Journal of Insect Physiology 117 (2019) 103900

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Journal of Insect Physiology

journal homepage: www.elsevier.com/locate/jinsphys

Chloroform and desflurane immobilization with recovery of viable

T
Drosophila larvae for confocal imaging
Duygu Cevika, Meryl Ackera, Pouya Arefia, Reza Ghaemib, Jimmy Zhanga, P.
Ravi Selvaganapathyb, Ian Dworkina,b, J. Roger Jacobsa,
a
Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada
b Department of Mechanical Engineering, McMaster University, Hamilton L8S 4L7, Canada

ARTICLE INFO ABSTRACT

Keywords: Imaging of living, intact Drosophila larvae is challenged if normal bodily function must be observed or when healthy larvae
Anesthesia must be recovered for subsequent studies. Here, we describe a simple and short protocol that employs transient airborne
Drosophila chloroform or desflurane (1,2,2,2-tetrafluoroethyl difluoromethyl ether) to effi-ciently immobilize larvae without the use of
Chloroform manipulation devices, vaporizers or imaging chambers. This non-lethal method allows the use of anesthetics while allowing
Desflurane
tracking of individual Drosophila into adulthood for follow-up experiments. At dosages sufficient to immobilize larvae,
Larvae
Desflurane, but not chloroform reduced the central nervous system response to auditory stimulus. Desflurane doses were
Immobilization
sufficient to arrest the heart, however significant rapid recovery was observed. With our method, chloroform provided more
rapid anesthesia but slower recovery than Desflurane. Without specialized hardware, this technique allows for repeated
imaging of living Drosophila larvae.

1. Introduction Sigrist, 2012; Zhang et al., 2010) or microfluidic devices (Ghannad-Rezaie et

In vivo microscopy and time-lapse of fluorescently-tagged proteins
enables scientists to observe developmental events at the cellular level in
Drosophila melanogaster embryos (Reed et al., 2009; Vanderploeg et al.,
2012). Even though larvae can be physically constrained using double sided
tape on a glass slide (Makhijani et al., 2011), the resolu-tion of this technique
in larvae is limited by ongoing peristaltic waves of contraction in the body
wall. Follow-up experiments on the same larvae are challenging, because
immobilization techniques such as an adhesive strip can stress larvae and
injure them upon removal. Previously we characterized a microfluidic device
which physically constrains the larvae, and allows efficient observation of
central nervous system (CNS) activity, however, recovery of healthy larva
was not assured (Ghaemi et al., 2015). Chloroform is the oldest anesthetic
drug, in the form of a colorless or light blue liquid (addition of dyes to
facilitate identification), also non-flammable or explosive. However, this
chloroform is very rarely used because, it can be easily oxidized under air and
light becomes very dangerous phosphates . Besides chloroform is also
hepatotoxic which can damage the liver (Andlauer and Segras)
Corresponding author.
E-mail address: jacobsr@mcmaster.ca (J.R. Jacobs).
al., 2012) that allow gases to be contained in a small space. This procedure
requires the use of such devices throughout the imaging process and creates
anesthetic venting issues. These devices must be customized for each imaging
technique such as confocal microscopy or OCT (optical coherence
tomography). Anesthetics such as chloroform have been introduced by
dissolving them in halocarbon oil and then immersing larvae in this medium
(Baqri et al., 2009), but it is chal-lenging to regulate the dosage and removal
of the anesthetic using this technique. Customized apparatus can be adapted
so that anesthetic vaporizers can better regulate and extend dose (Andlauer
and Sigrist, 2012).
Our aim was to develop a technically simple and short protocol that
allowed us to immobilize larvae quickly and did not cause lethality so that
follow-up experiments on the same organism can be performed. Our
technique does not require an imaging chamber or the immersion of larvae in
liquid or oil media in order to anesthetize larvae. We em-ploy calibrated doses
of airborne chloroform or desflurane gradually released from a cotton plug in
a closed chamber. The immobilized third instar larva can be observed for
extended periods after the larva is re-moved from the chamber. Chloroform,
or trichloromethane (CHCl3) is a fast acting anesthetic with a long history of
medical use (Wawersik,

https://doi.org/10.1016/j.jinsphys.2019.103900
Received 10 December 2018; Received in revised form 3 June 2019; Accepted 12 June 2019
Available online 13 June 2019
0022-1910/ © 2019 Elsevier Ltd. All rights reserved.
