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Electrophysiological Recording from Drosophila Larval Body-Wall Muscles

Bing Zhang and Bryan Stewart
Cold Spring Harb Protoc; doi: 10.1101/pdb.prot5487
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Protocol

Electrophysiological Recording from Drosophila

Larval Body-Wall Muscles
Bing Zhang and Bryan Stewart
INTRODUCTION
The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities
to synapses in other animals, including humans. These include the basic feature of synaptic transmission, as well as the molecular mechanisms regulating the synaptic vesicle cycle. Because of its large
size, easy accessibility, and the well-characterized genetics, the fly NMJ remains an excellent model
system for dissecting the cellular and molecular mechanisms of synaptic transmission. This protocol
describes the steps for performing intracellular recording from fly larval body-wall muscles and
explains how to record and analyze spontaneous and evoked synaptic potentials. Methods used
include larval dissection (filleting), identification of muscle fibers and their innervating nerves,
the use of a micromanipulator and microelectrode in penetrating the muscle membrane, and nerve
stimulation to evoke synaptic potentials.

RELATED INFORMATION
Details of how to assemble an electrophysiology rig can be found in Equipment Setup for Drosophila
Electrophysiology (Zhang and Stewart 2010a). This protocol includes equipment used in the Cold
Spring Harbor Laboratory (CSHL) course, Neurobiology of Drosophila, but equipment from
reputable commercial suppliers other than those mentioned here should work equally well. A method
for preparing microelectrodes is described in Fabrication of Microelectrodes, Suction Electrodes,
and Focal Electrodes (Zhang and Stewart 2010b). Those who are unfamiliar with the operation of
the amplifier and the software used for voltage-clamp experiments should see Electrophysiological
Recording from a Model Cell (Zhang and Stewart 2010c). Details for dissection of third-instar
larvae are found in Dissection of Drosophila Larval Body-Wall Muscles (Ramachandran and Budnik
2010). The Drosophila larval body wall muscle preparation was first used for electrophysiological
analysis in the 1970s (Jan and Jan 1976a,b). This preparation has become the gold standard for
studying neuronal excitability as well as synaptic transmission.

MATERIALS
CAUTIONS AND RECIPES: Please see Appendices for appropriate handling of materials marked with <!>, and

recipes for reagents marked with <R>.

Reagents
Drosophila melanogaster, wild-type strains (Canton-S or Oregon-R) and synaptic transmission
mutants (e.g., Syx3-69, snap-25ts, syt I, and lap), neuronal excitability mutants (e.g., eag, Sh
double mutant), or muscle excitability mutants (e.g., larvae that overexpress the inward
rectifier potassium channel Kir2.1 in body-wall muscles [referred to as the Kir mutant])

Adapted from Drosophila Neurobiology (ed. Zhang et al.). CSHL Press,

Cold Spring Harbor, NY, USA, 2010.
Cite as: Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5487
2010 Cold Spring Harbor Laboratory Press

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Maintain and raise all flies on cornmeal-based fly food at room temperature. Avoid crowding in the vials, so
that larvae can grow healthy and large.
<R>HL-3 saline (variable Ca2+)
Also prepare HL-3 saline without Ca2+, ice cold.

KCl, 3 M

Equipment
Dissection instruments
Electrophysiology rig (as described in Equipment Setup for Drosophila Electrophysiology
[Zhang and Stewart 2010a])
Microelectrodes, intracellular (as prepared in Fabrication of Microelectrodes, Suction
Electrodes, and Focal Electrodes [Zhang and Stewart 2010b])
Microscope
Petri dish (Sylgard-coated) and small insect pins
Alternatively, use a magnetic dish with paper clip pins (see Step 1).

