Targeted expression of truncated glued disrupts giant fiber synapse formation in Drosophila

Abstract

Glued(1) (Gl(1)) mutants produce a truncated protein that acts as a poison subunit and disables the cytoplasmic retrograde motor dynein. Heterozygous mutants have axonal defects in the adult eye and the nervous system. Here we show that selective expression of the poison subunit in neurons of the giant fiber (GF) system disrupts synaptogenesis between the GF and one of its targets, the tergotrochanteral motorneuron (TTMn). Growth and pathfinding by the GF axon and the TTMn dendrite are normal, but the terminal of the GF axon fails to develop normally and becomes swollen with large vesicles. This is a presynaptic defect because expression of truncated Glued restricted to the GF results in the same defect. When tested electrophysiologically, the flies with abnormal axons show a weakened or absent GF-TTMn connection. In Glued(1) heterozygotes, GF-TTMn synapse formation appears morphologically normal, but adult flies show abnormal responses to repetitive stimuli. This physiological effect is also observed when tetanus toxin is expressed in the GFs. Because the GF-TTMn is thought to be a mixed electrochemical synapse, the results show that Glued has a role in assembling both the chemical and electrical components. We speculate that disrupting transport of a retrograde signal disrupts synapse formation and maturation.

Fig. 1.

Top. Expression ofGl^Δ disrupts axon morphology in the GFs. A, Diagram depicting the morphology of the GFs in the CNS. Boxes indicate regions of the brain and thoracic ganglia shown in B–E. The soma and dendrites are located in the brain (box labeledB&D). The axon and presynaptic terminal are located in the second thoracic segment (box labeledC&E). B, C, Whole-mount preparations of the CNS from UAS–lacZ; A307 adult flies. Immunocytochemistry has revealed LacZ reporter protein in the GFs in the brain, including the cell bodies and the dendritic field (B), and in the thoracic ganglia where the GFs show their distinctive bends in the mesothoracic neuromere (C, arrowhead). D,E, Whole-mount preparations of the CNS from UAS–lacZ; A307/UAS–Gl^Δ adult flies. The dendritic field is unaffected byGl^Δ (D); however, the axon terminals exhibit large swellings and no bend (E, asterisk). Scale bar, 20 μm.

Fig. 4.

The strength of dye coupling is affected by the truncated Glued protein. A, Iontophoresis of neurobiotin into the TTM often led to dye filling of the GF in theGl¹/+ heterozygotes (arrow) just as is seen in the wild-type animals (Table3). B, Expression of the truncated subunit under the control of c17 had no detectable effect on dye coupling to the GF.C, Many fewer specimens showed dye coupling to the GF when the truncated Glued was expressed under the control of A307. See also Table 3. Scale bar, 20 μm.

Fig. 5.

The Glued poison subunit disrupts the physiology of the giant fiber system. A, Recordings from individual control (A1–3),Glued¹ (A4–6), and c17; UAS-Gl^Δ96B(A7–9) animals. A1, A4,A8, The latency for wild type is indicated inA1, the beginning of stimulus is indicated by the S, and the vertical linerepresents the wild-type response latency for TTM (0.8 msec) and is drawn through all recordings for comparison. The longer TTM latency seen when the poison subunit was driven in the GF is highlighted with an asterisk in A7. Note that the disynaptic pathway to DLM remains constant in all three genotypes (∼1.4 msec). Five responses are overlapped in each frame.A2, A5, A8, To determine the refractory period, twin stimuli were given with a different interstimulus interval (ISI) between the two (see Materials and Methods). Five different ISI responses are shown overlaid for each genotype with latencies of 10, 8, 6, 4, and 2 msec. A response to the first stimulation is always seen, and the minimal refractory period for TTM is 4 msec in controls, as well as inGl¹/+. No second response is seen at any of these frequencies in the c17; UAS-Gl^Δ specimens (A8, asterisk). A3,A6, A9, For following frequency, a single sweep of 10 stimuli with an ISI of 4 msec is shown. In the wild-type animals, the TTM follows 1:1 (A6, *), but in theGl¹/+ heterozygote and in the transgenic c17 animals no repetitive firing occurs (A9, *) Calibration: vertical scale bar, 20 mV for all traces; horizontal, 1 msec for Latency, 2 msec for Refractory Period, and 10 msec for Following Frequency (250 Hz). B, Schematic representing the identified neurons of the giant fiber system. Brain stimulation activates the GF, which in turn activates two follower neurons in the thorax: the tergotrochanteral motorneuron (TTMn) and the peripherally synapsing interneuron (PSI). The GF drives the tergotrochanteral muscle (TTM) through a monosynaptic pathway (GF–TTMn) and the flight muscles (DLMs) through a disynaptic pathway (GF–PSI–DLMns). Responses were recorded intracellularly from the TTM and a DLM.

Fig. 6.

Strong expression of the Glued poison subunit weakens or abolishes the GF–TTMn synapse but not the neuromuscular junction. A, An experimental A307; UAS–Gl^Δ96Bspecimen with a weakened synapse. The latency of the TTM synapse is longer that normal (A1, *), and the following frequency is much lower than normal (A2, *). When the motor neurons were stimulated directly, the latency of the TTM response was restored to a very short latency of 0.7 msec (A3,B2, #), showing that the TTMn and its neuromuscular junction are normal and the defect can be attributed to the GF–TTMn synapse. B, Some specimens exhibit no TTM response. In this specimen there was never a TTM response to GF stimulation (B1, top trace asterisk). B2 shows that direct stimulation can still activate the TTMn and its neuromuscular junction. C, Schematic representing the methods of stimulation to test for a weakened or absent GF–TTMn synapse. Calibration: vertical scale bar, 20 mV for all traces; horizontal, 1 msec for Latency and 10 msec forFollowing Frequency (250 Hz).

Fig. 7.

Glued¹ mutation compromises the chemical component of the GF–TTMn synapse.A, The response of a control TTM to repetitive GF stimulation at three frequencies. B, The response of the TTM to repetitive firing of the GF in aGl¹/+ specimen. Note the strong depression at 200 and 300 Hz. C, Driving the defective version of the tetanus toxin transgene has no affect on repetitive firing. D, Expression of the tetanus toxin light chain in the GF reduces the response to repetitive firing. Note the similarity between these curves and theGl¹/+ heterozygotes.