D. Cevik, et al.
1997). Its use in the laboratory is favored over other volatile anesthetics
because it is not flammable. Desflurane (C 3H2F6O) is another medical
anesthetic that has been used for live imaging studies of Drosophila larvae
(Andlauer and Sigrist, 2012; Füger et al., 2007; Zhang et al., 2010). Research
institutions without a veterinarian cannot easily obtain desflurane, and may be
constrained to chloroform use.
Here we assess and compare the efficiency of chloroform and des-flurane
vapors for reversible immobilization, and also characterize the physiological
effects of anesthetic exposure on third instar larvae by determining the larval
survival rate, heart rate and CNS response.
2. Materials and methods
2.1. Preparing larvae for immobilization
1) Plug a rayon ball into one end of an open ended glass tube. All our
experiments were done with a 40 mm ball of rayon pushed into a 28 mm
diameter, 60 mm high scintillation vial with the glass bottom removed, so
we suggest this standard. The rayon should not be closer than 35 mm to
the larva.
2) Place up to 5 third instar larvae on a glass slide. Do not use a plastic
surface. The larva can be water rinsed and dried gently with a lab tissue
before placement. Water treatments do not alter viability.
3) Place the tools from step 1 and 2 in a fume hood and follow the rest of the
protocol described below in 2.2 or 2.3.
2.2. Immobilizing third instar larvae with airborne chloroform
1) Pipette 500 μl of chloroform (ACP Chemicals C-3300, 99.8%) on the
rayon from step 1 of 2.1. Chloroform should be released into the middle of
the rayon, accessed from inside the tube.
2) Place the open-ended side of the tube on a surface for three seconds.
3) Place the open side of the tube on the glass slide that has larvae on it for
24 s. (Various exposure times and their physiological effects are discussed
below. Exposure times can readily be adjusted, but in-termittent exposures
are essential for reversible anesthesia.)
4) Repeat steps 2 and 3. Do not add fresh chloroform.
5) Remove the tube. The larvae are now immobilized. They may dry and
adhere to the glass slide if left in the same position for a long time, so
transfer them to moist surface such as a fruit-juice/agar plate if necessary.
Otherwise you can mount them on a slide and image them immediately.
2.3. Immobilizing third instar larvae using airborne desflurane
1) Cut a 7.5 × 1.5 cm filter paper (Whatman, 185 mm, qualitative filter paper)
and position it inside the scintillation vial (from step 2.1) so that it sits as a
collar above the thread of the narrow end of the vial. Dampen this paper
with double-distilled water.
2) Use a 1 ml syringe to put 500 μl of liquid desflurane (Suprane, Baxter
Corp. Mississauga ON) on the rayon from step 1 of 2.1. Desflurane should
be released into the middle of the cotton, accessed from in-side the tube.
3) Immediately place the open side of the tube on the glass slide that has
larvae on it for ten minutes.
4) Remove the tube and repeat step 2 and 3 so that larvae will receive a
second dose of desflurane.
5) Remove the tube. The larvae are now immobilized. They may dry and
adhere to the glass slide if left in the same position for a long time, so
transfer them to moist surface such as a fruit-juice/agar plate if necessary.
Otherwise you can mount them on a slide and image them immediately.
2.4. Fly stocks and anesthetization of the larvae for all experiments
Stocks were raised at room temperature on yeast fly food. 5 larvae at
2
Journal of Insect Physiology 117 (2019) 103900
a time were exposed to chloroform or desflurane in two doses with a break in
between as described in protocol 2.2 and 2.3. For chloroform, step 3 of
protocol 2.2 was modified to perform two doses of either 18, 24 or 30 s of
chloroform treatment. Larva handling artifacts were ad-dressed in controls by
placing larvae on a glass slide for the equivalent time, and covering larvae
with an equivalent, but un-dosed anesthetic cylinder. For all experiments,
migratory third instars were used.