Scissors (fine)
Suction electrode (as prepared in Fabrication of Microelectrodes, Suction Electrodes, and
Focal Electrodes [Zhang and Stewart 2010b])
Syringe, 1-mL or 5-mL
Tissue (e.g., Kimwipe)

METHOD
Dissecting Third-Instar Larvae
1. Dissect third-instar larvae as described in Dissection of Drosophila Larval Body-Wall Muscles

(Ramachandran and Budnik 2010). Use either a Sylgard-coated Petri dish and small insect pins or
a magnetic dish with paper clip pins. Perform the dissection in ice-cold, Ca2+-free HL-3 saline
(Stewart et al. 1994).
2. Using a pair of fine scissors, transect the segmental nerves loose from the base of the ventral nerve

cord so that the nerve end can be picked up with a suction electrode (Step 20).
3. Immediately following dissection, rinse the NMJ preparation at least three times with room

temperature HL-3 saline containing the desired level of Ca2+.

The purpose of these washes is to ensure that the bathing solution is free of any material that could clog the
suction electrode. We recommend using 0.8 mM Ca2+ in the HL-3 saline.
4. Position the NMJ preparation:
i.

Place the dish on the center of the microscope stage.

ii. Bring the preparation into focus and take note of the orientation, segments, and muscle

patterns.
iii. Place the preparation so that the posterior end of the larva points toward you.
This orientation gives enough space on the two lateral sides so that the electrodes can access the NMJ
without bumping into the insect pins.

Recording Membrane Potentials

5. Backfill a microelectrode with 3 M KCl solution:
i.

Fill the electrode approximately two-thirds full.

ii. Add one drop of the KCl solution at the end of the glass pipette so that the solution can be

drawn into the fine tip of the electrode through the built-in capillary (see Fabrication of
Microelectrodes, Suction Electrodes, and Focal Electrodes [Zhang and Stewart 2010b]).

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iii. Use a piece of soft tissue (e.g., Kimwipe) to remove any liquids outside of the electrode

so that salt crystals do not accumulate inside the electrode holder.

6. Position the microelectrode and microelectrode holder:
i.

Carefully insert the microelectrode into the holder.

ii. Tighten the screw cap slightly so that the microelectrode is held firmly in place.
Do not screw the cap on too tightly or the glass electrode may break.
iii. Make sure that the silver wire from the electrode holder is in contact with the KCl solution

inside the electrode lumen.

iv. Screw the electrode holder onto the head stage.
7. Place the reference (ground) wire into the bath saline (HL-3 saline).
8. Adjust the focal plane above your preparation and lower the microelectrode into contact with

the HL-3 saline.

The first sign that the electrode has entered the saline is that the digital voltmeter begins to display a steady
voltage.
See Troubleshooting.
9. Turn the Input offset knob in the ME1 panel until Vm reads 0 mV. Lock the knob if necessary to

avoid accidentally changing the setting.

10. Visually inspect the tip of the electrode for any obvious damage under the microscope. Replace it

if the electrode is broken or contains an excessive amount of debris or dirt.

11. Check the input resistance of the electrode by turning the toggle switch to the on position on

the Step Command panel and manually injecting 1 nA of current.

A good electrode for intracellular recording usually has an input resistance ranging from 15 to 25 M. Record
the electrode input resistance in a notebook to serve as a reference on which electrode to use in the future.
12. Perform bridge balance to cancel out the resistance of the electrode until the square wave

disappears (similar to those conducted in Fig. 1A-D). Use the Capacitance compensation knob
(with a yellow top) to increase the rise time.
Avoid overcompensation (i.e., when the trace will be ringing with high-frequency oscillations).
13. Lower the focal plane first and then the electrode so that the tip of the electrode is always within

the visual field. Repeat the focus down first and lower electrode next motion until the electrode
tip is just above the preparation.
A common mistake that most novices make is to lower the electrode too fast and too far, with the result that the
electrode is driven through the preparation!
14. Once the electrode is close to the muscle surface, switch the manipulator to the fine control

movement mode to position the microelectrode above a muscle fiber (e.g., muscle 6 in abdominal segment 4). Carefully lower the electrode above the preparation while adjusting the focal plane
toward the preparation.
15. Penetrate the muscle membrane slowly and gently. If you are using the Sutter P-250 motorized

manipulator, switch to the diagonal mode so that the electrode moves diagonally toward the
muscle, which makes membrane penetration easier.
Two hints that your electrode is touching or pressing on the muscle surface are that (1) the digital voltmeter
begins to show negative potentials up to 4 to 5 mV and (2) you may notice a dent on the muscle surface
at the tip of the electrode. The potential readings are a more reliable and sensitive indicator, especially if it is
difficult to clear the electrode tip. At this point, a slight advance or vibration of the electrode is sufficient to enter
the cell. There should be a rapid voltage drop to at least 45 mV.
16. Wait for the membrane potential to become more hyperpolarized. It should stabilize at 65 to

