Department of Biokinesiology and Physical Therapy, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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Bradley, Nina S.. Age-Related Changes and Condition-Dependent Modifications in Distribution of Limb Movements During Embryonic Motility. J. Neurophysiol. 86: 1511-1522, 2001. It has long been known that the chick initiates spontaneous motility early in embryogenesis, that the distribution of this activity is episodic, and that it varies both quantitatively and qualitatively with age. It is also well established that embryonic motility is controlled by spinal circuits and features of motility at early stages of development are likely the product of immature network properties. Over the course of embryonic development, however, the episodic distribution of motility becomes more variable. Because we are interested in determining whether movement experience in ovo is fundamental to the establishment of adaptive posthatching behaviors, this study examines the normal within-subject variability of episodic activity in embryos across ages under control and several experimental conditions. The distribution of activity, pause, and episode duration was obtained from video recordings of embryos prepared for electromyographic (EMG) and/or kinematic studies of motility in ovo at select ages (E9, E10, E12, E15, E18) under control conditions (control), acute reduction in buoyancy (ARB), ankle restraint (AR), thoracic spinal transection (spinal). Both control and ARB embryos exhibited significant age-related changes in the distribution of motility. Activity duration progressively increased with age and largely accounted for age-related increases in the variability of episodic behavior. Pause duration decreased markedly between E9 and E12 and did not appear to be a critical parameter in accounting for age-related changes in motility distribution. Activity duration was significantly lengthened in ARB embryos and decreased in spinal embryos. Pause duration was selectively lengthened in AR embryos. Collectively, age-related changes and selective effects of experimental preparations suggest that activity and pause duration are controlled by different mechanisms that operate independent of one another by E12. The results also suggest that the spinal network controlling motility becomes increasingly dependent on excitatory drive from supraspinal centers between E9 and E18. It is proposed that age-related increases in activity duration variability and condition-dependent effects on the distribution of activity are indicative of changing inputs weights for descending and sensory pathways and that they significantly impact spinal control of motility as the embryo's movement and posture are increasingly constrained by the fixed volume of the egg.
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INTRODUCTION |
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Although they credit
Visintini and Levi-Montalcini (1939) as the first to
recognize the periodicity of motility in chick embryos, Hamburger and Balaban (1963)
provided the first
analyses. Their analyses indicated that beginning embryonic day (E)
3.5, movement occurred in "cycles," each cycle consisting of an
"activity phase" lasting 5-15 s and containing 2-10 movements,
followed by an "inactivity phase" of 30-60 s. Findings indicated
that the duration of activity and inactivity was variable, and the
lengths of the two phases within a cycle were unrelated, yielding an
irregular periodicity. Over age, the activity phase lengthened and the
inactivity phase shortened until activity was nearly continuous at E13
(Hamburger 1963
) but remained cyclic through to E17
(Hamburger et al. 1965
). Hamburger (1963)
proposed that two mechanisms controlled motility, one turned it on and
another turned it off. Based on studies of chronic spinal embryos, he
and his colleagues argued that the structure of episodic motility
(activity plus inactivity phases) sat within the spinal cord but that
the brain and possibly other components (muscle, proprioception)
contributed to the control of motility with increasing age
(Hamburger et al. 1965
).
Subsequent studies of spinal embryos revisited the notion of two
separate control mechanisms and suggested that repetitive movements
within an activity phase were controlled by local spinal circuitry but
that supraspinal and propriospinal inputs regulated the temporal
distribution of activity by E9 (Bradley and Bekoff 1992;
Oppenheim 1975
). In the absence of descending input,
activity phase duration and cycle times for repetitive limb movements
were shortened to the point of producing tonic seizure-like activity by
E15. The precise regulatory mechanisms are not known, but anatomic and
physiologic studies suggest both descending and afferent inputs can
potentially impact motility during the latter two-thirds of embryonic
development. Reticulospinal pathways reach the lumbosacral spinal cord
by E5 (Okado and Oppenheim 1985
) and make synaptic contacts by E6-7 (Shiga et al. 1991
). Electrical
stimulation of the ventral pontine and medullary reticular formation or
bath application of N-methyl-D-aspartate (NMDA)
to the brain stem can evoke motor neuron responses in the lumbosacral
cord by E6 (Sholomenko and O'Donovan 1995
), and embryos
are responsive to cutaneous and proprioceptive stimuli by E7
(Oppenheim 1972
).