During CNS response and heart rate experiments, control larvae were
immobilized with double-sided tape (Scotch “Permanent” – 3 M) on a glass
slide. To keep conditions consistent, anesthetic exposed larvae were placed on
double-sided tape during these experiments.
Larvae used in CNS response experiments were ChaT-Gal4/CyO;
UAS-GCaMP5G/TM3 (Bloomington 56500). Confocal imaging was per-
cc0079
formed on HmldsRed, vkg /CyO larvae (created from (Buszczak
et al., 2007) and Hml -DsRed (Makhijani et al., 2011)). In all other
1 1118
experiments, y w (Bloomington 6598) were used.
2.5. Determining the optimal anesthetic dosage
Our objective was to optimize a protocol that allowed for 20 min of
imaging. Preliminary experiments determined that interrupted anes-thetic
exposure reduced the lethal effects of exposure. Therefore we optimized an
exposure protocol with a single interruption. For mobility tests, five larvae at
a time were exposed to 500 μl of chloroform or desflurane evaporated from a
cotton plug, with a single break between two exposures. With 10 repetitions,
50 larvae were sampled for each data point. Desflurane acted slowly with our
method, in contrast to use of a vaporizer. Larger doses did not efficiently
increase speed, so we opted for a longer dosing protocol. Ideal anesthetic
dosage was de-termined by quantifying the maximum immobilization time
and mini-mizing the toxic effects that increased anesthetic exposure might
have on larvae. From here onwards, treatment times will be indicated with s
(seconds) or m(minutes) and ×2(two doses). For example: 18 s × 2
chloroform (two doses of chloroform that were each 18 s long with a three
second break in between).
2.6. Assessing mobility
Following anesthetic exposure, larvae were put on a transparent fruit-
juice/agar plate. The initial positions of larvae were marked with a pen on the
surface of the plate. Larvae were observed for four hours: every five minutes
for the initial 30 min and every 30 min for the re-maining 3.5 h. It was
recorded if each larva was moving its body or mouth-hooks during ten
seconds of observation. If a larva had moved away from its initial position
during the time while not being observed we scored the larva for locomotion.
Immobilization scores were re-peatedly measured for each larva for four
hours, and normalized for each experimental group of five larvae. Complete
immobility = 3, mouth movement only = 2, body movement = 1, locomotion
= 0. Experiment was performed 10 times with 5 larvae each trial.
2.7. Videos of anesthetized larvae
Larvae exposed to 24 s × 2 chloroform, 10 m × 2 desflurane or handled as
a control were rolled on a slide with a thin layer of halo-carbon oil. Larvae
were imaged using the brightfield setting on a fluorescent stereo microscope
(Leica M165 FC) for 30 min after being anesthetized.
2.8. Quantifying survival into adulthood
Following the anesthetic or control treatment, larvae were kept in tubes at
room temperature with yeast fly media. Halocarbon groups (Fig. 2B) were
gently rolled on a glass slide with a thin smear of ha-locarbon 700
(Halocarbon Inc.) before being placed in the pairwise tubes.
D. Cevik, et al.
The number of emerging adults and dead larvae/pupae were scored for 13
replicates of five larvae. Analysis is described in 2.12.
2.9. Measuring CNS auditory response
In order to detect the possible changes in neural physiology due to
anesthesia, we measured the neural activity of the CNS in response to
auditory stimulus in larvae under anesthesia. To do so, we used a sound-
insulated box under a fluorescent microscope (Ghaemi et al., 2015) and the
transgenic stock ChaT-Gal4/CyO; UAS-GCaMP5/TM3. We sought to
visualize an aggregate CNS response to sound vibration with a fluorescent
reporter of intracellular calcium flux. GFP-tagged GCaMP5G emits
fluorescence in cells in neurons depolarized by ex-citation (Akerboom et al.,
2012). Employing Cha-GAL4 directed ex-pression of GCaMP5G in choline
acetyl transferase expressing cells (Salvaterra and Kitamoto, 2001), we
monitored the calcium response in the larval brain to acoustic vibration.