75 mV.
i.

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If the resting potential remains at -45 mV or is even more depolarized, withdraw the
electrode and try inserting it into the adjacent muscle or try a muscle cell in a different
segment.

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FIGURE 1. Bridge balance in a model cell. A problem one encounters during current injection is the serial resistance
from the microelectrode, which will mask the voltage response from the cell membrane following current injection.
Bridge balance allows one to determine the input resistance of the cell membrane by canceling out the serial resistance
of the microelectrode. (A-D) Performing bridge balance in the bath mode of a model cell. Following current injection,
the square waveform represents the voltage drop across the electrode resistor. (A) The peak amplitude (i.e., the plateau
portion) allows one to determine the input resistance of the electrode. Examples of under-adjustments (B) and overadjustments (C) of bridge balance. (D) A perfect bridge balance can be achieved by carefully canceling out the electrode
response. (E-H) Performing bridge balance in the cell mode of a model cell. (E) Before any bridge balance, current
injection results in a complex waveform, which has sharp rise and fall phases as well as slowly charging and discharging
components. The sharp components, which are essentially identical to the square wave in A, come from the electrode
resistor. The slow components are responses from the cell membrane. Examples of under-adjustments (F) and
over-adjustments (G) of the bridge. (H) Once the bridge is perfectly adjusted, the only remaining part is the response
from the cell membrane resulting from a parallel resistor-capacitor (RC) circuit. Membrane input resistance as well as
time constants can be readily determined from this waveform. Note the initial and tail sharp spikes (in B-D and F-H),
which result from capacitance currents of the electrode. The x-axis is in milliseconds.

ii. Continue this trial-and-error approach until a reasonably good resting potential is obtained.
Before inserting the electrode into another muscle cell, check its input resistance again, and replace the
electrode if its input resistance is dramatically altered.
See Troubleshooting.
17. Once a suitable and stable resting potential is obtained, cancel out the electrode resistance by

adjusting the bridge balance again:

i.

Inject a -1-nA current pulse for 400 msec duration at 1 Hz (Fig. 2A; Fig. 3A,B).

ii. Determine and record the muscle-input resistance (Rm) and the membrane time

constant . Record the Rm and values in the notebook.

Fly muscle Rm varies between 5 and 12 M, and is ~30 msec.

iii. Try a new muscle fiber if the input resistance of the recorded muscle is <5 M.
Readjusting the bridge balance is necessary because muscle membrane debris may alter the input
resistance of the electrode.

Synaptic Potentials
This section explains how to record synaptic potentials. Many of the previous steps are applicable here, but in addition,
a suction electrode must be properly positioned and used to stimulate motor nerves. Typically, the manipulator for
the suction electrode is placed on the side of the microscope opposite the recording electrode.

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FIGURE 2. Bridge balance in muscle cells. The examples shown here are intracellular recorded waveforms in response
to injection of -1-nA current square wave (400 msec duration) in body-wall muscles of a wild-type larva (A) and a larva
whose muscles express the human inward rectifying potassium channel Kir2.1 (Kir mutant) (B). Note the dramatic
reduction in both membrane input resistance and time constant in the Kir mutant. The electrode capacitance spikes
at the beginning and end of the current pulse are evident in these examples.

Positioning the suction electrode

18. Immerse the stimulating wire in saline:
i.

Lower the suction electrode just below the surface of the bath saline (HL-3 saline).

ii. Use a 1- or 5-mL syringe to gently apply negative pressure (suction) so that the HL-3 saline

fills one-half of the length of the suction electrode.