Studies in the acute isolated spinal cord have greatly advanced
understanding of the endogenous spinal control of motility. O'Donovan
and colleagues demonstrated that the repetitive bursts of motor neurons
responsible for limb movements (O'Donovan and Landmesser
1987) are accompanied by waves of neural activity beginning ventrolaterally and spreading dorsomedially across the spinal cord
(O'Donovan et al. 1994
). Because periodic
initiation of rhythmic activity persisted following an array of
ablations (Ho and O'Donovan 1993
), and recovered in the
presence of either excitatory or inhibitory pharmacological blockades
(Chub and O'Donovan 1998
), they proposed that both the
periodic initiation of activity and rhythmic excitation during activity
are emergent products of population dynamics (O'Donovan and
Chub 1997
). They proposed that the periodic initiation of activity is the product of recovery from (long-lasting)
activity-dependent depression of transmitter release within
ventrolateral neurons (see also Fedirchuk et al. 1999
);
whereas the duration of rhythmic activity is a function of the number
of neurons recruited by recurrent excitation and the time to recover
from short-lasting synaptic depression (O'Donovan
1999
). A mathematic model of these elements was recently
published (Tabak et al. 2000
).
A focus of work in our lab is to determine if movement-dependent
experience shapes attributes of motility in preparation for adaptive
posthatching behaviors, for sensory inputs can potentially impact
motility during the second to third embryonic week prior to hatching at
E21. Primary afferent synaptic efficacy is established by E7.5
(Davis et al. 1989; Lee et al. 1988
),
muscle afferent innervation is established by E11-E13 (Maier
1992
, 1993
), cutaneous coding of movement may begin as early as
E7 (Koltzenburg and Lewin 1997
; Oppenheim
1972
; Scott 1982
), and by E20 proprioceptive
input arising from neck flexion initiates hatching (Bekoff and
Kauer 1982
; Bekoff and Sabichi 1987
). Because
recent studies indicated that the distribution of activity is extremely
variable with increasing age (Ganley and Bradley 1999
;
Rose et al. 1998
), the purpose of this study was to
determine the normal within-embryo variability for motility between E9
and E18. Also, because activity distribution can be altered by a
reduction in buoyancy at E9 (Bradley 1997
; Chambers et al. 1995
), a second purpose of this study
was to determine if the reduction in buoyancy impacts activity
distribution at later ages. The distribution of activity was also
examined following application of an ankle restraint or chronic spinal
transection so as to compare data across preparations to gain further
insight into the neural control of episodic behavior in the intact
embryo. Preliminary findings were recently published (Bradley
2000
).
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METHODS |
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Video data were obtained from leghorn chicken embryos incubated
for experiments at E9, E10, E12, E15, or E18. Age was verified using
established staging criteria (Hamburger and Hamilton
1951). The samples included embryos drawn from an array of
earlier and more recent studies representing five experimental
preparations: embryos prepared as controls for electromyographic (EMG)
and/or kinematic studies (control); embryos that did not experience EMG or kinematic procedures (control II); embryos prepared for kinematic study during an acute reduction in buoyancy (ARB); embryos prepared for
kinematic study during mechanical restraint of ankle movement (AR); and
embryos prepared for kinematic study after chronic spinal transection
(spinal). Table 1 summarizes the number
of experiments per preparation and age and indicates those embryos from
published studies whose data were re-analyzed for inclusion in this
study. All procedures were approved by the University Institutional
Animal Care and Use Committee.
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Experiments were conducted in ovo and the eggs were maintained in a
temperature-controlled chamber (38°C) throughout preparation and
recording. A window was place in the shell to expose the embryo, and in
all groups, except control II, membranes were cut (for EMG and/or
kinematic preparation). In ARB experiments, 5-7 ml of amniotic fluid
was extracted at all ages except at E18; ARB experiments were not
performed at E18 due to difficulty extracting more than 2 ml of fluid.
In AR experiments, a rigid ankle restraint was secured to the right
lower leg at E9 or E12; space limitations as body size increased
prohibited application of the appliance at older ages. Spinal
experiments were conducted at E9 after removing three to four segments
of spinal cord between somites 18 and 22 at E2. Completeness of
thoracic transections was confirmed by 10-µm serial sections after
block silver stain (Shimizu et al. 1990). Spinal
experiments were not performed at later ages due to difficulty
maintaining viability. Pulse rate and rhythm were monitored throughout
experiments and total exposure was limited to 90 min to optimize
behavioral data (Chambers et al. 1995
).
