Peripheral multidendritic sensory neurons
are necessary for rhythmic locomotion
behavior in Drosophila larvae
Wei Song, Maika Onishi, Lily Yeh Jan, and Yuh Nung Jan*
Departments of Physiology and Biochemistry, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143
Contributed by Yuh Nung Jan, February 1, 2007 (sent for review January 3, 2007)
From breathing to walking, rhythmic movements encompass phys-
iological processes important across the entire animal kingdom. It
is thought by many that the generation of rhythmic behavior is
peripheral sensory input. Sensory feedback is, however, required
to modify or coordinate the motor activity in response to the
circumstances of actual movement. In contrast to this notion, we
report here that sensory input is necessary for the generation of
Drosophila larval locomotion, a form of rhythmic behavior. Block-
age of all peripheral sensory inputs resulted in cessation of larval
crawling. By conditionally silencing various subsets of larval pe-
ripheral sensory neurons, we identified the multiple dendritic (MD)
neurons as the neurons essential for the generation of rhythmic
peristaltic locomotion. By recording the locomotive motor activi-
ties, we further demonstrate that removal of MD neuron input
disrupted rhythmic motor firing pattern in a way that prolonged
the stereotyped segmental motor firing duration and prevented
the propagation of posterior to anterior segmental motor firing.
These findings reveal that MD sensory neuron input is a necessary
rhythmic behavior ? sensory feedback ? locomotion generation ?
motor pattern ? central pattern generator
behavior is that all rhythmic movements are generated by
specialized circuits within the CNS called central pattern gen-
erators (CPGs), which can operate without sensory inputs (3–5).
Nevertheless, a functional motor program also requires sensory
inputs reporting peripheral feedback due to the actual move-
ments to produce the correct behavior (6, 7). This model draws
support from reports of sensory feedback affecting the timing
and magnitude of motor activity generated by CPGs and from
studies based on surgical removal of afferent input (1, 4, 6, 7).
Whereas there are electrophysiological demonstrations of the
influence of sensory feedback on the output of CPGs, how
interactions between sensory neurons in the peripheral and
neurons in the CNS contribute to rhythmic behavior in an intact
organism is not known. It is therefore important to ask whether
sensory inputs are essential for rhythmic behavior and how
sensory inputs contribute to rhythmic behavior.
Drosophila is ideal for investigations of neural circuits under-
lying rhythmic behavior due to its amenability to genetic ma-
nipulation. Larval crawling, used for travel across various types
of terrain, is accomplished by repeated peristaltic wave-like
strides (8) and involves molecules mediating neuronal signaling
(9–12). Whereas one study suggests that embryonic assembly of
CPG for larval locomotion does not require sensory inputs, the
same study reports that interference with the sensory function
during embryogenesis severely disrupts the actual patterns of
locomotion (13). To explore how sensory inputs contribute to
larval locomotion, we conducted a Gal4/upstream activating
sequence (UAS)-based screen to identify components of the
hythmic behaviors comprise a cyclic, repetitive set of move-
ments, such as locomotion, respiration, and mastication (1,
neural circuitry for larval locomotion, revealing that sensory
neuron function is necessary for larval locomotion.
Results and Discussion
Drosophila Larval Sensory Neurons Are Necessary for Locomotion
Behavior. Drosophila larval crawling entails repeated, rhythmic,
peristaltic contraction. During each peristaltic stride, muscle
contractions are propagated from one end of the body to the
other end, passing through all 11 segments one by one [support-
ing information (SI) Fig. 6 and Movie 1]. To identify sensory
neurons important for larval locomotion, we crossed ?1,000
Gal4 driver lines to UAS-shibirets(UAS-shits) (‘‘ts’’ indicates
temperature sensitive) to transiently reduce the synaptic func-
tion of various subsets of Gal4 expressing neurons at the third
larval instar and looked for gross locomotion defects in peri-
stalsis rhythmicity and speed. Because shits, a temperature-
sensitive mutant form of dynamin, blocks synaptic vesicle recy-
cling and disrupts synaptic transmission only at restrictive
temperatures (?29°C) (14, 15), we could raise these larvae at
18°C and ensure normal development of the larval crawling
circuitry. We then subjected third instar foraging larvae to
restrictive temperature (37°C) for 15 min to silence neurons
expressing shitsand observed the locomotion behavior.
