Chun-Fang Wu

Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions

Neuromuscular preparations from third instar larvae of Drosophila are not well-maintained in commonly used physiological solutions: vacuoles form in the muscle fibers, and membrane potential declines. These problems may result from the Na∶K ratio and total divalent cation content of these physiological solutions being quite different from those of haemolymph. Accordingly haemolymph-like solutions, based upon ion measurements of major cations, were developed and tested. Haemolymph-like solutions maintained the membrane potential at a relatively constant level, and prolonged the physiological life of the preparations. Synaptic transmission was well-maintained in haemolymph-like solutions, but the excitatory synaptic potentials had a slower time course and summated more effectively with repetitive stimulation, than in standard Drosophila solutions. Voltage-clamp experiments suggest that these effects are linked to more pronounced activation of muscle fiber membrane conductances in standard solutions, rather than to differences in passive muscle membrane properties or changes in postsynaptic receptor channel kinetics. Calcium dependence of transmitter release was steep in both standard and haemolymph-like solutions, but higher external calcium concentrations were required for a given level of release in haemolymph-like solutions. Thus, haemolymph-like solutions allow for prolonged, stable recording of synaptic transmission.

High-quality life extension by the enzyme peptide methionine sulfoxide reductase

Cumulative oxidative damages to cell constituents are considered to contribute to aging and age-related diseases. The enzyme peptide methionine sulfoxide reductase A (MSRA) catalyzes the repair of oxidized methionine in proteins by reducing methionine sulfoxide back to methionine. However, whether MSRA plays a role in the aging process is poorly understood. Here we report that overexpression of the msrA gene predominantly in the nervous system markedly extends the lifespan of the fruit fly Drosophila. The MSRA transgenic animals are more resistant to paraquat-induced oxidative stress, and the onset of senescence-induced decline in the general activity level and reproductive capacity is delayed markedly. The results suggest that oxidative damage is an important determinant of lifespan, and MSRA may be important in increasing the lifespan in other organisms including humans.

Morphological Plasticity of Motor Axons in Drosophila Mutants with Altered Excitability

An anatomical and electrophysiological study of Drosophila mutants has been made to determine the effect of altered electrical activity on the development and maintenance of larval neuromuscular junctions. We examined motor axon terminals of (1) hyperexcitable mutants Shaker (Sh), ether a go-go (eag), Hyperkinetic (Hk), and Duplication of para+ (Dp para+); and (2) mutants with reduced excitability, no action potential (napts) and paralytic (parats 1). Nerve terminals innervating larval body-wall muscles were visualized by using anti-HRP immunocytochemistry, which specifically stains neurons in insect species. In wild-type larvae, motor axon terminals were distributed in a stereotypic fashion. However, in combinations of eag and Sh alleles, the basic pattern of innervation was altered. There was an increase in both the number of higher-order axonal branches over the muscles and the number of varicosities on the neurites. A similar phenomenon was found in the double mutant Hk eag and, to a lesser extent, in Dp para+ and Dp para+ Sh mutants. It is known that at permissive temperature the napts, but not parats 1, mutation decreases excitability of larval motor axons and suppresses the behavioral phenotypes of Sh, eag, and Hk. In the mutant napts (reared at permissive temperature), a slight decrease in the extent of branching was observed. Yet, when combined with eag Sh, napts completely reversed the morphological abnormality in eag Sh mutants. No such reversion was observed in parats 1 eag Sh mutants. The endogenous patterns of electrical activity at the neuromuscular junction were analyzed by extracellular recordings in a semi-intact larval preparation. Recordings from wild-type body-wall muscles revealed rhythmic bursts of spikes. In eag Sh mutants, this rhythmic activity was accompanied by or superimposed on periods of strong tonic activity. This abnormal pattern of activity could be partially suppressed by napts in combination with eag Sh.

A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.