Video recordings and analyses
Video recordings were continuous over the duration of an experiment. Video was collected at 30 frames/s and stored on VHS tape with a SMPTE time code for subsequent analyses. VHS recordings were reviewed frame-by-frame to determine the SMPTE code at onset and offset of all activity in the ipsilateral wing and/or leg (the right side is typically presented if the egg is not rolled in the 24 h preceding an experiment). No distinction in onset and offset was made between wing and leg movements in intact embryos; but in spinal embryos, the wing and leg were separately analyzed. Contralateral limbs were not considered because they could not be reliably analyzed. Any displacement, regardless of how brief, was defined as activity (Fig. 1). Inactivity less than 10.0 s was treated as part of the activity preceding and following it. Inactivity exceeding 10.0 s was defined as a pause. Combined, activity and the subsequent pause formed an episode, and episode duration was equivalent to the time between consecutive onsets in activity. Activity duration, pause duration, episode duration, and percent activity were calculated for each episode, and subject means were generated for within- and between-group comparisons. Standard deviations and coefficients of variation were calculated within subject and averaged within group to examination parameter variability. Linear regression statistics were performed to examine the relationships between variables. A total of 165 video recordings, ranging from 30 to 60 min in duration, were reviewed to determine the distribution of activity (Table 1). All videos were analyzed at least twice and nearly all videos were analyzed by two reviewers. Two- and one-way ANOVAs were used to test for significant main effects at the level of P < 0.05. In two-way ANOVAs, the group mean was substituted for one E9 missing cell. One-way ANOVA and one-tailed Student t-tests were used for post hoc comparisons, and the Bonferroni correction (P = 0.05/number of comparisons) was used to adjust the level of significance.
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RESULTS |
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Control data were first examined for age-related changes in the distribution of motility. Control data were then used to determine whether the distribution of activity is altered by mechanical manipulations that have been shown to alter limb excursions during motility at one or more ages. Finally, comparisons are made with motility following chronic spinal transection to examine the pattern of distribution in the absence of descending neural input.
Distribution in motility varied with age
The four parameters of motility, activity duration, pause duration, episode duration, and percent activity, varied with age. Examples of motility distribution for control embryos at each age studied are plotted in Fig. 2. At E9, activity duration and pause duration ranges were similar, rarely exceeding 60 s, and both parameters varied closely with episode duration. However, only activity duration varied with episode duration across ages, as the range in activity duration increased from more than 1 min at E10 to more than 5 min at E18. Also, at E18, longer sequences typically occurred in clusters preceded and followed by extended sequences of very brief activity (1-10 s) lasting 30-60 min. Two-way ANOVAs of control and control II data for activity, pause, episode durations, and percent activity established that EMG and kinematic procedures did not alter the distribution of motility, thus only control data are further considered.
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One-way ANOVA post hoc comparisons for control embryos confirmed that age-related changes were significant for each of the four motility parameters (Bonferroni corrections, P < 0.0125). Descriptive statistics indicated that activity duration increased between E9 and E12, peaked between E12 and E15, and decreased between E15 and E18 (Fig. 3). Pause duration decreased primarily between E9 and E12, and continued a downward trend in smaller increments between E12 and E18. The combined effect of these trends was a progressive decrease in episode duration between E9 and E18, the percent activity peaking at E12 and dropping to lowest levels at E18 (Fig. 3). One-way ANOVAs also indicated that linear regression statistics exhibited significant age-related trends (Table 2). The strength of co-variation for activity and episode duration increased between E9 and E12 then decreased slightly during E12-E18. Pause varied most closely with episode duration at E9 and decreased between E9 and E18. Activity duration did not vary significantly with the subsequent pause at any age and only modestly with the preceding pause at E9 (Table 2).
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Episode variability, or variability in periodic onset of activity, ranged 30-50 s across ages. Nonetheless, the coefficient of variation indicated episode variability was nearly 54% of mean episode duration at E9 and increased to nearly 90% at E18 (Fig. 4C). Both within-subject standard deviations and coefficients of variation indicated episode variability were primarily attributable to age-related increases in activity duration variability (Fig. 4, A and C). Pause duration variability, in contrast, progressively decreased with age, the coefficient of variation remaining unchanged, suggesting that pause variability had minimal impact on episode variability after E9.
