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First published online 16 October 2008
doi: 10.1242/dev.026773
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1 Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2
3EJ, UK.
2 Lehrstuhl für Genetik und Neurobiologie, Theodor-Boveri-Institut
Biozentrum, Julius-Maximilians-Universität Würzburg, Am Hubland,
97074 Würzburg, Germany.
* Author for correspondence (e-mail: sjc85{at}cam.ac.uk)
Accepted 26 September 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: Drosophila, Embryo, Movement, Muscle, Coordination
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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At the heart of the output machinery are central pattern generating
circuits whose outputs drive stereotyped patterns of movement such as
swimming, walking and breathing (Grillner
et al., 2005
). These rhythmic patterns form a large part of the
repertoire of animal movements. Part of the difficulty in studying how such
circuits develop and begin to function lies in the fact that they are embedded
within the central nervous system and do not have the explicit, often
two-dimensional, organisation that characterises the anatomy of many sensory
maps. To this anatomical problem are added technical difficulties inherent in
attempting to make a quantitative assessment of the maturing functional
properties of such circuits (such as coordination between multiple different
neurons) in the developing embryonic nervous system.
However, there are instances, such as the spinal cord in Xenopus
and zebrafish, where numbers of cells are small and the complete set of
different cell classes required for early movements can be defined
(Bernhardt et al., 1990;
Brustein et al., 2003;
Hale et al., 2001;
Roberts, 1990;
Roberts et al., 1998). This
catalogue, in combination with paired cell recordings in Xenopus
allows a very complete picture to be built up of how these cells operate in
the motor network that generates swimming
(Li et al., 2007;
Li et al., 2004). In both
cases, swimming emerges from earlier spontaneous movements driven by motor
outputs that begin shortly after functional endplates are formed on the
myotomes of the trunk (Kuwada et al.,
1990; Saint-Amant and Drapeau,
1998; van Mier et al.,
1989). Initial outputs in zebrafish appear to depend on periodic
depolarisation of electrically coupled neurons in the early spinal network
(Roberts and Perrins, 1995;
Saint-Amant and Drapeau, 2000;
Saint-Amant and Drapeau, 2001;
Soffe and Roberts, 1982).
These early outputs are interesting because they raise the obvious issue of
whether such precocious network activity is incidental to the sequential
assembly of motor circuitry or required for its normal development. Activity
is also periodic and organised into bursts in early spinal networks of chick
and mouse (Landmesser and O'Donovan,
1984; Suzue,
1996); recent work with the mouse indicates that
acetylcholine-mediated transmission during this phase is essential to the
development of normal patterns of rhythmic output from central pattern
generators controlling limb movements
(Myers et al., 2005).
Clearly therefore, many motor networks become active before they are required to generate patterned movements that contribute to normal behaviour and a key issue is whether this activity is part of a necessary developmental step in which functional properties of circuitry are validated and adjusted to ensure optimal performance. We have chosen to use Drosophila embryos as models to study the development of locomotor circuitry and coordinated movement. A major advantage of Drosophila is that we can use non-invasive techniques to monitor the outputs of the motor circuitry and to manipulate the network activity as it develops. To do this, we need to define the basic features of motor development and to show whether, as in other organisms, normal development includes a phase of early activity in which the network becomes periodically active before it is required for mature patterns of behaviour.
Here, we report the use of a novel, non-invasive method to study the onset
of motor activity in Drosophila. Using this method, we investigate
the beginnings of function in the neural network and the subsequent appearance
of coordinated movement. A previous study has suggested that neural control of
motor activity develops continuously and gradually from the earliest embryonic
muscle twitches to the well-orchestrated peristaltic waves that are
characteristic of larval crawling and that this reflects the progressive
maturation and increasing complexity of underlying motor circuitry
(Pereanu et al., 2007).
However, our experimental analysis shows that this is not the case and that
the earliest movements in Drosophila are myogenic in origin. In fact,
body wall muscles responsible for locomotion only come under neural control
late in embryogenesis. Our analysis defines exactly when this neural control
of movement begins and shows that early motor output is organised into
periodic bursts of activity. We also show that this activity is not triggered
as a reflex response to developing sensory inputs but probably results from
spontaneous activity that starts as electrical properties of neurons in the
central network mature. We also find that the onset of neural control is
accompanied by a phase of gradual improvement in behavioural performance from
muscle contractions that are uncoordinated to recognisable locomotor motifs
and the later emergence of complete sequences that resemble larval
crawling.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Imaging
Embryos carrying muscle markers G203 and ZCL2144 were
placed in saline between a gas permeable membrane (BioFolie, Grenier) and a
cover-glass and imaged in real time using a Hamamatsu ORCA-ER camera on a
Leica DM IRBE confocal microscope with a Yokagawa CSU-10 scanner and a
10x objective. Movies captured at 5 frames/second using the Perkin Elmer
Temporal Module image analysis system were analysed frame by frame in
QuickTime and muscle contraction on and offsets were documented in Microsoft
Excel.