The hemolymph-like HL3 saline () and standard saline () are two widely used bathing solutions for physiological recordings at the Drosophila larval neuromuscular junction. It has been established that longevity of larval preparations is better maintained in HL3 saline. However, HL3 can produce results that are inconsistent with previous findings in standard saline, particularly on temperature sensitivity and membrane excitability phenotypes. In wild-type larvae, the excitatory junctional potentials (EJPs) in standard saline (containing 4 mM Mg2+ and 1.8 mM Ca2+) were not blocked by a temperature increase up to 39–40°C, consistent with unimpaired larval locomotion below these temperatures. However, in HL3 saline (containing 20 mM Mg2+ and 1.5 mM Ca2+), EJPs were blocked at 30°C. As for temperature-sensitive mutants napts and parats, the EJP-blocking temperatures were decreased from about 29 and 33°C in standard saline to about 23 and 26°C in HL3, respectively. Compound action potential recordings confirmed that segmental nerve action potentials were more readily blocked by a temperature increase in HL3 than in standard saline. Axonal excitability was suppressed in HL3 even at room temperatures, as evidenced by a lengthened refractory period in wild-type larvae. Similar suppression occurred for the hyper-excitable double mutant eag Sh, which maintained high-frequency spontaneous EJPs in standard saline but showed a rapidly declining EJP frequency in HL3. Application of HL3 saline also strongly suppressed the prolonged transmitter release following removal of repolarization mechanisms by K+ channel blockers or by the eag Sh mutation previously described in standard saline. These discrepancies suggest that the high divalent cation content in HL3 may confer a surface charge screening effect to suppress nerve membrane excitability. We found that a minimal adjustment of the HL3 saline, decreasing the Mg2+ ion concentration from 20 to 4 mM, was sufficient to resolve the discrepancies. While retaining the longevity of the larval neuromuscular preparation, the modified HL3 saline (HL3.1) restored the established wild-type EJP properties as well as phenotypes of several widely used temperature-sensitive and hyper-excitable mutants previously documented in standard saline.

Alteration of four identified K+ currents in Drosophila muscle by mutations in eag

Voltage-clamp analysis of Drosophila larval muscle revealed that ether a go-go (eag) mutations affected all identified potassium currents, including those specifically eliminated by mutations in the Shaker or slowpoke gene. Together with DNA sequence analysis, the results suggest that the eag locus encodes a subunit common to different potassium channels. Thus, combinatorial assembly of polypeptides from different genes may contribute to potassium channel diversity.

Coordination and Modulation of Locomotion Pattern Generators in Drosophila Larvae: Effects of Altered Biogenic Amine Levels by the Tyramine β Hydroxlyase Mutation

Forward locomotion of Drosophila melanogaster larvae is composed of rhythmic waves of contractions that are thought to be produced by segmentally organized central pattern generators. We present a systematic description of spike activity patterns during locomotive contraction waves in semi-intact wild-type and mutant larval preparations. We have shown previously that TβhnM18 mutants, with altered levels of octopamine and tyramine, have a locomotion deficit. By recording en passant from the segmental nerves, we investigated the coordination of the neuronal activity driving contraction waves of the abdominal body-wall muscles. Rhythmic bursts of activity that occurred concurrently with locomotive waves were frequently observed in wild-type larvae but were rarely seen in TβhnM18 mutants. These centrally generated patterned activities were eliminated in the distal stumps of both wild-type and TβhnM18 larvae after severing the segmental nerve from the CNS. Patterned activities persisted in the proximal stumps deprived of sensory feedback from the periphery. Simultaneous recordings demonstrated a delay in the bursting activity between different segments, with greater delay for segments that were farther apart. In contrast, bilateral recordings within a single segment revealed a well synchronized activity pattern in nerves innervating each hemisegment in both wild-type and TβhnM18 larvae. Significantly, rhythmic patterns of bursts and waves could be evoked in TβhnM18 mutants by head or tail stimulation despite their highly irregular spontaneous activities. These observations suggest a role of the biogenic amines in the initiation and modulation of motor pattern generation. The technique presented here can be readily extended to examine the locomotion motor program of other mutants.