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Acute reduced buoyancy enhanced age-related changes in distribution of motility
Age-related trends were also significant and even more apparent in ARB than in control embryos (Fig. 3). Activity duration progressively increased with age as pause duration progressively decreased, thus percent activity also increased with age. An age-related trend for episode duration fell short of significance (P < 0.08). ANOVA comparisons indicated that the strength of co-variation between activity and episode duration also increased with age while the relationship between pause and episode duration decreased (Table 3). Variability of activity and episode duration also increased with age (Fig. 4, B and C).
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ANOVA comparisons indicated that activity duration was longer, pause duration shorter, and percent of activity greater for ARB than control embryos (Fig. 3). Post hoc comparisons for each of the four parameters were significant at E15 (Student t-test, Bonferroni correction, P < 0.0125), and percent activity was significantly greater at all ages except at E10. Group differences between ARB and control embryos were also apparent in individual recordings. For example, ARB plots typically included the longest activity durations per age and contained fewer episodes per unit time than control plots due to the overall increase in activity duration (Fig. 5). Further, the longer activity durations and shorter pause durations in ARB, as compared with control embryos, also appeared to account for the significantly stronger linear relationship between activity and episode duration as well as a weaker relationship between pause and episode duration (Table 3). An ANOVA comparison indicated activity duration variability was also greater in ARB than control embryos (Fig. 4B) and at E15 in specific (Bonferroni correction P < 0.0125). However, when subject standard deviations were normalized to their respective means, relative variability for ARB and control embryos was similar (Fig. 4C).
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Ankle restraint lengthened pause and episode duration
Due to inclusion of activity lasting less than 10 s, temporal
measures were substantially less than previously reported for AR
embryos, though age-related trends persisted (Bradley and
Sebelski 2000), and most parameters differed significantly from
those for control embryos. One-way ANOVAs for age indicated that
activity duration did not vary between E9 (28 s) and E12 (29 s),
whereas pause duration decreased (from 93 to 37 s), episode
duration decreased (from 120 to 65 s), and percent activity
increased (from 24 to 40%). One-way ANOVA comparisons for AR and
control embryos indicated that both activity duration and its
relationship to episode duration were similar between groups (Fig.
6). However, pause and episode duration
were significantly longer and percent activity was significantly less
for AR than control embryos; all post hoc Student t-tests were significant at E9 but fell short of the Bonferroni correction (P < 0.025) at E12. Nonetheless, as illustrated in
Fig. 6, pause and episode duration varied more closely in AR than
control embryos, linear coefficients averaging 0.95 ± 0.06 at E9,
0.77 ± 0.15 at E12, and post hoc tests were significant at both
ages. Additionally, activity duration was typically less variable and
pause duration more variable in AR than control embryos; post hoc tests
were significant at E9. Also, because brief movements were rarely noted in the earlier study, instances of activity less than 10.0 s were tabulated, and ANOVA comparisons confirmed that AR produced fewer brief
movements (4, E9; 6, E12) than control conditions (6, E9; 9, E12). Post
hoc comparisons (Bonferroni, P < 0.025) were
significant at E12 and fell just short of significant at E9
(P < 0.026).
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Thoracic spinal transection altered the distribution of motility caudal to the lesion
Thoracic spinal transection selectively reduced the absolute and relative activity duration of leg motility compared with both wing motility in spinal embryos and motility in control embryos. ANOVA comparisons indicated activity duration, episode duration, and percent activity were significantly less for spinal leg than wing motility, whereas pause duration did not differ between limbs (Fig. 7). Also, as illustrated in Fig. 8, linear regressions for activity and episode duration did not differ significantly between the leg (R = 0.76 ± 0.1) and wing (0.66 ± 0.13). However, regression statistics for pause and episode duration were significantly stronger for the leg (R = 0.93 ± 0.02) than wing (0.83 ± 0.06). Coefficients of variation were significantly greater by 25-44% for activity, pause, and episode duration in the leg compared with wing.