Visualising and recording embryonic movements before 16 hours AEL
To assess when movement begins, embryos were selected immediately after the
formation of the second midgut constriction [13 hours after egg laying (AEL)].
Dechorionated embryos were placed on slides and covered with a layer of
halocarbon oil to prevent dehydration. Five-minute video recordings of the
ventral surface were captured on digital tape every 15 minutes, using a Leica
M420 microscope, JVC TK-C1380 video camera and Sony DSR-309 digital
videocassette recorder. Denticle band movements in late embryos (>18.5
hours AEL) were also recorded in this way.
Testing embryonic reflex responses
To investigate the capacity of the embryo to produce larval-like behaviours
prior to hatching, we pierced the vitelline membrane of dechorionated embryos
with a glass needle to allow the animal to hatch prematurely. We tested the
touch response in wild-type embryos hatched prematurely 18.5-20.5 hours AEL.
Each embryo was allowed to emerge completely from the vitelline membrane
before testing (around 5 minutes) and then stroked gently on the anterior
segments using an eyelash. Each embryo was tested 10 times, with at least 30
seconds between each trial (to allow for recovery and to prevent adaptation).
To assess the ability of prematurely hatched embryos to self-right, we gently
rolled the newly hatched embryo (18.5-20.5 hours AEL) onto its dorsal surface
with forceps and measured the time taken for the embryo to right itself. Each
embryo was tested three times, with at least 2 minutes recovery time between
each trial.
Light stimulation in embryos expressing ChR2
Parental flies were fed yeast paste containing all-trans retinal (100
µM) for 2 days prior to collection of embryos carrying ChR2. ChR2 embryos
were raised in darkness prior to experiments. Light pulses (20 mseconds,
minimum required for contractile responses in late embryos expressing ChR2) of
specific wavelengths were delivered at 1 Hz (to avoid synaptic run down) using
a stimulator (Master-8, A.M.P.I.) to control the interlock on the
acousto-optic tuneable filter of a 488 nm laser (Melles-Griot, 534-A-A03).
Embryos were imaged without activating ChR2, by illuminating with long-pass
filtered visible light [Thorlabs long-pass filter (>550 nm)], on an Olympus
BX51 WI microscope (x10 objective).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). These authors suggest that a backward wave of peristalsis
leads to forward movement, but this is not the case. A forward crawl requires
a forward wave of contraction in the abdominal segments, and is initiated in
the abdomen by contraction of the most posterior segments (A8/9)
(Dixit et al., 2008).
Thereafter, the posterior end of the animal attaches to the substrate, and a
wave of contractions advances anteriorly as each segment (A7-A1) is
transiently lifted from the substrate, pulled forwards and then lowered. Each
abdominal segment engages with the substrate through an anterior belt of
cuticular denticles that act as anchorage points as neighbouring segments move
forwards in the peristaltic wave. As contractions begin in the abdomen, the
head and thorax are extended forwards and anchored by the mouth hooks. A
backward crawl reverses the peristaltic wave of contraction from A1 to A8/9,
and is initiated by a backwards contraction of the head and thorax.
A method for quantitative analysis of coordinated movements in intact embryos
We tracked the development of embryonic movement by monitoring contractions
of individual muscles non-invasively. Our method uses fly strains carrying
green fluorescent protein (GFP) traps in proteins
(Morin et al., 2001) expressed
at the Z-lines of somatic muscles. At 25°C embryogenesis in
Drosophila lasts 21 hours and embryos begin to perform coordinated,
crawling-like movements at about 18 hours after egg laying (AEL)
(Pereanu et al., 2007). To
study development of these movements, we combined two GFP trap lines
(w;G203;ZCL2144), which allowed us to image muscles from 16 hours AEL
onwards using spinning disc confocal microscopy. For imaging, we released
embryos from the egg case into saline and sandwiched them between a cover
glass and gas permeable membrane. Embryos treated in this way from 16 hours
AEL develop normally and patterns of movement parallel those seen in embryos
retained within the vitelline membrane. Embryos left for many hours in
sandwich preparations develop into normal larvae that crawl on agar plates,
feed and continue to develop (n>20).