For the majority of the analyzed Gal4 lines, incubation at
restrictive temperature resulted in enhanced locomotion speeds
as observed in wild-type larvae (Fig. 1a); faster crawling and
increased activity might be a natural avoidance response to high
temperatures (16, 17). We isolated 130 Gal4 lines that give rise
to different types of defects in larval locomotion (see Methods).
Among those 130 lines, UAS-shitsexpression driven by 10 Gal4
lines resulted in drastically disrupted peristaltic rhythm and
reduced locomotion speed. Using these 10 Gal4 lines to drive the
expression of UAS–mCD8–GFP to identify the neurons that
were silenced in the larvae with defective rhythmic locomotion
(18), we found that all 10 Gal4 lines showed expression in some
or all peripheral sensory neurons. In particular, the 5-40-Gal4
line drives gene expression in all larval peripheral sensory
neurons, but not in CNS neurons (Fig. 2). This Gal4 line
(SN-Gal4) allowed us to study the role of peripheral sensory
neurons in larval locomotion.
First, we subjected SN-Gal4/UAS-shitslarvae to restrictive
temperature (37°C) for different durations to examine more
M.O. analyzed data; and W.S., M.O., L.Y.J., and Y.N.J. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Abbreviations: CPG, central pattern generator; Kir, inwardly rectifying K?channel; MD,
multiple dendritic; shits, shibirets; TNT, tetanus toxin; ts, temperature sensitive; UAS,
upstream activating sequence.
*To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0700895104 PNAS ?
March 20, 2007 ?
vol. 104 ?
no. 12 ?
Locomotion was analyzed by using two methods. First, movies
were analyzed by using Digital Imaging Analysis Software
(DIAS) (Solltech, Oakdale, IA) as described in ref. 8. For each
frame, the position of the larval centroid was determined.
Centroid paths were generated by plotting the movement of the
larvae over the duration of the film. ‘‘Speed’’ was computed
automatically. Second, ‘‘peristalsis duration’’ and ‘‘peristalsis
frequency’’ were analyzed manually from QuickTime files. Peri-
stalsis duration was defined as the time required for the prop-
agation of muscle contractions to travel from the posterior to the
SN-Gal4;Gal80ts/UAS-Kir larvae were also raised at 18°C
until third instar to prevent the expression of Kir. To turn on the
expression of Kir, these larvae were exposed to restrictive
temperature (37°C) for 30 min to inactivate the inhibitory
function of Gal80 on Gal4.
Gal4 Screen. We observed a variety of different crawling defects
after blocking synaptic transmission within different sets of
neurons, including paralysis (53 lines), locomotion orientation
change (30 lines), peristalsis length and locomotion speed
change (37 lines), and peristalsis rhythmicity and locomotion
speed change (10 lines). To understand the mechanisms under-
lying peristalsis locomotion, we only focused on studying those
Gal4 lines that generated both peristalsis rhythmicity and loco-
motion speed change.
Electrophysiology. Third-instar foraging larvae were dissected in
Ca2?-free haemolymph-like (HL3) solution. The larva was
dissected through the dorsal midline, and the CNS, brain lobes,
ventral ganglia, and segmental nerves were left intact (22). Most
of the dissected larvae presented spontaneous locomotion abil-
ity, characterized by muscle contraction waves that were prop-
continuously superfused with oxygenated HL3 solution contain-
ing 1.5 mM Ca2?. An extracellular recording from segmental
nerves was recorded by using a suction electrode with a diameter
of 7–10 ?m (36). Voltage signals were amplified by an Axoclamp
200B (Axon Instruments, Foster City, CA) with bandwidth
filters set at 0.1 and 10 kHz and digitized directly to a disk with
Statistical Analysis. All data were analyzed by one-way ANOVA
by using Origin 7.0 (OriginLab, Northampton, MA).
We thank U. Heberlein for making the 1,000 Gal4 lines available to us;
T. Kitamoto, H. H. Lee, and Y. Xiang for providing fly stocks; L. E. Fox
for advice about extracellular recording of segmental nerve, S. B. Yang
for technical help; and members of the Y.N.J. Laboratory for discussion.
This work is supported by National Institutes of Health Grant
R01NS40929 (to Y.N.J.). Y.N.J. and L.Y.J. are investigators of the
Howard Hughes Medical Institute.
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