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ANOVA comparisons between spinal leg and control motility were similar to comparisons between spinal leg and wing (Bonferroni correction, P < 0.025). Activity duration and percent activity were significantly less for spinal leg than control motility, but pause duration was similar between groups (Fig. 7). Also, the linear regressions for activity and episode duration were similar, but regression statistics for pause and episode duration were significantly stronger for spinal leg than control motility (Fig. 8). Finally, coefficients of variation were significantly greater by 38-47% for activity, pause, and episode durations in spinal leg. Parameters for spinal wing did not differ from those for control embryos.
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DISCUSSION |
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Age-specific transformations in temporal distribution of limb movements
The distribution of motility was first described nearly 40 yr ago,
and despite differences in methodology, control embryos exhibited
age-related trends similar to those first reported by Hamburger
et al. (1965). Activity increased E9-E12 and decreased E15-E18; pause duration decreased primarily between E9 and E12, followed by smaller incremental decreases E12-E18; and percent activity peaked E12-E15, then dropped to lowest levels E18. [However, Bollweg and Sparber (1999)
, using electrophysiological
methods, reported increases in activity between E12 and E18.] Overall, control embryos appeared less active than observed by Hamburger and
colleagues (Hamburger et al. 1965
; Oppenheim
1975
). However, given the strong agreement between control and
control II data, the lower activity levels are likely due to longer
samples and/or our use of video to review past events (rather than
direct observation of ongoing events). It is possible the video image
obscures subtle visual perceptions; nonetheless, video permits multiple
reviews for greater consistency and sensitivity in statistical comparisons.
Results of this study also revealed that activity and pause duration
exhibited different relationships to the periodicity of motility over
age (Fig. 9A). Activity
duration closely co-varied with episode duration at E9, and the
strength of the relationship increased with age. Pause duration,
conversely, varied closely with episode duration at E9, but the
relationship dropped off precipitously with age. There was also a
modest relationship between activity duration and the preceding pause
at E9, but this too dropped off with age, and as first noted by
Hamburger and Balaban (1963), no relationship was found
between activity duration and the subsequent pause at any age. Finally,
age-related trends also indicated that variability in the episodic
distribution of activity was primarily attributable to activity
duration.
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Periodicity is a variable attribute of motility
Results are consistent with studies in both chick and rat,
indicating that the periodic initiation of motility is more variable than stereotypic (Hamburger and Balaban 1963;
Narayanan et al. 1971
). In our study, episode duration
ranges exceeded 100 s, and standard deviations of 30-50 s were
typical at all ages. Variable periodicity is observed in other immature
motor systems, such as the lobster stomatogastic ganglion, but
variability decreases with maturation of circuit elements
(Richards et al. 1999
). Periodic motility presents a
very different developmental profile. The periodic initiation of
activity was most stable at E9 and became more variable with age.
O'Donovan and Chub (1997) proposed that periodic
initiation of motility is an emergent product of population dynamics
attributable to immature neuronal properties. In their model, based on
the isolated spinal cord, activity is the product of recurrent
excitation and is dependent on the number and strength of functional
spinal synapses. The termination of activity and intervening pause are the product of synaptic depression within the network. The close co-variations for both activity and pause duration with episode duration at E9 in control embryos in our study is consistent with their
model and suggests that the two parameters contributed equally to the
episodic distribution of activity at E9. Also, activity duration
modestly co-varied with the preceding pause duration in control E9
embryos, suggesting that there was some shared element of control
underlying the two parameters that might account for the more stable
episode duration at younger ages. Tabak and O'Donovan (1998)
also found that activity duration co-varied with the
preceding pause in the isolated spinal cord and proposed that synaptic
depression regulates the strength of recurrent excitation. That is, the
longer time spent in recovery from depression, the greater number of functional excitatory synapses available to support the next activity phase. Thus based on their model (O'Donovan 1999
;
Tabak et al. 2000
), "slow" synaptic depression
appears to be the critical mechanism underlying the periodic
distribution of the activity at younger ages. The mechanisms
responsible for synaptic depression of the spinal network are not
currently known, but presynaptic transmitter depletion and postsynaptic
receptor desensitization have been proposed (Fedirchuk et al.
1999
). Given the rapid decrease in pause duration after E9 in
intact embryos, we propose that the increased episode variability with
age may be partially attributable to resolution of synaptic depression,
yielding a network more ready to respond to developing extra-network
sources (Fig. 9B).
