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Development of the larval NMJ:

• DEVELOPMENT OF DROSOPHILA LARVAE NMJ: SYNAPTIC STRENGTH

Project Summary- Communication between neurons and their target cells depends on their connections, specialized areas of contact which are called synapses. For proper function, the nervous system should be able to respond to changing circumstances and requirements during development. Maintaining a structural and functional matching between the pre- and postsynaptic cells is required as the animal grows to ensure synaptic efficacy. As a model system, Drosophila melanogaster provides experimental advantages for developmental studies. The simple innervation pattern of the body wall muscles is maintained during larval development. The easily identifiable muscle cells and motoneurons are amenable to physiological and morphological analysis.

To systematically pursue this investigation, five specific aims are being addressed:

1. Determine if there is a correlation between synaptic strength and input resistance (Rin) during development. Determine if the excitatory junction potentials from Ib & Is motor nerve terminals, recorded from muscles 6 & 7 change throughout larval development. If so, determine if the change is due solely to differences in Rin of muscle fibers 6 & 7 as they grow.

2. Determine if there is a correlation between the volume and surface area of muscles 6 & 7 and length of terminals and number of varicosities for both Is & Ib motoneurons. Determine if this correlation exists from 1st instar to early and late 3rd instar larvae.

3. Determine if the synaptic strength of Type Ib & Is varicosities at the most proximal and most distal ends of the nerve terminals changes during larvae development.

4. Elucidate the ultrastructure of both the most proximal and most distal ends of Type Ib & Is nerve terminal varicosities during each stage of larval development.

5. Measure locomotive function in relation to body size and mass from the 1st instar to early and late 3rd instar larvae.

The study is significant since it will provide pertinent information to address the degree in synaptic performance as measured directly at the release sites over developmental stages in the model system of Drosophila. Thus, the findings can be directly correlated to the underlying structural entities which are responsible for efficacy in chemical synaptic transmission in general. This proposed study also addresses the neuronal component of locomotive behaviors needed to move an enlarging body mass over or through a substrate. This will provide information addressing changes in synaptic performance during development. Direct measurement at synaptic release sites at different developmental stages of Drosophila will be made. Thus, there is promise that understanding the fundamental basics of synaptic transmission in this model system will be directly relevant to all neural systems, including humans.

Effects of 20-HE on growth:

• MAINTAINING SYNAPTIC EFFICACY AT THE NEUROMUSCULAR JUNCTION IN DROSOPHILA LARVA DURING NORMAL DEVELOPMENT AND PROLONGED LIFE WITH THE ECDYSONELESS MUTANT

ABSTRACT- Hormonal regulation in development and maintenance of synaptic transmission involves examination of both the presynaptic and postsynaptic components and a system in which the hormones can be controlled. We used the ecdysoneless heat-sensitive mutation (l(3)ecd1/l(3)ecd1) of Drosophila to provide the ability to regulate endogenous ecdysone production at various larval stages. In conjunction we used the neuromuscular junctions of Drosophila since they offer the advantage of assessable preparations for both morphological and physiological measures. The growth in the Ib & Is motor nerve terminals and the corresponding muscle 6 in segment 4 of the larval Drosophila throughout the 3rd instar stage in the presence of normal and a much reduced endogenous ecdysone level was investigated. Muscle 6 and the motor nerve terminals parallel in growth throughout the 3rd instar. The nerve terminal increase in length and varicosity number, thus providing an increase in the number of synaptic release sites. The ecdysoneless larva also show an increase in muscle size, however the Is & Ib motor nerve terminals do not mature to the extent as the wild type ecdysone producing flies. The motor nerve terminal length is shorter with fewer numbers of varicosities per terminal. In spite of a shorter nerve terminal and fewer varicosties, with an increasing muscle fiber, the compound EJP of Ib & Is in the ecdysoneless flies are larger, which is suggestive of synaptic structural modification. This study demonstrates ecdysone's role in modifying nerve terminal development and neuromuscular junction function.

20-HE.... Dopamine..

