Murphey, Rodney K.

In Drosophila melanogaster, the GFS is synaptically coupled to the
Tergotrochanteral motoneurons; these neurons form a signaling pathway from the brain to
the jump muscles (Thomas and Wyman, 1983). Part of this signaling is done through gap
junctions, and placement of these gap junctions was partially shown to be regulated by the
binding of Netrin, a class of guidance molecule (Orr et al., 2014). In the present study we
investigate the role of Netrin's receptor Frazzled in the placement of gap junctions in
Drosophila at: 1) Presynaptic neurons (Giant Fibers [GF]), 2) Postsynaptic neurons
(Tergotrochanteral motoneurons [TTMn]), and 3) Presynaptic + Postsynaptic neurons
simultaneously. Effects of Frazzled were tested using Frazzled RNAi and a combination
of electrophysiological recordings and imaging of the GF-TTMn synapse. The results from
this study show that presynaptic and postsynaptic knockdown of Frazzled delayed
muscular responses and altered the anatomy of both the GF's and TTMn's.

In the developing CNS, presynaptic neurons often have exuberant overgrowth and
form excess (and overlapping) postsynaptic connections. Importantly, these excess
connections are refined during circuit maturation so that only the appropriate connections
remain. This synaptic rearrangement phenomenon has been studied extensively in
vertebrates but many of those models involve complex neuronal circuits with multiple
presynaptic inputs and postsynaptic outputs. Using a simple escape circuit in Drosophila
melanogaster (the giant fiber circuit), we developed tools that enabled us to study the
molecular development of this circuit; which consists of a bilaterally symmetrical pair of
presynaptic interneurons and postsynaptic motorneurons. In the adult circuit, each
presynaptic interneuron (giant fiber) forms a single connection with the ipsilateral,
postsynaptic motorneuron (TTMn). Using new tools that we developed we labeled both
giant fibers throughout their development and saw that these neurons overgrew their targets and formed overlapping connections. As the circuit matured, giant fibers pruned
their terminals and refined their connectivity such that only a single postsynaptic
connection remained with the ipsilateral target. Furthermore, if we ablated one of the two
giant fibers during development in wildtype animals, the remaining giant fiber often
retained excess connections with the contralateral target that persisted into adulthood.
After demonstrating that the giant fiber circuit was suitable to study synaptic
rearrangement, we investigated two proteins that might mediate this process. First, we
were able to prevent giant fibers from refining their connectivity by knocking out
highwire, a ubiquitin ligase that prevented pruning. Second, we investigated whether
overexpressing Netrin (or Frazzled), part of a canonical axon guidance system, would
affect the refinement of giant fiber connectivity. We found that overexpressing Netrin (or
Frazzled) pre- & postsynaptically resulted in some giant fibers forming or retaining
excess connections, while exclusively presynaptic (or postsynaptic) expression of either
protein had no effect. We further showed that by simultaneously reducing (Slit-Robo)
midline repulsion and elevating Netrin (or Frazzled) pre- & postsynaptically, we
significantly enhanced the proportion of giant fibers that formed excess connections. Our
findings suggest that Netrin-Frazzled and Slit-Robo signaling play a significant role in
refining synaptic circuits and shaping giant fiber circuit connectivity.

The ubiquitin ligase Highwire is responsible for cell-autonomously promoting
synapse formation in the Drosophila Giant Fiber system. highwire mutants show defects
in synaptic function and extra branching at the axon terminal, corresponding to transient
branching that occur in the course of giant synapse formation during metamorphosis. The
MAP kinase pathway, including Wallenda and JNK/Basket, plus the transcription factor
Jun, act to suppress synaptic function and axon pruning in a dosage sensitive manner,
suggesting different molecular mechanisms downstream of the MAP kinase pathway
govern function and pruning. A novel role for Highwire is revealed, regulating the giant
fiber axon’s ability to respond to external cues regulated by Fos. When expression of the
transcription factor Fos is disrupted in the post-synaptic TTMn or surrounding midline
glia of highwire mutants, the giant fiber axons show a marked increase in axon overgrowth and midline crossing. However, synaptic function is rescued by the cell nonautonomous
manipulation of Fos, indicating distinct mechanisms downstream of Highwire regulating synaptic function and axon morphology.

The classic guidance molecules, Netrin and its receptor Frazzled (Fra), dictate the strength of
synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating
gap junction localization in the pre-synaptic terminal. In Netrin mutant animals the synaptic
coupling between a giant interneuron and the jump motor neuron was weakened. Dye-coupling
between these two neurons was severely compromised or absent. These mutants exhibited
anatomically and physiologically defective synapses between the giant fiber (GF) and
tergotrochanteral motor neuron (TTMn). In cases where Netrin mutants displayed apparently
normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses.
Dye-coupling between the giant fiber and the motor neuron was reduced or eliminated,
suggesting that gap junctions were disrupted in the Netrin mutants. When we examined the gap
junctions with antibodies to Shaking-B Innexin (ShakB), they were significantly decreased or
absent in the pre-synaptic terminal of the mutant GF. This data is the first to show that Netrin and
Frazzled regulate placement of gap junctions pre-synaptically at a central synapse. In the Drosophila Giant Fiber System, we demonstrate a mechanism that ensures the monoinnervation of two homologous motor neurons by two homologous interneurons. In a scenario where both interneurons could synapse with both motor neuron targets, each interneuron exclusively synapsed with only one target and the circuit functions at normal physiological levels. This innervation pattern depended on the ratio of netrin-to-frazzled expression. When Netrin was over expressed in the system, shifting the ratio in favor of Netrin,
both interneurons synapsed with both target motor neurons and physiological function was reduced. This resulted in the polyinnervationof a single target. In contrast, when Frazzled was over expressed in the system, one interneuron innervated both targets and excluded the remaining interneuron from making any synaptic contact. This resulted in a single interneuron mono-innervating both motor neurons and physiological function was mutant. The orphaned interneuron made no synaptic contact with either motor neuron target. Physiological function was only normal when the Netrin-Frazzled ratio was at endogenous levels and each GF monoinnervated one motor neuron. When we examined the gap junctions at this synapse in experimental animals, there was a significant reduction of gap junction hemichannels in the presynaptic terminal of axons that deviated from normal innervation patterns. While the synapse dyecoupled, the reduction in gap junction hemichannels reduced function in the circuit.