This ensures that the stimulating silver wire inside the electrode is deeply immersed in the saline. Avoid using
a larger (e.g., 10-mL) syringe because the suction it generates is too strong and difficult to control.

FIGURE 3. Intracellular recording of synaptic potentials from larval body-wall muscles. (A) An illustration of the thoracic
(T) and abdominal (A) segments and the repeated pattern of musculature arrangements (the brain and the ventral nerve
cord are removed in this image) in a dissected, flattened, and fixed third-instar larva. (Adapted, with permission, from
Bellen and Budnik 2000.) Major ventral, lateral, and dorsal muscles are labeled with numbers. The ventral midline is
indicated by the blue arrow. (B) One hemisegment of the body-wall muscles is illustrated here, with ventral (green),
lateral (black), and dorsal (blue) muscles marked (Hoang and Chiba 2001). Major surface muscles are identified by their
numbers. (Dashed red line) The ventral midline. A suction electrode is shown to pick up a segmental nerve and used to
stimulate the motor axons within the nerve. A microelectrode is used to record minis and evoked synaptic potentials
from muscle 6. Segments 4 and 5 are often used for synaptic transmission studies because the nerve leading to these
muscles is long and easier to pick up by the suction electrode. Ventral longitudinal muscles such as muscles 6, 7, 12,
and 13 are typically used for intracellular recordings, largely because of their large sizes and ventral positions. (C) Without
nerve stimulation, the muscle constantly displays spontaneous miniature synaptic potentials (called minis or mEJPs).
Following a single stimulus, the whole muscles response to transmitter released from the nerve terminal is recorded as
a compound EJP, which is more generally known as an excitatory postsynaptic potential (EPSP). (For color figure, see
doi: 10.1101/pdb.prot5487 online at www.cshprotocols.org.)

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iii. Place the silver wire near the shank of the suction electrode so that the wire remains in

touch with the saline.

iv. Stop suctioning by applying a small amount of positive pressure into the suction electrode.
This action prevents the suction electrode from continuing to suck and picking up nerves randomly along
its path. Avoid pushing the HL-3 saline out of the electrode. It is possible to maintain a steady state by
gently balancing the positive and negative pressures inside the electrode.
19. Lower the suction electrode to the segment where recordings from muscles will occur (e.g.,

abdominal segment 4, muscles 6 and 7).

Keep in mind that the nerve enters each segment from the anterior end, near the denticle belt area.
20. Once the open tip of the suction electrode is positioned near the nerve entry point, gently apply

a negative pressure until the nerve is pulled into the electrode lumen.
Avoid applying too much pressure into the suction electrode. Excessive pressure will cause the nerve to stretch
and pull away from the muscle, resulting in a partial or a complete failure to evoke synaptic responses.

Recording synaptic potentials

21. Record spontaneous minis:
i.

Once the suction electrode is in place, position the intracellular electrode and impale a
muscle of interest (e.g., muscle 6) (Fig. 3A,B).

ii. Perform a routine check of the input resistance and time constant, balance the bridge,

and perform capacitance compensation.

iii. Record spontaneous minis, which occur randomly at 2-5 Hz with average amplitude

ranging from 0.6 to 1 mV in wild-type larvae (Fig. 3C).

iv. Continue to record minis for 45 sec to 1 min.
These recordings can be used to determine the average amplitude of the minis as well as the average mini
frequency (see Step 30).
22. Evoke excitatory junction potentials (EJPs):
i.

Stimulate the motor nerve by delivering a current pulse of 150-200 sec duration,
gradually increasing the stimulus strength until a synaptic potential (commonly called an
EJP) is evoked.

ii. Once one EJP has been observed, increase the stimulus strength to excite another axon

(Kurdyak et al. 1994) because muscles 6 and 7 are dually innervated.