Neuromodulation:

1. 20-HE

• DEPRESSION OF SYNAPTIC EFFICACY IN HIGH- AND LOW-OUTPUT DROSOPHILA NEUROMUSCULAR JUNCTIONS BY THE MOLTING HORMONE (20-HYDROXYECDYSONE) DURING INTERMOLT

ABSTRACT- The molt-related steroid hormone, 20-hydroxyecdysone (20-HE), was applied to muscle 6 and muscle 7 of 3rd instar larval of Drosophila melanogaster neuromuscular junction preparations to examine if rapid, non-genomic responses could be observed as was recently shown to occur in crustacean neuromuscular junctions. At a dose of 10 µM, the excitatory junction potentials were reduced in amplitude within minutes. In order to elucidate the site of action of the hormone, focal-macropatch recordings of synaptic currents were obtained over the neuromuscular junctions. The results showed that the high-output (Is) and the low-output (Ib) motor nerve terminals, that innervate muscles 6 and 7, released fewer synaptic vesicles for each stimulation while exposed to 20-HE. Since the size and shape of synaptic currents from spontaneous releases did not change, the effects of the 20-HE are presynaptic. The rapid effects of this hormone may account in part for the quiescent behavior associated with molts among insects and crustaceans. (see - Marvin E. Ruffner et al., 1999, J Neurophysiol, 81:788-794)

2. Dopamine

• DOPAMINERGIC MODULATION OF MOTOR NEURON ACTIVITY AND NEUROMUSCULAR FUNCTION IN DROSOPHILA MELANOGASTER

ABSTRACT- Dopamine is found in both neuronal and non neuronal tissues in the larval stage of the fruit fly, Drosophila melanogaster, and functions as a signaling molecule in the nervous system. Although dopaminergic neurons in the central nervous system (CNS) were previously thought solely to be interneurons, recent studies suggest that dopamine may also act as a neuromodulator in humoral pathways. We examined bath application of dopamine on intact larval CNS-segmental preparations and isolated neuromuscular junctions (NMJs). Dopamine rapidly decreased the rhythmicity of the CNS motor activity. Application of dopamine on neuromuscular preparations of the segmental muscles 6 and 7 resulted in a dose-responsive decrease in the excitatory junction potentials (EJPs). With the use of focal, macropatch synaptic current recordings the quantal evoked transmission showed a depression of vesicular release at concentrations of 10 µM. Higher concentrations (1mM) produced a rapid decrement in evoked vesicular release. Dopamine did not alter the shape of the spontaneous synaptic currents, suggesting that dopamine does not alter the postsynaptic muscle fiber receptiveness to the glutaminergic motor nerve transmission. The effects are presynaptic in causing a reduction in the number of vesicles that are stimulated to be release due to neural activity.

[This project is in colaboration with Dr. W.S. Neckameyer, Department of Pharmacology & Physiological Sciences, St. Louis University, Medical School,St. Louis, MO]

EPSP Fluctuation Project:

• ASSESSING VARIANCE OVER TIME IN SYNAPTIC TRANSMISSION USING CONTROL CHARTS

ABSTRACT- Rapid synaptic transmission occurs in response to a Ca2+ transient generated in presynaptic nerve terminal by depolarization. There is substantial inherent variation among various types of synapses in the magnitude of the synaptic currents which leads in part to the variation of the evoked excitatory postsynaptic potentials (EPSPs). As a model system, crayfish and Drosophila melanogaster provides experimental advantages for such studies because of the genetic manipulability of particular proteins known to play a role in synaptic transmission. This implies that these findings can be directly correlated to the underlying structural entities, which are responsible for efficacy in chemical synaptic transmission in general. The EPSPs have nonlinear trends, which were summarized using non-parametric smoothing techniques. Using the residuals from smoothing analysis, the variability is able to be rapidly assessed over time with the help of control charts. Control limits were derived for median and interquartile range charts based on resampling techniques. These control limits provide an index to the fluctuations that can be used to determine how experimental manipulation or various therapies alter synaptic properties over time. There is promise that understanding the fundamental basics in analysis of synaptic transmission will be directly relevant to all neural systems, including humans.

[This project is in colaboration with Dr. Arnold J. Stromberg, Department of Statistics, Univ. of Kentucky, Lexington, KY, USA]