We find that, in principle, the contraction-relaxation cycle of every muscle can be recorded in animals moving freely over a substrate (Fig. 1) providing a precise, non-invasive method for monitoring motor outputs during behaviour. For our study, we focussed on the segmentally repeated ventral longitudinal muscles that form a major part of the larval musculature used in crawling. We recorded images of these muscles at five frames per second to obtain precise records of their contractions throughout the period during which coordinated movements first develop. This continuous record of muscle activity in many segments allows for a detailed description of the sequence of motor development and for quantitative comparisons between control and genetically manipulated animals.
The normal sequence of behavioural development in Drosophila embryos
We first describe the development of movement and the acquisition of simple
reflex behaviours. These descriptions refine and augment earlier accounts
(Pereanu et al., 2007) and are
a prerequisite for our experimental investigation of the mechanisms that
underlie the emergence of coordinated patterns of motor output in the
embryo.
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Although sequences resembling peristaltic crawling now occur, embryos do
not hatch and crawl until some 3 hours later at 21 hours AEL. We made records
of contractile activity throughout these final 3 hours of embryogenesis, which
reveal that periodic bursts of activity continue until 
30 minutes before
hatching (Fig. 3). Backward
peristaltic waves consistently appear after the first few waves of forward
peristalsis and embryos perform complete sequences of forward and backward
peristaltic contractions, interspersed with incomplete, partial waves and
other contraction patterns. Shortly before hatching, bursts and partial waves
cease. Instead, there are now occasional complete sequences of forward and
backward peristalsis that culminate in specialised movements that break the
vitelline membrane (Siekhaus and Fuller,
1999), at which point the larva crawls out over the substrate and
begins to exhibit mature patterns of behaviour.
To show whether normal patterns of behaviour can begin with the onset of
peristaltic contractions, or whether, as we expected, there would be a
progressive acquisition of more mature patterns of movement as development
continued, we hatched embryos prematurely at intervals after the first onset
of peristaltic sequences, by releasing them from the vitelline membrane. Such
embryos will move over an agar surface and we tested them for their ability to
perform two characteristic patterns of larval behaviour: the reflex response
to touch and the `righting' response. Larvae respond to anterior touch in a
characteristic fashion, incorporating one or more of the following movements:
head withdrawal, head turning and reverse peristaltic waves
(Kernan et al., 1994). The
righting response is seen when a larva is placed upside down on its dorsal
surface and consists of a sequence of movements that rapidly turn it the right
way up with its ventral surface in contact with the substrate.
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). We find that the response increases steadily, indicating
that changes in sensorimotor processing are occurring as the embryo develops
(Fig. 4A). Although there is
almost always some response to touch, even at 18.5 hours AEL, and embryos at
this stage sometimes display backward peristaltic waves spontaneously,
indicating that the separate elements of the complete reflex are present, the
full response of a backwards wave of contractions rarely occurs. We conclude
that sensory inputs only become fully integrated with circuits controlling
motor output during later stages of embryogenesis, so that increasingly mature
motor responses are triggered.
To assess the ability of prematurely hatched embryos to self-right, we
gently inverted prematurely hatched embryos onto their dorsal surface with
forceps and measured the time that these embryos required to right themselves.
When mature larvae are deliberately rolled onto their dorsal surface, they
contract their muscles strongly, in a circumferential sequence that throws the
body into a curve and rolls them back rapidly onto their ventral surface
(
The complete sequence, from the earliest movements, through the onset of coordinated sequences of contraction, and the later integration of touch and righting reflexes is shown in Fig. 5.
The onset of bursting represents a transition from myogenic movements to muscle contraction that is under neural control
The earliest contractions seen in the embryo, that is the isolated twitches
from 14 hours AEL and the later unilateral waves of contraction, occur well
before neurons have developed the electrical properties that allow them to
generate propagated action potentials (17 h AEL)
(Baines and Bate, 1998) (A.
Nair, PhD thesis, University of Cambridge, 2005). It seems therefore that
early muscle contractions in the Drosophila embryo are not driven by
the firing of motoneurons, but are likely to be myogenic in origin.
