Crayfish and Drosophila NMJs.

Many synaptic studies have utilized the experimental advantages of the Arthropod NMJ and the most prominent preparations have been the crayfish and Drosophila larval NMJs. Early cellular studies in the crayfish established the framework for later molecular studies in Drosophila. The two neuromuscular systems are compared including the advantages presented by each preparation for cellular analysis.

Parvalbumin expression affects synaptic development and physiology at the Drosophila larval NMJ.

Presynaptic Ca appears to play multiple roles in synaptic development and physiology. We examined the effect of buffering presynaptic Ca by expressing parvalbumin (PV) in Drosophila neurons, which do not normally express PV. The studies were performed on the identified Ib terminal that innervates muscle fiber 5.

The essential oil of and its components affect behavior and synaptic physiology.

is a flowering shrub in the verbena family and its essential oil (EO) is known for its sedative, antidepressant and analgesic properties. In the Amazon region of Brazil, it is used in aquaculture to anesthetize fish during transport. Many of the specialized metabolites found in EOs presumably evolved to protect plants from herbivores, especially insects.

Increased postsynaptic Ca reduces mini frequency at the Drosophila larval NMJ.

Repetitive stimulation of the Drosophila larval NMJ can produce a reduction in the frequency of miniature excitatory postsynaptic currents. By buffering postsynaptic Ca , it was shown that the decrease in "mini" frequency was due to an increase in postsynaptic Ca .

Regulation of quantal currents determines synaptic strength at neuromuscular synapses in larval Drosophila.

Studies of synaptic homeostasis during muscle fiber (MF) growth in Drosophila larvae have focused on the regulation of the quantal content of transmitter release. However, early studies in crayfish and frog suggested that regulation of quantal current size may be an integral mechanism in synaptic homeostasis. To examine this further in Drosophila, we compared the electrical properties, miniature excitatory postsynaptic potentials (minEPSPs) and miniature excitatory postsynaptic currents (minEPSCs) in different-sized MFs in third-instar larvae and for a single MF during larval growth.

Synaptic excitation is regulated by the postsynaptic dSK channel at the Drosophila larval NMJ.

In the mammalian central nervous system, the postsynaptic small-conductance Ca(2+)-dependent K(+) (SK) channel has been shown to reduce postsynaptic depolarization and limit Ca(2+) influx through N-methyl-d-aspartate receptors. To examine further the role of the postsynaptic SK channel in synaptic transmission, we studied its action at the Drosophila larval neuromuscular junction (NMJ). Repetitive synaptic stimulation produced an increase in postsynaptic membrane conductance leading to depression of excitatory postsynaptic potential amplitude and hyperpolarization of the resting membrane potential (RMP).

Characterization of postsynaptic Ca2+ signals at the Drosophila larval NMJ.

Postsynaptic intracellular Ca(2+) concentration ([Ca(2+)](i)) has been proposed to play an important role in both synaptic plasticity and synaptic homeostasis. In particular, postsynaptic Ca(2+) signals can alter synaptic efficacy by influencing transmitter release, receptor sensitivity, and protein synthesis. We examined the postsynaptic Ca(2+) transients at the Drosophila larval neuromuscular junction (NMJ) by injecting the muscle fibers with Ca(2+) indicators rhod-2 and Oregon Green BAPTA-1 (OGB-1) and then monitoring their increased fluorescence during synaptic activity.

Ca²+ buffering at a drosophila larval synaptic terminal.

A quantitative analysis of Ca²+ dynamics requires knowledge of the Ca²+-binding ratio (κ(S) ); this has not been measured at Drosophila synaptic terminals or any invertebrate synaptic terminal. We measured κ(S) at a Ib motor terminal in Drosophila larvae comparing single-AP Ca²+ transients in synaptic terminals that contained varying concentrations of the Ca²+ indicator, Oregon Green 488 BAPTA-1 (OGB-1). Using a linear single-compartment model, κ(S) was calculated based upon the effect of [OGB-1] on the time constant (τ(decay) ) for the decay of intracellular free Ca²+ concentration ([Ca²+](i)).

Intracellular Ca2+ regulates free-running circadian clock oscillation in vivo.

