Source: Swale, D. R., et al. Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster. J. Vis. Exp. (2018)
The video demonstrates the extracellular recording of neuronal activity from the central nervous system (CNS) of Drosophila larvae. The transected CNS is positioned in a recording chamber, and the electrical activity from a descending nerve is recorded to establish the baseline firing frequency. A test agent is then introduced, and the change in firing frequency is recorded to determine the agent's excitatory or inhibitory effect.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Dissect and Prepare the Larval Drosophila CNS
NOTE: Methods for larval CNS dissection are clearly illustrated in Hafer and Schedl, but these previously published methods reduce the length of the descending neurons that are important for measuring spike frequency. Here, an additional method is outlined to excise the larval CNS that maintains long, intact descending neurons.
2. Extracellular Recording of Drosophila CNS.
Figure 1: Method for excising the CNS from third-instar maggots. (A) Intact maggot submerged in 200 µL of saline. The arrow indicates the mouth hooks that are used for separation of the body wall. (B) Two pairs of forceps are placed in the middle of the maggot and on the mouth hooks to begin separation of the body wall. (C) Body wall is separated by applying slight and continuous pressure to expose the viscera. (D) CNS is clearly visible (white arrows) and is occasionally intertwined with the viscera. The scale bar represents 1000 µm, 750 µm, 500 µm, and 200 µm for panels A, B, C, and D, respectively.
Figure 2: Disruption of the blood brain barrier by transecting the CNS. (A) Intact CNS with descending nerves clearly visible at the caudal end of the ventral ganglia. The red line indicates the location of transecting the CNS to disrupt the BBB. (B) A transected CNS with the caudal end of the ventral ganglia still exposing long descending neurons. The ventral ganglia can be discarded. The scale bar represents 200 µm for both panels.
Figure 3: Neurophysiological recordings from the CNS of third-instar larvae of D. melanogaster. Representative nerve discharge traces before and after exposure to (A) DMSO, (B) propoxur, and (C) GABA. Initial firing frequencies in spikes/s (Hz) for each experiment are given to the left of each trace. Concentration-response curves for propoxur (D) and GABA (E) to CNS nerve discharge of D. melanogaster larvae from replicated recordings (n = 3-5 concentrations per curve, with each concentration replicated at least 5 times). Arrows represent point of drug application. Data points represent mean percent increase of baseline firing rate and error bars represent standard deviation. When error bars are absent, it is because they are smaller than the size of the symbol.
The authors have nothing to disclose.
Drosophila melanogaster (strain OR) | Bloomington Drosophila Stock Center | 2376 | |
Vibration isolation table | Kinetic Systems | 9200 series | |
Faraday Cage | Kinetic Systems | N/A | |
Dissecting Microscope on a Boom | Nikon | SMZ800N | Multiple scopes can be used; boom stand is critical |
AC/DC differential amplifier | ADInstruments | AM3000H | The model 1700 can be used instead of the model 3000 |
audio monitor | ADInstruments | AM3300 | |
Hum Bug Noise Eliminator | A-M Systems | 726300 | |
Data Acquisition System (PowerLab) | ADInstruments | PL3504 | Multiple PowerLab models can be used. |
Lab Chart Pro Software | ADInstruments | N/A – Online Download | |
Fiber Optic Lights | Edmund Optics | 89-740 | Different light sources can be used, but fiber optics are the most adaptable |
Micromanipulator | World Precision Instruments | M325 | |
Microelectrode Holder | World Precision Instruments | MEH715 | Different models are acceptable |
BNC cables | World Precision Instruments | multiple based on size | |
Glass Capillaries | World Precision Instruments | PG52151-4 | |
Microelectrode Puller | Sutter Instruments | P-1000 | Also can use Narashige PC-100 |
Black Wax | Carolina Biological Supply | 974228 | |
Non-coated insect pins, size #2 | Bioquip | 1208S2 | |
Fince Forceps | Fine Science Tools | 11254-20 | |
Vannas Spring Scissors | Fine Science Tools | 15000-03 |