synaptic failure at neuromuscular junctions, as might be expected, but is the result of a failure of locomotor circuit operation at a higher integrative level.

Action Potentials↗
PubMed2002

Enhancement of presynaptic performance in transgenic Drosophila overexpressing heat shock protein Hsp70.

Prior heat shock confers protection to Drosophila synapses during subsequent heat stress by stabilizing quantal size and reducing the decline of quantal emission at individual synaptic boutons. The major heat shock protein Hsp70, which is strongly induced by high temperatures in Drosophila, may be responsible for this synaptic protection. To test this hypothesis, we investigated synaptic protection and stabilization at larval neuromuscular junctions of transgenic Drosophila which produce more than the normal amount of Hsp70 in response to heat shock. Overexpression of Hsp70 coincides with enhanced protection of presynaptic performance, assayed by measuring mean quantal content and percentage success of transmission. Quantal size was not selectively altered, indicating no effects of overexpression on postsynaptic performance. Thus, presynaptic mechanisms can be protected by manipulating levels of Hsp70, which would provide stability to neural circuits otherwise susceptible to heat stress.

Animals↗
PubMed2002

Diversification of synaptic strength: presynaptic elements.

Synapses are not static; their performance is modified adaptively in response to activity. Presynaptic mechanisms that affect the probability of transmitter release or the amount of transmitter that is released are important in synaptic diversification. Here, we address the diversity of presynaptic performance and its underlying mechanisms: how much of the variation can be accounted for by variation in synaptic morphology and how much by molecular differences? Significant progress has been made in defining presynaptic structural contributions to synaptic strength; by contrast, we know little about how presynaptic proteins produce normally observed functional differentiation, despite abundant information on presynaptic proteins and on the effects of their individual manipulation. Closing the gap between molecular and physiological synaptic diversification still represents a considerable challenge.

Animals↗