Although circadian oscillation in dynamics of intracellular Ca2+ signals has been observed in both plant and animal cells, it has remained unknown whether Ca2+ signals play an in vivo role in cellular oscillation itself. To address this question, we modified the dynamics of intracellular Ca2+ signals in circadian pacemaker neurons in vivo by targeted expression of varying doses of a Ca2+ buffer protein in transgenic Drosophila melanogaster. Intracellular Ca2+ buffering in pacemaker neurons results in dose-dependent slowing of free-running behavioral rhythms, with average period >3 h longer than control at the highest dose.

Ca2+ dynamics along identified synaptic terminals in Drosophila larvae.

Changes in intracellular Ca2+ concentration ([Ca2+]i) play an important role in the function and plasticity of synapses. We characterized the changes in [Ca2+]i produced by action potentials (APs) along two identified motor terminals found on separate muscle fibers in Drosophila larvae and examined factors that influence the amplitude and duration of the residual Ca2+ signal. We were able to measure Ca2+ transients produced along terminals by both single APs and AP trains using Oregon Green 488 BAPTA-1 and streaming images at 20-50 Hz.

Sexual differentiation of identified motor terminals in Drosophila larvae.

In Drosophila, we have found that some of the motor terminals in wandering third-instar larvae are sexually differentiated. In three out of the four body-wall muscle fibers that we examined, we found female terminals that produced a larger synaptic response than their male counterparts. The single motor terminal that innervates muscle fiber 5 produces an EPSP that is 69% larger in females than in males.

Regenerating crayfish motor axons assimilate glial cells and sprout in cultured explants.

Phasic and tonic motor nerves originating from crayfish abdominal ganglia, in 2-3-day-old cultured explants, display at their transected distal ends growth zones from which axonal sprouts arise. The subcellular morphology of this regenerative response was examined with thin serial-section electron microscopy and reveals two major remodeling features. First, the external sprouts that exit the nerve are a very small part of a much more massive sprouting response by individual axons comprising several orders of internal sprouts confined to the nerve.

Ca2+ clearance at growth cones produced by crayfish motor axons in an explant culture.

Intracellular free Ca2+ concentration ([Ca2+]i) plays an important role in the regulation of growth cone (GC) motility; however, the mechanisms responsible for clearing Ca2+ from GCs have not been examined. We studied the Ca2+-clearance mechanisms in GCs produced by crayfish tonic and phasic motor axons by measuring the decay of [Ca2+]i after a high [K+] depolarizing pulse using fura-2AM. Tonic motor axons regenerating in explant cultures develop GCs with more rapid Ca2+ clearance than GCs from phasic axons.

Effects of chronic lead exposure on the neuromuscular junction in Drosophila larvae.

Long term or chronic exposure to lead is associated with cognitive and other deficits in humans, which may reflect lead-induced changes in synaptic development and function. We believe that Drosophila has great potential as a model system for studying such changes. To test this, we compared the structure of single, identified synapses between identified axons (axons 1 and 2) and muscle fibers (fibers 6 and 7) in untreated 3rd instar larvae, and in larvae reared on medium made with 100 microM lead acetate in distilled water.

Effect of reduced impulse activity on the development of identified motor terminals in Drosophila larvae.

In Drosophila larvae, motoneurons show distinctive differences in the size of their synaptic boutons; that is, axon 1 has type Ib ("big" boutons) terminals and axon 2 has type Is ("small" boutons) terminals on muscle fibers 6 and 7. To determine whether axon 1 develops large boutons due to its high impulse activity, we reduced impulse activity and examined the motor terminals formed by axon 1. The number of functional Na(+) channels was reduced either with the nap(ts) mutation or by adding tetrodotoxin (TTX) to the media (0.

Activity-dependent plasticity of calcium clearance from crayfish motor axons.

Previous studies of a crayfish explant culture demonstrated that regenerating motor axons with high impulse activity develop more rapid clearance of cytoplasmic free Ca(2+) than those with low impulse activity. We examined whether Ca(2+) clearance in mature axons also showed activity-dependent plasticity. We studied the phasic and tonic axons of the motor bundle innervating the crayfish closer muscle that display large differences in impulse activity.