1Department of Biological Sciences, Florida Atlantic University, 2Department of Chemistry & Biochemistry, Florida Atlantic University
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Mejia, M., Heghinian, M. D., Busch, A., Marí, F., Godenschwege, T. A. Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System. J. Vis. Exp. (62), e3597, doi:10.3791/3597 (2012).
Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on Dα7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.
1. Electrophysiology Rig Set-up
2. Nanoinjection Set-up
3. Drosophila melanogaster Preparation
4. Paired Nanoinjection/electrophysiology
Note: The electrophysiology traces shown in the video do not correspond to the effects of pure dye injection.
5. Representative Results
Effect of an antagonist on the PSI to DLM synapse of the Giant Fiber System
Methyllycaconitine citrate (MLA) is a nAChR antagonist that is specific for α7 nAChR subtypes. The PSI to DLMn synapse in the GF-DLM pathway is dependent on the Dα7 nAChR subtype for proper function, while genetic removal of Dα7 nAChR subtype has no effect on the GF-TTM pathway5,6. In order to demonstrate the specificity and sensitivity of our assay we injected MLA at different concentrations (0, 0.02, 0.04, 0.08, 0.12 ng/mg, 46 nl injected) into the head of the animal (n=10 per compound treatment; n=15 for saline treatment). Only male flies (of the wild type genotype wild 10E) were used, and the effect of the compound was monitored for a total of 15 minutes after injection.
Figure 5 depicts the difference between baseline recordings obtained before injection and those obtained after injection in response to MLA and saline control solution. We found that injection of MLA resulted in the inability of the GF-DLM pathway to follow one-to-one at 100 Hz by stimulations of the GFs in the brain while the GF-TTM pathway remained unaffected. (Figure 5, Top and middle trace, t-test performed between saline controls [0 ng/mg] and the different concentrations of MLA at each time point unless the data is non-parametric [normality and equal variances tested], otherwise we use a Mann-Whitney Rank Sum Test. *p<0.001). However, a one-to-one response of the DLM was observed when the motor neurons were stimulated directly (Figure 5, bottom trace), demonstrating that the NMJ function of the DLM and TTM is not affected by MLA. MLA appeared to reach its maximum effect 1 minute after injection for 0.04, 0.08 and 0.12 ng/mg of MLA injected, as no further significant changes were noted during the following 15 minutes of testing period. Moreover, the compound reached a maximum effect at 0.08 ng/mg since stronger responses were not observed with the higher dosage of 0.12 ng/mg.
Figure 1. The Giant Fiber System Diagram of the Giant Fiber System (GFS). The Giant Fibers (GFs, shown in red) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI, shown in green) and the Tergo Trochanteral Muscle neuron (TTMn, shown in yellow)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron, shown in blue) connection is dependent on Dα7 nAChR subtype6. Finally the neuromuscular junction (NMJ) of the TTMn and the DLMn onto the jump (TTM, shown in purple) and flight muscles (DLM, shown in purple) is glutamatergic.
Note: The GF to PSI connection is both electrical and chemical. However, in shakB mutants (which lack gap junctions), no response can be recorded from the DLM upon stimulation of the GFs in the brain, demonstrating that the chemical component in the absence of electrical connections is not sufficient to evoke an action potential in the PSI 5,16-18. Because the GF to PSI connection is gap junction dependent, this figure shows only the GAP junction at the synapse for simplicity reasons.
Figure 3. Beveled injection micropipette. A diagram of a properly beveled micropipette is shown here. The electrode opening should be beveled at a 45 degree angle and have an opening between 11 to 17 μm. A proper beveled injection micropipette is crucial for a smooth injection with minimal damage to the fly.
Figure 4. Overall scheme of the nanoinjection/electrophysiology protocol. A representative diagram of the overall scheme for the nanoinjection/electrophysiology protocol. Start by obtaining a baseline recording by stimulating the Giant Fibers (GFs) at 100 Hz with 10 trains of 10 stimuli each (only one train shown here). Before injection, begin the 1 Hz stimulations one second apart. During injection time (while injector is plugged in to the control box), you will observe significant background noise; however, do not discontinue the recordings. After injection (and injector is unplugged from control box), continue the 1 Hz stimulation for about 1 more minute. Finally, proceed to stress the GFs with 10 trains of 10 stimuli at 100 Hz and continue to test the function of the GF pathways with this paradigm every 5 minutes up to 15 minutes. Note: recordings were manipulated to create the overall scheme and do not represent a specific result obtained. Not to scale, not all traces are shown. Click here for larger image.
Figure 5. The effects of MLA in the GFS.
The nanoinjection/electrophysiology bioassay presented here allows for a rapid screening of compounds in the nervous system of the fruit fly. This is a novel in vivo technique that requires small quantities of a compound to elicit an effect on a variety of molecular targets in a well-characterized neuronal circuit. This method can be used to test the bioactivity of different compounds, from unknown toxins to commercially available pharmacological agents.
