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Using Optogenetics to stimulate neuromuscular junctions in Drosophila

melanogaster

Brett Fields14SS Neurophysiology Laboratory 4010 Section 003

AbstractThe rapidly growing field of Optogenetics offers unique and innovative ways to open ion channels and

cause depolarization in post-synaptic cells. We investigated the neuromuscular junction of Drosophilamelanogasterlarvae by stimulating the ChR2 light-gated ion channel with controlled blue light. We wanted to

investigate the effect of varying voltage, duration, and frequency on the fly larvae neuromuscular junction. Wefound no significant correlation between voltage and post-synaptic cell response but recommend further

investigation into this relationship. During extended periods of blue light stimulus, the cells depolarized every

18.4!3.9 ms. This translates to a post-synaptic cell maximum firing frequency of 54.5 Hz. For the frequency

tests, we found no significant trend of facilitation or depression between the first and second peaks but we did

find statistically significant depression between the first and fifth response. This depression seemed to increaseas frequency increased. Overall, our experiment provided us with a better understanding of the techniques and

applications of Optogenetics as well as post-synaptic responses in theDrosophilaneuromuscular junction.

IntroductionDrosophila melanogaster, or fruit flies, have

been used for research purposes for decades. This isdue to the species high rate of turnover and easily

manipulated genetic code. Drosophila can also be

easily anaesthetized for close investigation. Thefruit fly nervous system also shows severalsimilarities to the human nervous system. This

makes Drosophila a great candidate forneuroanatomical studies. Specifically, fruit flies are

most similar to the neurotransmitter systems andchannels of humans.

Because of their easily manipulated geneticsequence, fruit flies can be genetically modified to

express certain genes that they do not normallypossess. By using tissue-specific genetic expression

systems, scientists can express transgenes that allowfor precise and easily reversible manipulation of

neural activity. One particularly interesting systemis the GAL4-UAS system. Exploiting this

expression system allows for control of ionchannels and vesicle trafficking proteins that are

gated by light and temperature.

One of the most novel and innovative ways ofactivating these channels is the light-gated ion

channel channelrhodopsin-2 (ChR2). This channelis originally found in green algae but can be added

to the Drosophila genome. ChR2 is only activatedby blue light. When this channel is added to the fly

neurons and is activated, it causes depolarizationand an action potential. This method of activating

neurons is known as Optogenetics.At the synaptic level, Optogenetics does little to

change the fundamental principles of theneuromuscular junction. Synaptic plasticity

continues to allow for post-synaptic potentials(PSPs). These PSPs can be excitatory or inhibitoryExcitatory PSPs (EPSP) occur due to the process of

facilitation which happens when there is an increasein the number of readily available vesicles in the

pre-synaptic cell. When the number of availablevesicles decreases, depression is observed which

results in inhibitory PSPs (IPSP).By using Optogenetics, we attempted to study

the ChR2 channel in Drosophila. Specifically, wewere interested in studying the effects of prolonged

light stimulation, varying voltages, and rapidrepeated stimulation. By investigating the effect that

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A

B C

D

E

these variables have on the ChR2 channel, we hopeto gain a better understand of the use of

Optogenetics in research as well as the fundamentalprinciples of synaptic dynamics.

MethodsWe used Drosophila melanogaster larvae that

had been genetically modified to include the ChR2channel at the neuromuscular junction (NMJ).

Larvae were anaesthetized with ice and thendissected along the midline to expose the

neuromuscular junction. A recording electrode wasthen inserted into available cells. After dissection,

the larvae were kept in a bath of Calcium Chlorideto evoke stronger synaptic responses.

Our experimental rig consisted of a microscope,

a recording electrode which was connected to ananalog-to-digital (A-D) board, a blue LED lightwhich was also connected to the A-D board, and a

computer which connected to and interfaced withthe A-D board. The A-D board and computer

interface allowed us to have complete control of theblue LED light. The computer could specifically

engineer pulses of blue light to investigate variousaspects of the NMJ. Figure 1 shows the

experimental rig and its various components thatwere used to investigate aspects of the NMJ.

Figure 1. Experimental rig setup. A: Computer. B:

Analog-to-digital board. C: Blue LED light connected to

heat sink. D: Microscope with 2x lens. E: Recording

electrode filled with KCl.

ResultsTo investigate the use of Optogenetics on the fly

larvae NMJ, we used the computer to change

various aspects of the blue LED light such asvoltages, durations, and frequencies. Figure 2

depicts several examples of the post-synaptic cellrecordings that occurred during these light

variations.

Figure 2. Example recordings of three light variations

A: Cell response to 8V of blue light. B: Cell response to

prolonged duration (80ms). C: Cell response to five

sequential pulses (5Hz).

First, we investigated the effect that varyingvoltages had on the NMJ. We were unable to elicit

A.

B.

C.

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During this period of extended stimulus, wemeasured the rate at which the post-synaptic cell

fired. The cell depolarized every 18.4!3.9 ms. This

translates to a post-synaptic cell maximum firing

frequency of 54.5!2.3 Hz.

To investigate how the NMJ is affected duringrepeated short pulses of light, we measured the

responses for three cells at varying frequencies.Tested frequencies were 5Hz, 10Hz, 20Hz, 30Hz,

and 40Hz. Five pulses of blue light were deliveredat each frequency interval. Between the first and

second peak, the average change in amplitude was

-0.125 ! 0.4mV and the average proportional

difference was -0.0027!0.1mV. Between the firstand last peak, the average change in amplitude was

-1.26 ! 0.86mV and the average proportional

difference was -0.086!0.15mV. Table 4 displaysaverage peak strengths of the first and second peak

as well as a comparison of the difference betweenthese peaks. Table 5 displays average peak strengths

of the first and last peak as well as a comparison ofthe difference between these peaks.

Table 4. Average peak strengths of first and second

peak with amplitude difference.

Table 5. Average peak strengths of first and second peak

with amplitude difference.

Figure 5 displays the relative proportionaldifferences between the first and second peak and

the first and last peak as well as a trendline to helpdetermine the trend and correlation of the data.

Figure 5. Relative proportional differences between

peaks. A: Difference between first and second peak

Slope = -0.0005. R2 = 0.0042. B: Difference between

first and last peak. Slope = -0.0016. R2= 0.017.

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