Age-related transformations in the distribution of motility
Age-related changes in the distribution of motility likely reflect
both the maturation-dependent changes in spinal network behavior and
increasing influence of extra-spinal projections. At E9, the overall
similarity in periodic activation of motility in spinal leg, spinal
wing, and control data, i.e., the co-variation of both activity and
pause duration with episode duration, supports the view first posed by
Hamburger (1963) and later Oppenheim
(1975)
that motility distribution in the intact E9 embryo is
primarily controlled by local spinal circuits. However, the reduction
in both absolute and relative duration of activity in spinal leg, compared with both spinal wing and control data, suggests that recurrent excitability of spinal circuitry is somewhat dependent on
extra-spinal inputs by E9. Further, the extended pauses observed in the
isolated cord (2-30 min, Tabak et al. 2000
; 12 min,
Fedirchuk et al. 1999
), compared with longest pauses in
intact embryos (11-70 s), suggest that network depression is also
influenced by extra-network circuitry, such as descending and/or
afferent pathways, by E9. However, similarities in pause duration
across spinal leg, spinal wing and control raise the possibility that
the prolonged depression observed in the isolated cord is influenced by
additional variables unique to the preparation.
Age-related changes in distribution of motility beyond E12 appear to be
primarily attributable to maturational processes governing the
excitatory drive of spinal circuits. One, pause duration substantially decreased with age, suggesting that network depression has limited impact on the initiation and duration of activity during the second half of embryonic development. Two, activity duration variability increased with age as the range in activity duration lengthened and
included the increasing incidence of very brief movements (Fig. 2).
Thus it is likely that the variability is due to drive of the spinal
network for motility by multiple sources of excitation that are yet
immature. Some excitatory drive is likely from intra-spinal and/or
afferent sources, for activity phases lengthened between E11 and E17 in
chronic spinal embryos (Hamburger et al. 1965). Nonetheless the chronic spinal embryos exhibited a reduction in activity duration, compared with age-matched controls, leading Hamburger and colleagues to argue that brain input also provided an
excitatory drive to spinal motility circuits. The precise pathways driving motility circuits are not known, but electrical stimulation of
medullary gigantocellular neurons can trigger motor activity and
application of NMDA to the brain stem can decrease pause duration by
several minutes in the isolated brain stem-spinal cord by E6 (Sholomenko and O'Donovan 1995
), suggesting that
descending pathways are potentially available to provide some
excitatory drive to spinal networks soon thereafter. It also appears
that dopaminergic pathways may begin to impact activity distribution
between E13 and E16 (Chub 1991
; Sedlack
1992
). Additionally, age-related increases in very brief
movements observed in the present study appeared to accompany
respiratory-like chest movements, raising the possibility that motility
circuits are also driven by the developing respiratory center. Finally,
Hamburger et al. (1965)
speculated that proprioceptive reflexes might also lengthen activity phases, and our findings in ARB
embryos provide evidence for this view, as discussed in the following text.
Mutability of motility parameters
For some time, we have been interested in whether the in ovo
environment and/or resulting movement experiences imposed by it
contribute to attributes of motility, and here consider findings across
conditions, as summarized in Fig. 9A. In two previous
studies, we observed several kinematic changes in wing and leg
movements indicative of mechanical dampening plus a reduction in pause
duration in E9 ARB embryos (Bradley 1997;
Chambers et al. 1995
). In this study of E9-E15 ARB
embryos, pause duration was again decreased, plus an increase in
activity duration was found. Studies of similar mechanical constraints
in mammals (oligohydramnios) have reported dampening of select limb
movements, but the net level of activity did not appear to be altered
(Robinson and Smotherman 1992
; Sival 1993
). However, it appears that the fluid reduction was coupled with a reduction in total free space in utero; this is thought to have
an inhibitory effect on activity during normal late-stage development
because rat fetuses exhibit increased levels of activity when freed
from uterine constraint (Robinson and Smotherman 1992
). In the ARB preparation, on the other hand, total free space appeared to
be slightly increased by the fluid extraction, owing to rigid (shell)
rather than muscular (uterine) walls and resulted in more extended limb
postures compared with control conditions (unpublished observations).
Conversely, extreme movement restriction of the ankle in AR embryos
lengthened pause duration. Further, AR embryos failed to exhibit the
age-related increase in activity duration at E12 and exhibited fewer
brief movements than control embryos. It is possible that longer pauses
were the result of mechanically dampening brief movements that would
have parceled the pauses into smaller segments. However, because wing
movements were not mechanically restrained but remained coupled with
leg movements (Bradley and Sebelski 2000
), it appears AR
conditions exerted a central effect on motility distribution.
