Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster (original) (raw)

Abstract

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Figures (6)

Figure 2: Electrophysiology setup that is used to perform extracellular recordings. (A) Faraday cage; (B) vibration table; (C) dissecting microscope; (D) AC/DC differential amplifier; (E) audio monitor; (F) noise eliminator; (G) data acquisition system; (H) computer running lab chart pro software; (I) fiberoptic cable with external illumination source; (J) micromanipulator; (K) microelectrode holder with pressure port with glass electrode and preparation wax dish. Please click here to view a larger version of this figure. Figure 1: Excised CNS from third-instar Drosophila melanogaster. Arrows point to various anatomical structures of the CNS that correspond to the labels. The scale bar represents 250 um. Please click here to view a larger version of this figure.

Figure 2: Electrophysiology setup that is used to perform extracellular recordings. (A) Faraday cage; (B) vibration table; (C) dissecting microscope; (D) AC/DC differential amplifier; (E) audio monitor; (F) noise eliminator; (G) data acquisition system; (H) computer running lab chart pro software; (I) fiberoptic cable with external illumination source; (J) micromanipulator; (K) microelectrode holder with pressure port with glass electrode and preparation wax dish. Please click here to view a larger version of this figure. Figure 1: Excised CNS from third-instar Drosophila melanogaster. Arrows point to various anatomical structures of the CNS that correspond to the labels. The scale bar represents 250 um. Please click here to view a larger version of this figure.

Figure 3: Method for excising the CNS from third-instar maggots. (A) Intact maggot submerged in 200 uL of saline. The arrow indicates th mouth hooks that are used for separation of the body wall. (B) Two pairs of forceps are placed at 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 um, 750 um, 500 um, and 200 um for panels A, B, C, and D, respectively. Please click here to view a larger version of this figure.

Figure 3: Method for excising the CNS from third-instar maggots. (A) Intact maggot submerged in 200 uL of saline. The arrow indicates th mouth hooks that are used for separation of the body wall. (B) Two pairs of forceps are placed at 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 um, 750 um, 500 um, and 200 um for panels A, B, C, and D, respectively. Please click here to view a larger version of this figure.

Figure 4: Disruption of the blood brain barrier by transecting the CNS. (A) Intact CNS with descending nerves clearly visible at the cauda 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 um for both panels. Please click here to view a larger version of this figure.

Figure 4: Disruption of the blood brain barrier by transecting the CNS. (A) Intact CNS with descending nerves clearly visible at the cauda 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 um for both panels. Please click here to view a larger version of this figure.

Figure 5: 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. Please click here to view a larger version of this figure.

Figure 5: 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. Please click here to view a larger version of this figure.

Figure 6: Increased penetration of tacrine into the nervous system after transection of the CNS. Representative recordings of (A) intact and (B) transected larval CNS exposed to monomeric tacrine, which was applied at the arrow. Initial firing frequencies in spikes/s (Hz) for each experiment are given to the left of each trace. Please click here to view a larger version of this figure.

Figure 6: Increased penetration of tacrine into the nervous system after transection of the CNS. Representative recordings of (A) intact and (B) transected larval CNS exposed to monomeric tacrine, which was applied at the arrow. Initial firing frequencies in spikes/s (Hz) for each experiment are given to the left of each trace. Please click here to view a larger version of this figure.

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