To test this idea directly, and to define the precise moment in
embryogenesis when the nervous system first generates a motor output, we
compared the developing patterns of movement in normal embryos with those that
occur in embryos where evoked synaptic transmission has been blocked by the
expression of tetanus toxin (using the pan neuronal driver elav-Gal4)
(Sweeney et al., 1995). We
reasoned that myogenic movements would be unaffected by blocking synaptic
transmission and therefore common to the two classes of embryos, whereas
neurally controlled muscle contractions would be seen only in embryos with
functional synapses.
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We were interested to see whether spontaneous, i.e. non-evoked release of
neural transmitter (glutamate) at the neuromuscular junction might contribute
to early embryonic movement. To test this, we performed two experiments.
First, we analysed the movements of embryos in which presynaptic terminals
were removed from the muscles by expressing the cell death gene grim
(Wing et al., 1998)
ectopically in all neurons, so that they, and their axons, degenerated during
embryogenesis. There is no bursting activity in these embryos but myogenic
contractions persist at a frequency that is not significantly different from
that seen when evoked synaptic transmission is blocked
(Fig. 7). This suggests that
spontaneous release of transmitter does not make a major contribution to
myogenic activity. In the second set of experiments, we analysed movement in
embryos homozygous for a null mutation in the muscle-specific subunit of the
glutamate receptor (GluRIII) (Marrus et
al., 2004). Again, there is no sign of bursting activity and
characteristically myogenic contractions persist
(Fig. 7). However, in the
absence of the receptor, the frequency of these contractions is significantly
reduced in comparison with denervated embryos (elav-GAL4;UAS-grim)
and embryos without synaptic transmission (elav-Gal4; UAS-TNT-G).
This sensitivity of myogenic movement to the removal of the receptor may
indicate that non-neuronal sources of glutamate such as the haemolymph
(Chen et al., 1968) can
contribute to embryonic muscle contractions.
The onset of motor activity is not triggered by sensory input
We next asked why the motor network begins to burst at a particular point
in development. One possibility is that the network fires as a reflex response
to rising levels of activity in sensory neurons as they mature
(Sanes et al., 2006). Sensory
activity could either be spontaneous or evoked in response to myogenic
contractions occurring prior to the first burst. To show whether this is
indeed the case, we raised embryos in which sensory input was blocked by the
selective expression of tetanus toxin in sensory neurons using the
P0163-Gal4 driver (Hummel et al.,
2000; Suster and Bate,
2002). Expression of PO163 begins at stage 12 in the precursors of
sense organs, well before any movement begins
(Wolf and Schuh, 2000) and
such embryos are completely insensitive to mechanical stimulation
(Suster and Bate, 2002).
Remarkably, despite the loss of all sensory input, these embryos made a normal
transition on schedule from myogenic to bursting, neurally controlled
contractile activity (Fig. 8).
We conclude that early activity of the motor network is an autonomous property
of the central network that does not require input from the sensory system.
However, the frequency of subsequent bursts is reduced in embryos that lack
sensory input (3.0±0.3 bursts/hour compared with 4.6±0.3
bursts/hour in controls, n=5 in each group, Student's
t-test, P=0.004), and although coordinated sequences
resembling peristalsis are eventually generated [as expected from earlier work
(Suster and Bate, 2002)], they
are considerably delayed (74±13 minutes, P<0.0005).
Bursts of activity are not a feature of spontaneously active motoneurons, but are network derived
As an alternative to reflex triggering of motor output in the embryo,
bursting activity might be a network phenomenon, which arises as embryonic
neurons mature and become spontaneously active. In this view the
interconnectedness of immature neurons might be sufficient to trigger repeated
firing (resulting in a burst of activity) through recurrent excitation as
levels of spontaneous firing rise to some threshold level.
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) (also
Richard Baines, personal communication), we targeted tetanus toxin to these
neurons using the Cha-Gal4 driver
(Salvaterra and Kitamoto,
2001). These embryos show no bursting activity and we conclude
that bursts are driven by activity in the central network. Interestingly, however, at 17 hours AEL, the frequency of muscle contractions when motoneurons are simply deprived of their inputs is greater than that seen when all synaptic transmission, including that from motoneurons, is blocked [in embryos that are Cha-GAL4;UAS-TNT-G, each ventral longitudinal (VL) muscle contracts 5.4% of the time (s.e.m.=0.3%, n=4), whereas in embryos that are elav-GAL4;UAS-TNT-G, each VL muscle contracts on average just 3.3% of the time (s.e.m.=0.3%, n=4), Student's t-test, P=0.002] (see Fig. S1 in the supplementary material). This suggests that neurons such as motoneurons do indeed become spontaneously active at the stage when normal bursting would begin. However, this spontaneous activity is not itself bursting.
