Electrophysiological recordings from the Drosophila giant fiber system (GFS)

Abstract

The giant fiber system (GFS) of Drosophila is a well-characterized neuronal circuit that mediates the escape response in the fly. It is one of the few adult neural circuits from which electrophysiological recordings can be made routinely. This protocol describes a simple procedure for stimulating the giant fiber neurons directly in the brain of the adult fly and obtaining recordings from the output muscles of the GFS: the tergotrochanteral "jump" muscle (TTM) and the large indirect flight muscles (dorsal longitudinal muscles, or DLMs). It is a relatively noninvasive method that allows the investigator to stimulate the giant fibers in the brain and assay the function of several central synapses within this neural circuit by recording from the thoracic musculature.

Figure 1

The giant fiber system: neurons and muscles A) Schematic indicating the neurons and connections of the GFS. For clarity, only one half of the bi-lateral circuit is shown. The giant fiber (GF, Red) relays information from the brain to the thoracic ganglia where it makes an electro-chemical synapse to the tergotrochanteral motorneuron (TTMn, Blue) which innervates the tergotrochanteral muscle (TTM). It also makes an electro-chemical synapse to the peripherally synapsing interneuron (PSI, Green) which, in turn, makes chemical synapses to the dorsal longitudinal motorneurons (DLMns, Yellow) that innervate the dorsal longitudinal muscles (DLMs). The relative positions of the stimulating and recording electrodes are indicated. Adapted from Allen et al., (2006). B) Artists impression of the GFS showing the CNS within the fly’s body. The neurons and muscles of the GFS are shown in their approximate positions and the best positions for the stimulating and recording electrodes are depicted.

Figure 2

Electrophysiology of the Giant fiber system. A) Components of the electrophysiological rig. 1: Stimulator (S48 Square Pulse Stimulator, Grass instruments); 2: Stimulation isolation unit (SIU5 RF Transformer Isolation Unit, Grass instruments); 3: Two Intracellular amplifier (Model 5A Microelectrode Amplifier, Getting Instruments); 4: Data acquisition system (Digidata 1440A, Molecular devices) & Computer with software (not shown); 5: Storage Oscilloscope 5111A (Tektronix); 6: Stereomicroscope (Wild M5) on a boom stand; 7: Vibration isolation table (TMC); 8: Light source (Fostec). 9: Recording platform with 5 manual multi axis micromanipulators (Narashigi, Sutter and World Precision Instruments). B) Magnification of 9 in Figure 2A. Around the recording tray are arranged: Two stimulation electrodes (sharp tungsten electrodes), two recording electrodes (glass electrodes filled with saline) and one ground electrode (sharp tungsten electrodes). C) Drosophila melanogaster impaled with stimulation electrodes through the eyes in the brain and a ground in the abdomen. Two glass electrodes are placed the thorax for recording of responses from the TTM and DLM. D) Sample electrophysiological traces from recordings of the TTM and DLM upon brain stimulation of a wild type fly. The response latency of the GF-TTM pathway is 0.8 ms and it can follow stimuli one to one at 200 Hz. In contrast, the response latency of the GF-DLM pathway is 1.2 ms and responses are not seen after every stimulus when given 10 stimuli at 200 Hz.

Figure 3

Identification of the TTM and DLM localization using bristles and illumination. A) There are six pairs of indirect flight muscles, but only the Dorsal Longitudinal Muscle pair 45a (also called the dorsal median muscle or muscle number 6) are innervated by the DLM motor neurons that receive input via the PSI from the contra-lateral GF (Demerec 1994). The attachment site of the DLM 45a muscles are under the cuticle between the anterior Dorso-Central setae (yellow arrows) and the midline of the animal (yellow circle indicates site of left DLM). B) The TTM is underneath the cuticle, just dorsal of the anterior and posterior Supra-Alars setae (yellow arrows) as indicated by the area circled by a dotted line (Demerec 1994). C) As the TTM fibers are running along the dorsal-ventral axis they can be nicely visualized when a light source is placed underneath the fly (black arrows).