The threshold voltage needed to evoke EJPs is ~0.6 V. An example of a compound EJP on muscle 6 on
segment A4 is shown in Figure 3C.
See Troubleshooting.
23. Stimulate the motor nerve at a low rate (0.1-0.2 Hz) and record at least five different EJPs.
The ratio of the average EJP amplitude to the average mini amplitude determines the quantal content (see Step
30). The average amplitude of the EJPs will not be determined accurately if the nerve is stimulated at too
high a frequency (1 Hz), because frequent stimulation depletes the releasable pool of synaptic vesicles,
causing the EJP amplitude to gradually reduce with consecutive stimuli. This will lead to an underestimate of
quantal content.
To gain more experience recording synaptic potentials and performing quantal analysis, take recordings from
other well-established mutant flies. Compare the observations of wild-type larvae with the mutants and with
data from the published literature.

[CA2+]-DEPENDENT SHORT-TERM SYNAPTIC PLASTICITY

Once intracellular recording and nerve stimulation skills have been mastered, there are many other exciting areas of synaptic transmission to explore. These include testing the dependence of transmitter release
on extracellular [Ca2+] (Jan and Jan 1976b), investigating the effect of repetitive nerve stimulation on the
rate of synaptic vesicle pool depletion (Delgado et al. 2000), and studying activity- and [Ca2+]-dependent
short-term plasticity (Zhong and Wu 1991; Rohrbough et al. 1999; Bao et al. 2005).

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24. Design a stimulation paradigm that can deliver two pulses separated by 100 msec (10 Hz),

50 msec (20 Hz), 20 msec (50 Hz), and 10 msec (100 Hz), respectively. Observe the relative
amplitude of two evoked EJPs in HL-3 saline containing 0.6 mM Ca2+.
This is a classic exercise that is often referred to as twin-pulse or two-pulse facilitation (TPF).
25. Repeat the above experiments in NMJ preparations based in HL-3 saline containing 1, 1.5, and

2 mM Ca2+, respectively.
Examples of short-term synaptic plasticity at 30 Hz at 0.6 mM Ca2+ and 1.5 mM Ca2+ are shown in Figure 4A
and Figure 4B, respectively.

ELECTRODE CLEANUP
26. At the end of the day, rinse the suction electrode thoroughly with H2O and leave the electrode

to air-dry to prevent bacterial growth. When rinsing the suction electrode, always suck H2O in
and do not push the liquid out, because small bits of debris may clog the tip of the electrode,
rendering it useless.
A suction electrode, properly cared for, can be used repeatedly for as long as it works (6 mo or longer).

27. Discard the intracellular microelectrode, and turn off the electronics.

DATA ANALYSIS
28. Using Clampfit, analyze the input resistance and time constant of the muscle membrane, and the

amplitude of EJPs.
29. Use Mini Analysis to analyze the mini frequency and amplitude.
30. Estimate quantal content of a synapse by calculating the ratio of the average EJP amplitude and

the average mini amplitude, provided that the resting potential of different recorded muscles is
similar and that the [Ca2+] is relatively low.
Otherwise, nonlinear summation should be taken into consideration in determining quantal content (Martin
1976; Stevens 1976; Feeney et al. 1998).

TROUBLESHOOTING
Problem: No voltage can be detected when the microelectrode is placed in the HL-3 saline bath

solution.
[Step 8]
Solution: There may be an open circuit. Consider the following:
1. Look for broken wires, and replace any that are found.
2. Confirm that the ground wire is in the bath.

FIGURE 4. Short-term synaptic plasticity at the larval NMJ. Synaptic release of transmitter is not fixed but, rather,
plastic, depending on the amount of extracellular [Ca2+] and nerve activity patterns. (A) At low [Ca2+] (0.6 mM), the basal
amplitude of EJPs is small. In response to twin-pulse stimulation at 30 Hz, the amplitude of the second EJP is larger
compared with the amplitude of the first one, revealing the ability of the synapse to facilitate. (B) At high [Ca2+]
(1.5 mM), the basal amplitude of EJPs is large. Instead of facilitation, now the same synapse displays short-term
synaptic depression following twin-pulse nerve stimulation.