Bursts of activity induce network depression, with slow kinetics of recovery
If bursting activity truly depends on spontaneous activity reaching a
threshold value, we reasoned that this might be detectable by comparing the
number of muscle contractions occurring just before and just after each burst.
Although activity levels before and after bursts were highly variable, it is
clear from our quantitative analysis that a period of heightened spontaneous
activity precedes each episode, and that levels of activity are much reduced
after an episode (Fig. 9A, part
i). Continuous analyses of muscle contractions occurring after a burst of
activity show a steady increase in the frequency of contractions before the
next burst occurs (Fig. 9A,
parts ii, iii).
The reduction in spontaneous activity after each episode could represent
the effect of an intrinsic activity-dependent depression of neuronal firing or
synaptic transmission. We tested this hypothesis directly by recording the
embryonic response to stimulation at defined intervals after a burst. To
stimulate neurons without the need for direct access with electrodes, we
expressed channel rhodopsin 2 (ChR2) in all neurons using elav-GAL4
(Schroll et al., 2006). ChR2
is a light-activated cation-selective ion channel from the green alga
Chlamydomonas reinhardtii. In the presence of all-trans retinal (an
essential co-factor), neurons expressing ChR2 fire action potentials in
response to a light stimulus of the appropriate wavelength (488 nm). We used
long-pass filtered visible light (>550 nm), which does not activate ChR2,
to identify bursts of activity occurring naturally in such embryos between
17.5 and 18.5 hours AEL. At varying intervals after the end of a burst (5, 6,
7, 8, 9 and 10 minutes), we stimulated with a series of 488 nm light pulses of
increasing duration (25, 50, 100, 200 and 400 mseconds separated by 30 second
intervals). We recorded the length of the minimum pulse required to evoke a
vigorous response (contraction within 15 seconds of stimulus, lasting over 15
seconds and involving most, if not all, segments, i.e. resembling a naturally
occurring burst). Any embryo performing a burst before the stimulation
protocol was applied was excluded. These responses were then translated into a
score (response to 25 mseconds scores 5, 50 mseconds scores 4, 100 mseconds
scores 3, 200 mseconds scores 2, 400 mseconds scores 1 and no response to any
of the pulses scores 0). Embryos were tested only once because the stimulation
protocol could produce synaptic rundown, and this in turn could affect the
outcome of subsequent trials. Embryos were pulsed with longer wavelengths of
light (568 nm and 638 nm), which do not activate ChR2, to obtain control
response scores (vigorous movements occurring soon after one of the test
pulses, but which are naturally occurring rather than stimulated by ChR2
opening, i.e. the rate of false positives).
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).
Naturally occurring inter-burst intervals in wild-type embryos (dechorionated
but not devitellinised) are on average 12.0 minutes long (and show a tight
distribution, s.e.m.=0.3 minutes, n=7). Interestingly, the shortest
interval that we recorded between spontaneous bursts was 6.6 minutes, and this
is very similar to the shortest interval we found before a second burst of
activity could be triggered by stimulation.
Inhibitory neurotransmission is not required for early episodic activity in Drosophila embryos
The activity-dependent depression that operates in the embryonic nervous
system could conceivably be mediated by the recruitment of inhibitory neurons.
We examined embryos in which fast inhibitory neurotransmission through GABA
receptors is disrupted by a mutation in Rdl, a gene coding for a
subunit of a GABA-gated Cl- ion channel
(ffrench-Constant, 1993;
Lee et al., 2003).
Rdl is expressed in the embryonic CNS and embryos that are homozygous
for the Rdl1 allele lack detectable Rdl
transcripts, do not hatch and die around 24 hours AEL
(Stilwell et al., 1995).
Rdl1 embryos show bursting activity from 17 hours AEL. From 17 to 18 hours AEL, the frequency of bursts in these embryos is not significantly different from that seen in embryos with completely intact synaptic transmission (Fig. 10). After 18 hours AEL, the bursts of activity in the mutant embryos lengthen and activity gradually becomes nearly continuous, with rapidly propagated, repeated waves of contraction. Thus, at least initially, GABAergic transmission is not required for the organisation of activity into bursts.
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Strigini et al., 2006).