Within 20 min after the anesthetic exposure, larvae were subjected to a single
500 Hz, 105 dB acoustic signal for five seconds. Previous research
demonstrated a robust GCaMP5G response to auditory stimulus in third instar
brains under normal conditions (Ghaemi et al., 2015). All groups were
immobilized on a glass slide using tape during imaging. Larvae that were
imaged later than one hour were hydrated on an apple juice agar plate until
imaging. No repeated measurements were performed. Using ImageJ
(Schneider et al., 2012), the percent change in response was determined from
the ratio of GFP fluorescence intensity produced during the au-ditory stimulus
to the baseline intensity observed two seconds before the stimulus. Ten larvae
were used per treatment.
2.10. Measuring heart rate
Larvae were filmed under a compound microscope immediately after
anesthesia, or after one to nine hours. After anesthetic treatment, larvae were
kept on an apple juice agar plate until filming. Larvae were positioned dorsal
side up on a glass slide with double sided tape during filming, which was done
within 20 min. No repeated measurements were done. Heart rate was
determined from subsequent video analysis with ImageJ. Ten larvae were
used per treatment.
2.11. Confocal imaging
A mounting slide was set up beforehand to minimize the time be-tween
the anesthesia treatment and imaging. Mounting slides were prepared with
two supports made of a stack of two coverslips layered with two-sided tape
(see graphic abstract). After placing the larvae between the two supports, they
were anesthetized with 24 s × 2 chloroform or 10 m × 2 desflurane. A thin
smear of halocarbon was added on a third coverslip, which was then
positioned over the larva, supported by the two coverslip stacks. The third
coverslip can be re-moved later for reuse of the mounting slide. Single
sections were ob-tained on an upright Leica SP5 at 63× within 20 min after
anesthetic, with a scan speed of 100 Hz, line averaging and zoom of one or
three times.
2.12. Statistical analysis
All statistical analyses were fit in R 3.5.0 (R Core Team, 2018), using the
lmer function in the lme4 library (Bates et al., 2015). As the re-sponse
variable was continuous, but bounded by 0 and 1, model re-siduals were
checked to confirm that they conformed to approximate normality. To
evaluate the overall model, a type II analysis of variance was used using the
Anova function in the car library (Fox and Weisberg, 2011). Coefficients and
their 95% confidence intervals were extracted using the lsmeans function in
the lsmeans library (Lenth, 2016). Pair-wise comparison among model
coefficients (including adjustments for multiple comparisons using the Tukey
method) was done using cld
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Journal of Insect Physiology 117 (2019) 103900
function in emmeans (Lenth, 2016), which adjusts for multiple com-parisons
via the glht function in the multcomp library (Hothorn et al., 2008).
For the immobilization data, the maximal model fit was a linear mixed
model, used with time, treatment and their interactions as fixed effects and
with a random effect of grouping structure that allowed both the overall
intercept and time component (i.e. random slopes model for the temporal
effects) to vary among grouping structure given the longitudinal (“repeated
measure”) design of this experiment. The im-mobilization data was
partitioned into early and late (before and after the 25 min evaluation time
point to separately examine the intended period of observation and the period
of recovery). For the early data (before 25 min), the model fit was singular
(attempted with 4 different optimizers) and as such a simpler linear mixed
model was fit with the same fixed effect structure as discussed above, but
where time was not allowed to vary according to grouping structure (sets of
individuals tested nested within treatment). Pairwise comparisons among
treat-ment groups were performed and adjusted as discussed above.
For the survival data, a logistic regression was used, modeling sur-vival
on treatment groups. A separate analysis model was performed for the
experiment with halocarbon, and without. Analysis of variance and pairwise
contrasts of estimates were performed as described above.
For the CNS auditory response, a linear model (ANOVA) was per-formed
to examine whether there was an immediate effect each for chloroform and
desflurane. Only the desflurane treatment was re-measured one hour later to
track recovery.
For the heart rate measures for the desflurane exposure experiment a
simple linear model was used with treatment, recovery and their in-teractions.
For the chloroform exposure experiment a similar linear model (with
continuous predictors) for time and treatment (in seconds) was estimated.