Concomitant alterations of physiological and developmental plasticity in drosophila CaM kinase II-inhibited synapses

Ca2+/calmodulin-dependent protein kinase II (CaM kinase) has been implicated in neural plasticity that underlies learning and memory processes. Transformed strains of Drosophila, ala1 and ala2, expressing a specific inhibitor of CaM kinase are known to be impaired in an associative conditioning behavioral paradigm. We found that these transformants had altered short-term plasticity in synaptic transmission along with abnormal nerve terminal sprouting and directionality of outgrowth. These results represent an interesting parallel with the activity-dependent regulation of synaptic physiology and morphology by the cAMP cascade in Aplysia and Drosophila. In contrast to the learning mutants dunce and rutabaga, which are defective in the cAMP cascade, inhibition of CaM kinase in ala transformants caused increased sprouting at larval neuromuscular junctions near the nerve entry point, rather than altering the higher order branch segments. In addition, synaptic facilitation and potentiation were altered in a manner different from that observed in the cAMP mutants. Furthermore, synaptic currents in ala transformants were characterized by greater variability, suggesting an important role of CaM kinase in the stability of transmission.

Complete separation of four potassium currents in drosophila

A number of voltage-activated and Ca2+ activated K+ currents are known to coexist and play a major role in a wide variety of cellular processes including neuromuscular phenomena. Separation of these currents is important for analyzing their individual functional roles and for understanding whether or not they are mediated by entirely different channels. In Drosophila, we have now been able to manipulate four different K+ currents, individually and in combination with one another, by a combined use of mutations and pharmacological agents. This allows analysis of the physiological and molecular properties of different K+ channels and of the role of individual currents in membrane excitability.

Reversible inhibition of endocytosis in cultured neurons from the Drosophila temperature-sensitive mutant shibirets1.

The Drosophila mutant, shibirets1 (shits1), is paralyzed at restrictive temperatures (greater than 29 degrees C) by a reversible block in synaptic transmission. Heat pulses deplete synaptic vesicles in nerve terminals and inhibit endocytic internalization of plasma membrane in garland cells and oocytes. In dissociated cultures of larval central nervous system (CNS), a temperature-sensitive defect is also expressed in shits1 neurons: at 30 degrees C, growth cone formation is retarded and neurite outgrowth is arrested. We now report that we have examined constitutive endocytosis in Drosophila CNS culture and have demonstrated directly an endocytic defect in shits1 neurons. At the permissive temperature, 20-22 degrees C, both shits1 and wild-type neurons actively endocytosed fluorescein-labelled dextran (40 KD, 5%) or horseradish peroxidase (HRP, 1%). Within 5 min, HRP was seen in vesicles, cup-shaped bodies, tubules and multivesicular bodies in neurites and cell bodies. In contrast, endocytosis was inhibited in cultures derived from the temperature-sensitive paralytic shits1 by a 15 min heat pulse (30 degrees C). Even after 30 min of HRP exposure at 30 degrees C, HRP-containing membranes were absent from almost all shits1 neurites; a minority of cell bodies had a few HRP-containing vesicles. The temperature-dependent block in endocytosis was readily reversed at 20 degrees C. Interestingly, the block was overcome by high concentration of external cations: shits1 neurons in culture actively took up HRP in numerous vesicles at 30 degrees C if 18 mM Ca2+ or Mg2+ was added to the medium. Our results support the notion that membrane recycling plays a critical role in regulating neurite outgrowth. This study also provides baseline information for further mutational analysis of the mechanism underlying the membrane cycling process in cultured neurons.

Neuronal Activity and Adenylyl Cyclase in Environment-Dependent Plasticity of Axonal Outgrowth in Drosophila

The development of the nervous system is influenced by environmental factors. Among all environmental factors, temperature belongs to a unique category. Besides activating some specific sensory pathways, it exerts nonspecific, pervasive effects directly on the entire nervous system, especially in exothermic species. This study uses mutants to genetically discover how temperature affects nerve terminal arborization at larval neuromuscular junctions of Drosophila. It is known that hyperexcitability in K+ channel mutants leads to enhanced ramification of larval nerve terminals. Elevated cAMP levels in dunce mutants with reduced phosphodiesterase activity also cause enhanced arborization. These genetic alterations are thought to perturb mechanisms relevant to activity-dependent neural plasticity, in which neuronal activity activates the cAMP pathway, and consequently affect nerve terminal arborization by regulating expression of adhesion molecules. Here we demonstrate the robust influence of rearing temperature on motor nerve terminal arborization. Analysis of ion channel and cAMP pathway mutants indicates that this temperature-dependent plasticity is mediated via neuronal activity changes linked to mechanisms controlled by the rutabaga-encoded adenylyl cyclase.