However, in normal embryos the earliest motor outputs are clearly driven by
the concerted, periodic firing of many neurons in the motor network. This
characteristically rhythmic firing, together with the fact that activity
begins several hours before it is required for hatching and larval crawling
suggests that bursting may be important for the proper maturation of the
network and the emergence of coordinated movements.
There is considerable circumstantial evidence to support this idea:
rhythmic oscillations occur in different parts of developing vertebrate
nervous systems, notably spinal cord
(Landmesser and O'Donovan,
1984) and retina (Wong et al.,
1993), and in retina this precocious activity is essential for
development and refinement of normal patterns of connectivity
(Gnuegge et al., 2001;
Katz and Shatz, 1996). The
stomatogastric network of the embryonic lobster also becomes active early in
development before feeding can occur, and spectrographic analysis shows that
these rhythms are irregular and may represent immature phases in the
development of fully adult outputs (Rehm
et al., 2008).
Significantly, we find that output from Drosophila network `improves' during bursting, in that motifs begin to appear in the record of contractions that resemble elements of normal crawling sequences - contractions that are coordinated across the midline with delays between segments. These partial sequences are followed by complete, but still imperfect waves of contractions from which well coordinated forward and later backward peristaltic sequences will develop. The gradual appearance of coordination is certainly consistent with the idea that rhythmic activity drives activity-dependent adjustment and tuning of the developing motor network. An alternative is that bursting begins before all cellular components of the motor circuitry are present and that progressive appearance of more mature contraction patterns simply reflects growth and addition of further essential elements to the motor network. Experiments to distinguish between these two alternatives are now planned.
We not only find striking similarities between temporal characteristics of
episodic activity in Drosophila embryos and motor outputs recorded
from chick spinal cord, but also analogous network properties. Two properties
essential for spontaneous episodic activity in spinal networks are
hyperexcitability and cell firing modulated according to recent network
history (Fedirchuk et al.,
1999). We find evidence for autonomous increases in levels of
activity occurring shortly after neurons acquire a mature complement of
currents (allowing action potential propagation for the first time), and this
increase coincides with the appearance of oscillatory, bursting activity in
normal embryos. In addition, GABA expression in embryos is low when motor
output first begins (Kuppers et al.,
2003), and removing GABAergic transmission has little effect on
early bursting activity. The combination of spontaneous firing and low levels
of the major inhibitory neurotransmitter could create a `hyperexcitable' state
in the embryonic network. We also find that, as in vertebrates, episodic
activity is a network property, rather than a feature of individual
neurons.
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;
Tabak et al., 2000). We deduce
from patterns of muscle contraction seen, using ChR2 to stimulate the
embryonic nervous system, that neuronal firing rates are modulated by the
recent history of the network in Drosophila. Such a direct
demonstration of activity-dependent depression with slow kinetics of recovery
has only been achieved in one other developing network - the chick spinal
cord. Here, stimulation experiments have revealed both depression of synaptic
potentials after an episode of bursting activity, with slow recovery during
the inter-episode interval (Fedirchuk et
al., 1999) and depression of the whole network, as indicated by
briefer duration and lower cycling frequency (indicators of excitability)
within evoked episodes that were artificially elicited soon after a
spontaneous episode when compared with later in the inter-episode interval
(Tabak et al., 2001).
Although episodic activity is characteristic of embryonic movement it is
not appropriate to the behaviour of hatched larvae, which perform sustained
bouts of forward crawling as they search for food. Not surprisingly,
therefore, we find that bursting activity ceases shortly before hatching and
this forms part of a sequence of behavioural maturation during the late stages
of embryogenesis. Shortly after the first crawling-like movements (18.25 hours
AEL), embryos are relatively unresponsive to touch and unable to perform
righting reflexes, but by the time bursting ceases and the animal is about to
hatch, touch responsiveness has increased markedly and righting reflexes are
present. The animal is now ready to emerge and it is likely that specialised
movements of hatching and, perhaps, other aspects of behavioural maturation
are triggered hormonally, as loss of Amontillado or PHM (proteins required in
neuroendocrine biosynthesis) prevents or delays the hatching sequence
(Jiang et al., 2000;
Siekhaus and Fuller, 1999). We
have documented several hours of activity in the embryonic nervous system
prior to this sequence, accompanied by progressive acquisition of more mature
patterns of behaviour. We now plan to investigate the role of this precocious
activity to show whether it is incidental to or essential for the normal
development of coordinated behaviour.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/22/3707/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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