3. Results
3.1. Airborne chloroform and desflurane immobilize larvae
Our objective was to optimize an anesthetic protocol that could provide
approximately 20 min of observation time-sufficient for col-lecting video or
confocal data. Here we characterize the degree and time-course of
immobilization subsequent to differing exposures to anesthetic.
Immobilization was evaluated over a 4-hour period by quantifying complete
immobility, mouth movement only, body move-ment and locomotion (Fig. 1).
Controls show that under normal con-ditions, larvae move less when they are
first placed on a new surface, but they become motile within a minute.
Supplementary Table S1 provides a statistical comparison how an-esthetic
dosage affected mobility over time. All of the chloroform or desflurane treated
larvae were significantly less motile than the controls during the first 25 min
(Table S1A, B). Higher doses of chloroform (24 s × 2, 30 s × 2) were
associated with higher immobilization rates than the lower dose (18 s × 2)
during this period. Additionally, des-flurane treated larvae were significantly
more immobilized for the first 25 min than chloroform treatment, except 30 s
× 2 (Table S1A). Video tracking of the first 30 min of 24 s × 2 chloroform or
10 m × 2 des-flurane treatment are shown in Fig. S1. We noticed intermittent
pharynx movement during chloroform anesthesia that may limit assay length
of CNS activity (Fig. S1).
Between 30 and 240 min, higher doses of chloroform were asso-ciated
with higher immobilization rates. Larvae treated with 18 s × 2 or 24 s × 2 of
chloroform regained full mobility in 3 h, whereas 30 s treatment group fails to
recover completely after 4 h. The effect of desflurane on mobility was not
observed beyond 60 min.
3.2. 30 s × 2 chloroform treatment increases larval/pupal lethality
High survival rates of anesthetized larvae into adulthood (eclosure)
D. Cevik, et al.
Fig. 1. Normalized immobilization scores over 4 h subsequent to chloroform exposure. Treatments include two exposures of the indicated time: 2 doses of 18–30 s of chloroform or 2
doses of 10 min of desflurane. Higher doses of chloroform were associated with higher immobilization scores overall. Desflurane treatment had a higher immobilization score for the first
25 min. Error bars indicate s.e.m. 50 larvae per data point. Statistical model in Table S1.
Fig. 2. Eclosion of yw flies subsequent to chloroform or desflurane exposure shown in
beeswarm plots. (A) 2 doses of 30 s long exposures to chloroform (asterisk) was
associated with a significant decrease in eclosion compared to the control when not
covered in halocarbon oil (ANOVA, α = 0.05). (B) Rate of eclosion was not significantly
different between the control, 24 s × 2 chloro-form or 10 m × 2 desflurane when
anesthetized larvae were covered with ha-locarbon oil.
can enable researchers to perform longitudinal studies where imaged larvae
are tracked to adulthood. Moreover, survival rates can suggest treatment
toxicity. Normal rates of eclosion vary with experimenter’s experience in
larva handling, and ranged from 70 to 90%. Similar rates of eclosion were
seen for all treatments except the 30 s chloroform group (Fig. 2A; Table
S2A). Additionally, we tested if covering the anesthetized larvae with
halocarbon oil would affect the eclosion rates
4
Journal of Insect Physiology 117 (2019) 103900
(Fig. 2B; Table S2B). There was no statistical significance in the dif-ference
between the eclosion rates of controls, 24 s × 2 chloroform or 10 m × 2
desflurane groups when larvae were covered with small amounts of
halocarbon oil. However, we noticed that larvae do not recover from this
treatment if their spiracles were dipped in oil. This can be avoided by gently
rolling the anesthetized larva on a glass slide that has a thin layer of
halocarbon spread on it.
3.3. CNS auditory response was compromised after 30 s × 2 chloroform
and 10 m × 2 desflurane exposure
Previously, it was shown that diethyl ether anesthetic decreases larval
CNS response to auditory stimulus (Ghaemi et al., 2015). We sought to
determine whether chloroform or desflurane acted similarly, by employing a
fluorescent reporter of intracellular calcium as a proxy for neural activity
(Akerboom et al., 2012). Chloroform treatments of 18 s × 2 or 24 s × 2 did
not reduce CNS responses to vibration (Fig. 3; Table S3A, B). The response
was visually apparent in false color (Fig. 3A, B) and quantitatively in Fig. 3C.