Here we demonstrated the function of our assay using MLA, which had an effect on the Giant Fiber System (GFS) of the fruit fly (Figure 5). We found that it selectively disrupted the GF to DLM pathway but not the GF to TTM pathway. Activating the motor neurons directly via thoracic stimulation demonstrated that the defect in the GF to DLM pathway was not due to a dysfunction at the neuromuscular junction (NMJ) but it was consistent with the antagonistic effect of MLA at the Dα7 nAChR subtypes present at the PSI-DLMn synapse (Figure 1). Although the GF to TTMn connection was shown to be cholinergic, it is unknown whether Dα7 nAChR subunits are present at this synapse. Furthermore, the genetic absence of the choline acetyltransferase (Cha) gene or the Dα7 nAChR subtype (Dα7) gene does not disrupt the function of the GF-TTMn connection because of the concurrent presence of an electrical junction 5,6,17,19,20, which makes the pathway unlikely to be affected by MLA.
After compound injection, the solution should immediately immerse the entire nervous system of the animal due to its open circulatory system21. If properly injected, the compound usually reaches the thorax and abdomen within seconds, but a homogenous dispersion can take up to a minute. However, if the compound is not injected properly into the hemolymph (i.e. injecting the micropipette too deep going into the brain tissue) then slower dispersion throughout the animal is observed. While dye may be used to practice a proper injection technique as shown in the video, it is not recommended to co-inject the blue food coloring with a compound to be tested as it may alter the properties of the compound and thus its bioactivity. Additionally, since most solutions used as solvent are clear in color (saline, DMSO, etc), it is difficult to see whether or not the compound was ejected from the injection needle. Therefore, when dissolving a specific compound it is important to ensure that it goes completely into solution; otherwise undissolved particles will rapidly clog the injection tip, preventing from any ejection of fluid. Furthermore, although compound dispersion may be immediate throughout the hemolymph, reaching the targets in the central nervous system, as well as reaching its maximum dosage, may take longer based on the compound's chemical properties, such as size and polarity, and its ability to permeate the fly's blood brain barrier.22 Thus, it is important to monitor potential effects of unknown compounds several minutes after injection because different compounds can have variation in times of onset effects, which may increase over time in some cases. Strong and immediate effects of the compound that completely block the function of neurons can already be seen with the triggered responses at 1 Hz, while stimulation of the GFS at higher frequencies (100 Hz) is used to detect more subtle effects due to lower dosage or potency of a compound. If no effects are observed after compound injection it can be due to either small drug dosage or the fact that the compound's specific molecular target is not present in the GFS.
Moreover, when using the bioassay presented here as a screening tool for novel compounds (such as conotoxins) it is important to note that the assay is restricted to the molecular targets found in the GFS of the fly. Although the assay itself does not permit locating the actual molecular targets of the compound injected, it does allow for the narrowing down of potential targets within the GFS. Additional tests, such as patch clamp on neurons or muscles or genetic interaction studies with Drosophila melanogaster mutants, can be done to determine the specific target of these compounds. Finally, the presented recording protocol was designed to detect antagonistic effects on the function of the GFS. However, the recording protocol may be easily adjusted to monitor for agonistic effects by passively monitoring for responses induced by the compound rather than testing if the GFS is not able to respond reliably when the circuit is stimulated by the experimenter.
No conflicts of interest declared.
We would like to acknowledge the members of the Mari lab and the Godenschwege lab, in particular Aline Yonezawa, for comments and help with this protocol. This work was funded by the National Institute for Neurological Disorders and Stroke grant R21NS06637 to F.M. and T.A.G.; A.B. was funded by the National Science Foundation award number 082925, URM: Integrative Biology for future researchers.
|Recording glass electrodes: borosilicate glass capillaries||World Precision Instruments, Inc.||1B100F-4||1.0mm OD, 0.58mm ID|
|Stimulator||Grass Technologies||Model S48|
|Amplifier||Getting Instruments, Inc.||Model 5A|
|Data acquisition Software: Digidata||Molecular Devices||Model 1440A|
|Data collection software: pCLAMP||Molecular Devices||Version 10|
|Stereomicroscope with fiber optic microscope ring illuminator||AmScope||SM-4T Model HL250-AR|
|Dissecting scope for mounting||AmScope||SM-2TZ|
|Kite Manual Micromanipulator & Tilting Base||World Precision Instruments, Inc.||Model # M3301 Kite: Model # KITE-M3-L|
|Drosophila melanogaster Wild 10E genotype (wild type strain)||Bloomington Stock center||Stock # 3892|
|Vertical pipette puller||David Kopf Instruments||Model 700c|
|Injection glass micropipettes: Borosilicate glass capillaries||World Precision Instruments, Inc.||Catalogue # 4878||1.14mm OD, 0.5mm ID|
|Silicon oil||Fisher Scientific||Catalogue # S159-500|
|Beveler||Sutter Instrument Co.||K.T. Brown Type Model # BV-10|
|Nanoliter2000||World Precision Instruments, Inc.||Catalogue # B203XVY|
|Blue food coloring||McCormick & Co.||N/A||Ingredients: Water, Propylene Glycol, FD&C Blue 1, and 0.1% Propylparaben (preservative).|
|Methyllycaconitine citrate (MLA)||Tocris Bioscience||Catalogue # 1029|
|Plastic wax sticks||Hygenic Corporation (Akron Ohio USA)|