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3. Look for a large air bubble inside the electrode tip that got there when the microelectrode was

backfilled.
4. Determine whether the silver wire inside the electrode holder is contacting the golden pin.
Problem: Resting potentials of the impaled muscle cell never hyperpolarize beyond 35 to 45 mV.
[Step 16]
Solution: Wild-type larval body-wall muscles have a resting potential from 65 to 75 mV. A poor rest-

ing potential usually means that the muscle has been damaged during dissection or penetration
with the microelectrode. Consider the following:
1. Healthy muscles have a translucent appearance, whereas degenerating muscles are brownish or

visibly tearing apart from the muscle attachment points in the denticle belt area. This tends to
occur if the preparation is left at warm temperatures. Keep and work with muscle preps in a cooler
room or cooled microscope stage (18C to 20C).
2. Physical tearing or damage to muscles should be avoided at all costs during dissection or electrode

impalement. However, electrode-caused damage is difficult to detect by eye. One way to detect
any damage is to measure the muscle input resistance and time constant. Both of these parameters will reduce dramatically in a damaged muscle. Thus, it is always a good idea to routinely monitor muscle input resistance and time constants in all of your experiments.
Problem: Although the resting potentials of the muscle and minis were normal, no evoked synaptic

potentials were detected when the segmental nerve was stimulated.

[Step 22]
Solution: Consider the following when evoking synaptic potentials:
1. The stimulator must be turned on and the voltage output level must be high enough to evoke

action potentials. Gradually increase the amplitude of the stimulus strength until an evoked potential is seen.
2. Check to see if the polarity of the stimulus was reversed. Switch the polarity of the stimulus isola-

tor and test again.

3. Determine if the suction electrode contains enough HL-3 saline so that the stimulating wire is in

contact with the saline. (If the wire is not in contact with the saline, then no current can pass
through the suction electrode.) Gently push the nerve ending off the suction electrode, raise the
suction electrode away and above the NMJ, and refill it with HL-3 saline. It is important to raise
the suction electrode so that no nerves are picked up during refilling. Lower the electrode and pick
up the nerve ending again.
4. Inspect the nerve for damage that may have occurred during dissection. If the forceps have

pinched the nerve, you will not get normal EJPs. Start over with a new dissection.
5. If too much pressure is applied to the suction electrode when picking up the nerve, it can cause

disruption of the synaptic contact. Switch the suction electrode to a different segment and try to
gently pick up the nerve.
6. Verify that the HL-3 saline contains Ca2+, which is essential to trigger exocytosis. Prepare HL-3

saline containing Ca2+ and repeat the experiment.

Problem: Instead of an EJP with peak amplitude of 25 mV, an odd-looking EJP was obtained having

much smaller amplitude and unusually slow rising and falling rates.
[Step 22]
Solution: Most likely the motor nerve from a neighboring segment was stimulated. Larval body-wall

muscles are electrically coupled by gap junctions with homologous muscles in the neighboring segments (Gho 1994). EJPs as well as minis can passively invade the neighboring muscles resulting in
EJPs or minis with much slower kinetics. The minis with slow rising and falling phases should be
excluded in determining mini frequency and amplitude.

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ACKNOWLEDGMENTS
We thank the members of our laboratories for their important contributions to our research programs.
B.Z. especially thanks Hong Bao for her expert assistance with the Neurobiology of Drosophila
course and for providing some of the figures in the chapter from which this article was adapted.
Research in the Zhang laboratory was supported by grants from the National Science Foundation
(NSF), National Institutes of Health (NIH), Oklahoma Center for the Advancement of Science and
Technology (OCAST), and research funds from the University of Oklahoma. B.Z. also thanks Phillip
Vanlandingham, Rudhof Bohm, and Hong Bao for their constructive comments. B.S.s research was
supported by a Canada Research Chair and grants from the Canadian Institute for Health Research,
the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation
for Innovation. An extensive citation of all publications related to the topics covered in this protocol
was not possible because of space limitations. We therefore focused on select initial publications and
apologize to our colleagues whose publications were not cited.

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