However, the 30 s × 2 dose of chloroform was associated with a loss of
response to acoustic vibration, for at least 20 min. In contrast, 10 m × 2
desflurane exposure sig-nificantly decreased the CNS response to acoustic
vibration, although recovery was rapid.
3.4. Heart rate is transiently decreased subsequent to chloroform and
desflurane exposure
Exposure to chloroform or desflurane produced strong, transient
depression of heart rate in all groups, followed by a longer, lesser de-cline in
heart rate (Fig. 4, Table S4A, B). Desflurane was more potent, triggering
cardiac arrest in some cases. Drosophila has a myogenic heart rate that can be
accelerated by neural input (Sláma and Farkas, 2005). It is possible that the
transient depression reflects the short-term loss of neural input, followed by a
protracted recovery of neural regulation.
3.5. Imaging of hemocytes in chloroform anesthetized larva
Subsequent to 10 m × 2 desflurane treatments (Fig. 5A, B) or
24 s × 2 chloroform or treatments (Fig. 5C, D), motility of the larvae and
peristalsis were reduced sufficiently to image intact larvae with GFP tagged
Collagen IV (vkgGFP) and hemocytes labeled with HmldsRed
D. Cevik, et al. Journal of Insect Physiology 117 (2019) 103900

Fig. 3. Change in calcium induced fluorescence intensity with auditory stimulus. (a) Pre stimulus fluorescence in false color. (B) Fluorescence intensity immediately subsequent to a 500
Hz dB stimulus. Expression of GCaMP5G in choline acetyl transferase expressing neurons was directed by Cha-GAL4 in control larva. (C) Box-whisker plot showing the CNS auditory
response of the control, chloroform and desflurane treated groups immediately and 1 h after the anesthetic treatment. 30 s × 2 chloroform or 10 m × 2 desflurane exposures cause a
significant (*) decrease in the CNS response compared to the control. Desflurane group recovers within an hour (simple linear model, α = 0.05) n ≥ 10 larvae/treatment.

(Fig. 5). Hemocytes accumulated around hepatocyte-like cells called
oenocytes (Gutierrez et al., 2007; Makhijani et al., 2011), outlined by
VkgGFP signal. Larval health was confirmed by monitoring heartbeat at the
end of imaging. The use of halocarbon oil as a mounting medium is
discretionary, and can increase image resolution and survival.
In some cases there was a brief heart arrest right after the desflurane
treatment. If a coverslip was positioned on the larva before the heart resumed
beating, the recovery of the heart was considerably delayed. Experimenters
should ensure there is little pressure on the heart during imaging and ensure
that the heart resumes contraction before imaging.

Fig. 4. Box-whisker plot showing the heart rate of third instar larvae that were twice exposed to chloroform or desflurane. In chloroform groups, heart rate was measured immediately or
after 1, 4 or 9 h following the chloroform exposure. In desflurane group, heart rate was measured immediately or after 1 h. Heart rates were lowest immediately after anesthetic
treatments, but recovery was observed with both anesthetics. n = 10–13. Statistical model in Tables S2 and S3.

5
D. Cevik, et al.
Fig. 5. Lateral view of vkg-GFP, hmldsRED/CyO third instar. Larvae anesthetized with desflurance (10 min × 2; A, B) and with chloroform (24 s × 2; C, D) Dorsal is at top and anterior
is at left. Hemocytes (red) accumulate around oenocytes (asterisk) that are outlined by Collagen (green) structures visible t hrough the cuticle and the ectoderm of the intact larva. Scale
bar: (A, C) 25 μm; (B, D) 10 μm.
4. Discussion
Previous studies document how specialized imaging chambers and
sustained anesthetic delivery can provide sub-cellular resolution for periods
longer than an hour (Andlauer and Sigrist, 2012; Zhang et al., 2010). Here we
report the feasibility of a simplified, higher throughput approach, allowing
efficient documentation or screening for shorter periods at lower resolution.
