Morphological plasticity of motor axons in Drosophila mutants with altered excitability

Abstract

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.

Ultrastructure of neuromuscular junctions in Drosophilal: Comparison of wild type and mutants with increased excitability

“…Some of the most characterized muscles, both anatomically and physiologically, are the abdominal ventral longitudinal muscles 6, 7, 12, and 13 (Jan and Jan, 1976a;Wu and Ganetzky, 1988;Johansen et al, 1989a,b;Budnik et al, 1990;Sink and Whitington, 1991a,b,c). These muscles are innervated by several motorneurons, each one of which establishes synaptic contacts with the muscle fiber at multiple sites over the length of the muscle (multiterminal innervation) characteristic for each muscle fiber (Johansen et al, 1989a).…”

“…Samples were then processed for anti-HRP immunocytochemistry according to Budnik et al, 1990. As a secondary antibody, a rhodamine-conjugated goat anti-rabbit antibody (Cappel) at a 1:100 dilution was used.…”

“…In particular, the body wall neuromuscular junction has recently received attention as a promising system in which to study synapse function and development (reviewed in Keshishian et al, 1993). This system is composed of a relatively simple pattern of identifiable muscles which are innervated by a few motoneurons whose synapses are accessible for both physiological and developmental studies (Wu and Ganetzky, 1988;Johansen et al, 1989a,b;Budnik, Zhong, and Wu, 1990;Halpern et al, 1991;Sink and Whitington 1991a,b,c;Chiba et al, 1992;Nose, Mahajan, and Goodman, 1992;Zhong, Budnik, and Wu, 1992;Broadie and Bate, 1993). …”

“…In Drosophila, electrical activity is also thought to regulate body wall muscle innervation (Budnik et al, 1990;Zhong et al, 1992;Jarecki et al, 1992). For example, it has been shown that in the potassium channel double mutant eag Sh, in which neurotransmitter release and motoneuron firing is dramatically increased (Ganetzky and Wu, 1983;Zhong and Wu, 1991), there is an increase in the number of synaptic boutons and motor axon terminal branching (Budnik et al, 1990;Zhong et al, 1992).…”

“…For example, it has been shown that in the potassium channel double mutant eag Sh, in which neurotransmitter release and motoneuron firing is dramatically increased (Ganetzky and Wu, 1983;Zhong and Wu, 1991), there is an increase in the number of synaptic boutons and motor axon terminal branching (Budnik et al, 1990;Zhong et al, 1992). In addition, TTX application to cultured embryonic body wall muscles produces an increase in the number of processes on developing synapses and changes in growth cone morphology during neuromuscular junction formation (Jarecki et al, 1992).…”