Collectively across conditions, the findings suggest that the
distribution of activity is largely determined by the spinal network
for motility up through approximately E9-E10 and becomes increasing
dependent on other sources for excitatory drive as intrinsic network
excitability decreases and synaptic depression resolves (Fig.
9B). The shorter activity duration exhibited by spinal
embryos suggests descending inputs provide at least some excitatory
drive by E9. The progressive increases in activity duration with age in
control embryos may be attributed in part to maturation of descending
reticulospinal pathways (Glover and Petursdottir 1988).
The progressive increases in activity duration variability between E12
and E18 may be partly due to mechanisms controlling the emergence of
breathing movements (Akiyama et al. 1999
) and circadian
rhythms (Akasaka et al. 1995
) in the final days before hatching.
We propose that the increasing variability with age is also
attributable to maturing somatosensory pathways and changes in the bias
of these inputs as the embryo increases in size and the fixed space in
ovo imposes an increasingly flexed posture. Given the greater
age-related increases in activity duration in ARB compared with control
embryos, somatosensory inputs likely begin to enhance the level of
excitability in spinal circuits between E9 and E12. However, their
effects are likely modest at this time, for elimination of
motion-dependent feedback between E10 and E12 does not appear to alter
motility as it re-emerges from blockade (Oppenheim et al.
1978). Nonetheless given the increase in pause duration in AR
embryos, postural context and/or extent of constraint may also be
increasingly important and account for some of the variability in
motility distribution during the later half of development. In support
of these views, cutaneous afferents may contribute to modest ARB and AR
affects at younger ages (Bradley and Sebelski 2000
;
Koltzenburg and Lewin 1997
; Scott 1982
),
and propriospinal afferents may impact parameters by E12-E15
(Maier 1992
, 1993
), for they initiate vigorous hatching
by E20 (Bekoff and Kauer 1982
; Bekoff and Sabichi
1987
). We speculate that afferent inputs associated with
flexion and extension postures may differently impact the control of
motility and that the normal reduction in activity during the final
days of normal development in ovo may be at least partially
attributable to the extreme flexed posture and constrained excursions
imposed by the shell wall. Somewhat related views regarding late-stage
constraints have been previously raised (for review, see
Oppenheim 1973
). However, in 5-min observations of
E18-E19 embryos, after partial exteriorization of the head and upper
body, Oppenheim (1973)
did not find a difference in the
total number of movements manually counted, suggesting there was
neither a decrease in hatching behaviors nor a net increase in activity
as observed in exteriorized rat fetuses (Robinson and Smother
1992
). Exteriorization of the chick may have introduced confounding effects not observed in the rat, but it is not possible to
reconcile the differences here. Nonetheless given the common observation of reduced activity across animals during late gestation as
they out-grow their embryonic space (ten Hof et al.
1999
), further study into the role of posture on distribution
of embryonic movements is warranted.
In sum, age-related changes in the distribution of motility and
selective effects of experimental preparations suggest that activity
duration and pause duration operate independent of one another with
increasing age. Results also suggest that transformations in the
control of pause duration are generally complete by E12, whereas
control of activity duration, initially somewhat stereotypic, becomes
more variable with age. Activity duration also appears to be influenced
by environment-related conditions with increasing age, and this may
partially account for the age-related increases in activity duration
variability. Supporting ideas first put forward by Hamburger et
al. (1965), results suggest that the age-related trends in
activity duration are a result changing input weights for descending
and afferent sensory contacts at spinal levels. The precise
contributions of these extraspinal inputs to the form and distribution
of motility over embryonic development remain to be elucidated.
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ACKNOWLEDGMENTS |
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Special thanks to D. Wong, C. Sebelski, K. Ganley, and D. Rose for assistance in data analyses. Review of an earlier draft and suggestions offered by Dr. Auke Ijspeert were of tremendous value.
This work was funded by National Science Foundation Grant IBN-9616100.
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FOOTNOTES |
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Address for reprint requests: Dept. of Biokinesiology and Physical Therapy, University of Southern California, 1540 E. Alcazar St., CHP155, Los Angeles, CA 90033 (E-mail: nbradley{at}usc.edu).
Received 15 March 2001; accepted in final form 4 June 2001.
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