We have demonstrated that transient chloroform or desflurane anesthesia is a
simple and effective im-mobilization technique that allows larvae to be
imaged for at least 20 min with or without using halocarbon oil as a mounting
medium. Anesthesia and handling can be performed with a minimum of spe-
cialized equipment. 10 m × 2 desflurane offers the most efficient im-
mobilization for the first 25 min following anesthetic treatment, but it is not
ideal for longer imaging studies, or if preparation time must be short (eg.
subsequent to experimental treatment), as anesthesia requires 20 min unless a
vaporizer is used. For those cases, chloroform can be used because larvae are
anesthetized in a few minutes, and although recovery is slower, it is still
complete. We converged on an optimal exposure of 24 s × 2 for chloroform,
because it was efficient and the survival rate was not affected. Longer
exposure time to chloroform (30 s × 2) should be avoided as it results in a
higher rate of side effects such as delayed recovery, lethality and CNS
depression.
Desflurane provides a stronger anesthetization and a faster recovery than
chloroform. At the recommended doses, desflurane (10 m × 2) resulted in
more profound short-term depression of CNS activity and
6
Journal of Insect Physiology 117 (2019) 103900
cardiac arrest. For CNS imaging experiments, the level of anesthetiza-tion
achieved by chloroform may not be sufficient for recording three-dimensional
stacks or extended time-lapse video. However, for simpler applications, for
which a shallower level of anesthetization is sufficient, use of chloroform, as
described here, offers a faster, inexpensive and efficient alternative. For
example, heart contraction dynamics can be visualized for a longer period in
larvae immobilized with chloroform, whereas the heart is arrested at ideal
doses of desflurane.
The long-term effects of the recommended dose of chloroform (24 s × 2)
or desflurane on the CNS or heart rate were minimal. These results indicate
that physiological effects of transient anesthesia are largely reversible and do
not affect viability. This allows for repeated observation of individual
Drosophila, including recovery of adults, without custom hardware.
Acknowledgements
Thanks to Megan Collie, Jake Szamosi, James St. Pierre and Dr. Ben
Bolker and to Dr. Ozan Erdem for their valuable contributions to sta-tistical
testing and data representations. Supported by NSERC Discovery 46651 to
JRJ and Canada Research Chair award to PRS.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jinsphys.2019.103900.
D. Cevik, et al.
References
Akerboom, J., Chen, T.-W., Wardill, T.J., Tian, L., Marvin, J.S., Mutlu, S., Carreras Caldéron,
N., Esposti, F., Borghuis, B.G., Sun, X.R., Gordus, A., Orger, M.B., Portugues, R., Engert,
F., Macklin, J.J., Filosa, A., Aggarwal, A., Kerr, R.A., Takagi, R., Kracun, S., Shigetomi,
E., Khakh, B.S., Baier, H., Lagnado, L., Wang, S.S.-H., Bargmann, C.I., Kimmel, B.E.,
Jayaraman, V., Svoboda, K., Kim, D.S., Schreiter, E.R., Looger, L.L., 2012. Optimization
of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840.
https://doi.org/10.1523/JNEUROSCI.2601-12.2012.
Andlauer, T.F.M., Sigrist, S.J., 2012. Building an imaging chamber for in vivo imaging of
Drosophila larvae. Cold Spring Harb. Protoc. 476–480. https://doi.org/10.1101/pdb.
prot068585.
Baqri, R.M., Turner, B.A., Rheuben, M.B., Hammond, B.D., Kaguni, L.S., Miller, K.E.,
2009. Disruption of mitochondrial DNA replication in Drosophila increases mi-
tochondrial fast axonal transport in vivo. PLoS One 4, e7874. https://doi.org/10.
1371/journal.pone.0007874.
Bates, D., Martin Maechler, M., Bolker, B., Walker, S., 2015. Fitting linear mixed-effects
models using lme4. J. Stat. Softw. 67, 1–48. https://doi.org/10.18637/jss.v067.i01.
Buszczak, M., Paterno, S., Lighthouse, D., Bachman, J., Planck, J., Owen, S., Skora, A.D.,
Nystul, T.G., Ohlstein, B., Allen, A., Wilhelm, J.E., Murphy, T.D., Levis, R.W., Matunis,
E., Srivali, N., Hoskins, R.A., Spradling, A.C., 2007. The carnegie protein trap library: a
versatile tool for drosophila developmental studies. Genetics 175, 1505–1531.
https://doi.org/10.1534/genetics.106.065961.
Carton, Y., David, J.R., 1985. Relation between the genetic variability of digging behavior of
Drosophila larvae and their susceptebility to a parasitic wasp. Behav. Genet. 15, 143–154.
Chaudhary, et al., 2017. On chip cryo-anesthesia of Drosophila larvae for high resolution in
vivo imaging applications. Lab. Chip 17, 2303–2322.
Core Team, R., 2018. R: A Language and Environment for Statistical Computing. R
Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/.
Fox, J., Weisberg, S., 2011. An R Companion to Applied Regression, second ed. Sage,
Thousand Oaks CA. URL: http://socserv.socsci.mcmaster.ca/jfox/Books/Companion.
Füger, P., Behrends, L.B., Mertel, S., Sigrist, S.J., Rasse, T.M., 2007. Live imaging of sy-napse
development and measuring protein dynamics using two-color fluorescence
Journal of Insect Physiology 117 (2019) 103900
recovery after photo-bleaching at Drosophila synapses. Nat. Protoc. 2, 3285–3298.
https://doi.org/10.1038/nprot.2007.472.
Ghaemi, R., Rezai, P., Iyengar, B.G., Selvaganapathy, P.R., 2015. Microfluidic devices for
imaging neurological response of Drosophila melanogaster larva to auditory stimulus. Lab.
Chip 15, 1116–1122. https://doi.org/10.1039/C4LC01245C.
Ghannad-Rezaie, M., Wang, X., Mishra, B., Collins, C., Chronis, N., 2012. Microfluidic chips
for in vivo imaging of cellular responses to neural injury in Drosophila larvae. PLoS One 7,
e29869. https://doi.org/10.1371/journal.pone.0029869.
Gutierrez, E., Wiggins, D., Fielding, B., Gould, A.P., 2007. Specialized hepatocyte-like cells
regulate Drosophila lipid metabolism. Nature 445, 275–280. https://doi.org/10.
1038/nature05382.
Hothorn, T., Bretz, F., Westfall, P., 2008. Simultaneous inference in general parametric
models. Biometrical J. 50, 346–363.
Lenth, R.V., 2016. Least-squares means: the R package lsmeans. J. Stat. Softw. 69, 1–33.
https://doi.org/10.18637/jss.v069.i01.
Makhijani, K., Alexander, B., Tanaka, T., Rulifson, E., Brückner, K., 2011. The peripheral
nervous system supports blood cell homing and survival in the Drosophila larva.
Development 138, 5379–5391. https://doi.org/10.1242/dev.067322.
Reed, B.H., McMillan, S.C., Chaudhary, R., 2009. The preparation of Drosophila embryos for
live-imaging using the hanging drop protocol. J. Vis. Exp., e1206. https://doi.org/
10.3791/1206.
Salvaterra, P.M., Kitamoto, T., 2001. Drosophila cholinergic neurons and processes vi-
sualized with Gal4/UAS-GFP. Gene Expr. Patterns 1, 73–82. https://doi.org/10.
1016/S1567-133X(01)00011-4.
Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of
image analysis. Nat. Methods 9, 671–675. https://doi.org/10.1038/nmeth.2089.
Sláma, K., Farkas, R., 2005. Heartbeat patterns during the postembryonic development of
Drosophila melanogaster. J. Insect Physiol. 51, 489–503. https://doi.org/10.1016/j.
jinsphys.2004.11.016.
Vanderploeg, J., Paz, L.L.V., Macmullin, A., Jacobs, J.R., 2012. Integrins are required for
cardioblast polarisation in Drosophila. BMC Dev. Biol. 12, 8.
Wawersik, J., 1997. History of chloroform anesthesia. Anesthesiol. Reanim. 22, 144–152.
Zhang, Y., Füger, P., Hannan, S.B., Kern, J.V., Lasky, B., Rasse, T.M., 2010. In vivo
imaging of intact Drosophila larvae at sub-cellular resolution. J. Vis. Exp. 43, e2249.
https://doi.org/10.3791/2249.