1School of Biological Sciences, University of Kentucky, Lexington 40506-0225; and 2Graduate Center for Toxicology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40506-0305
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ABSTRACT |
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Li, Hao, Doug Harrison, Grace Jones, Davy Jones, and Robin L. Cooper. Alterations in Development, Behavior, and Physiology in Drosophila Larva That Have Reduced Ecdysone Production. J. Neurophysiol. 85: 98-104, 2001. We investigated behavior, physiology, sensitivity to exogenous application of ecdysone, and nerve terminal structure for differences between the reduced ecdysone genotype, ecd1/ecd1, and wild-type control ecd1/TM6B animals during the early and late third instars when raised at 25°C. The ecd1 mutants were able to survive through larval development and form pupae. However, the results demonstrate that the time to pupation is lengthened by about 50 h for the ecd1/ecd1 as compared with the wild-type control siblings. In addition to the lengthened larval cycle in the mutant, ecd1/ecd1 animals, they also display behavioral differences as compared with controls. The rate of body wall contraction and mouth hook movements are reduced in the early third instar of ecd1/ecd1 as compared with controls. The physiological measure of excitatory junction potential amplitude for the combined Is and Ib terminals did not reveal any differences among the two genotypes during the early third instar but the synaptic strength is reduced in the late third instars for controls. Application of exogenous ecdysone is still effective during the late third instar for the ecd1/ecd1 but not the controls. This suggests that endogenous production of ecdysone have already taken place in the wild-type but not the ecd1/ecd1 larvae, thus the rapid nongenomic responses could still be observed in the late third ecd1/ecd1 larvae. Structurally the number of varicosities and the terminal length showed significant differences between ecd1/ecd1 and the wild-type ecd1/TM6B genotype in the late third instars.
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INTRODUCTION |
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Hormones have essential
roles in developmental changes in the life of Drosophila
melanogaster, from the larval instars to the adult form.
20-hydroxyecdysone (20-HE), currently regarded as the active form of
ecdysone, is associated with developmental changes during metamorphosis
(Baehrecke 1996; Cayre et al. 2000
; Farka
and
utáková 1998
, 1999
;
Henrich et al. 1993
, 1999
; Riddford 1985
;
Steel and Davey 1985
). This particular hormone is
important in causing the behavioral and physical changes during the
development stages of each molt (Truman 1996
). In
Drosophila, high titer levels of ecdysteroid are reached
during the third instar larva, between the late feeding stage and
prepupa, and in the pupal stage (Kim et al. 1999
;
White et al. 1997
). It has been observed that steroids
cause both physiological and anatomical effects on neurons
(Jacobs and Weeks 1990
; Levine and Weeks
1996
; Thummel 1996
). The majority of currently
described actions of ecdysteroids are genome based (Levine and
Weeks 1996
; Segraves 1994
; Thummel
1996
). There is also a substantial accumulating literature
documenting nongenomic effects, especially of membrane-bound steroid
receptors that cause relatively rapid changes in cellular processes
(Baulieu 1997
; Benten 1999
; Chang
and Chang 1999
; Christ et al. 1999
;
Hanaya et al. 1997
; Schmidt et al. 1998
;
Watson and Gametchu 1999
). We have investigated the
development and maintenance of motor neuron structure, function, and
sensitivity to exogenous application of ecdysone using the
ecdysoneless mutant strain of Drosophila,
ecd1, which contains a recessive,
temperature-shock-sensitive allele of a gene required for ecdysteroid
production. The homozygote ecd1/ecd1 is
larval lethal at 29°C, but at 25°C, it will survive to the late
third instar (Henrich et al. 1993
), thus allowing
behavioral, anatomical, and physiological studies.
Surprisingly, there are few studies of nongenomic actions of
molt-related steroid (i.e., 20-HE) compounds in crustaceans and insects
(Cooper and Ruffner 1998; Cromarty and Kass-Simon
1998
; Ruffner et al. 1999
). Behavior of the
Drosophila larvae changes immediately before pupation, but
the mechanisms modulating behavior are poorly understood. On exposure
of an isolated crayfish or early third instar Drosophila
nerve-muscle preparation to 20-HE, there is a pronounced reduction in
the size of the excitatory junction potentials (EJPs) recorded in the
muscle (Cooper and Ruffner 1998
; Ruffner et al.
1999
). These studies also showed a quick change in the quantal
release properties of synaptic transmission in response to 20-HE
(Cooper and Ruffner 1998
). The rapid rate of response
within the presynaptic terminal and the lack of transcriptional regulation, since the neuron is anucleated, implies nongenomic action.
The purpose of this study using Drosophila is to investigate both the behavior of the whole animal and the physiology and morphology of the NMJs of third instar under the conditions of reduced ecdysone production.
Segments of this work have appeared in abstract form (Cooper et
al. 2000; He et al. 1998
, Li et al.
1999a
,b
).
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METHODS |
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Husbandry
Dr. Vincent C. Henrich, University of North Carolina at
Charlotte, supplied the
ecd1/TM6B, Tubby
(Tb) fly genotype (Lindsley and Zimm 1992)
used for these experiments. Eggs from
ecd1/TM6B, Tb flies were
collected for 2-h periods on apple juice-agar plates with yeast paste.
The eggs were allowed to hatch and develop at 25°C until they reached
the third instar larval stage.
Developmental and behavioral assays
The second instar larvae were collected as they molted and
examined for the presence of nontubby morphology (indicating the ecd1/ecd1
genotype) approximately 48 h after the eggs were laid. Sibling animals with the TB mutant morphology (Craymer 1980) are
ecd1/TM6B and were used as
controls in all experiments because they are wild type for the
ecd, due to the ecd+ allele
carried on the TM6B chromosome. Thirty to 50 second instar larvae of
ecd1/ecd1 and
wild-type ecd1/TM6B,
Tb siblings from each plate were transferred separately into
vials containing standard cornmeal-dextrose-agar-yeast medium. The time
and number of white pupa that formed were determined.
Feeding and locomotory behavior was assessed in early third instar
larvae of both genotypes as described in Neckameyer
(1996). The number of body contractile motions and mouth hook
contractions were counted for 1 min. Ten animals were assayed in each
of six independent experiments.
Electrophysiology
The preparations were taken from early and late third instar
larvae staged as described in the preceding text. The larval dissections were performed as described in Cooper et al.
(1995b). The physiological solution used is the same as
previously described (Stewart et al. 1994
).
Intracellular recording were made with microelectrodes filled with 3 M
KCl (30-60 m). The responses were recorded with a 1× LU head stage
and an Axoclamp 2A amplifier to a VHS tape (Vetter, 400) as well as
on-line to a PowerMac 9000 via a MacLab/4 s interface (ADInstruments).
All events were measured and calibrated with the MacLab Scope software
version 3.5.4.
Anatomy
With the use of a fluorescent anti-HRP primary antibody and
confocal microscopy, the Type I endings of the two major axons (Is and
Ib) can be distinguished on the basis of their range of bouton size and
total bouton complement (Atwood et al. 1993). With
confocal microscopy, the quantitative data of bouton number, terminal
length, and muscle dimensions were obtained. Fluorescent images of the
nerve terminals were viewed with a Leica DM RE upright fluorescent
microscope using a ×40 water-immersion objective with appropriate
illumination. The composite images of Z-series were collected with a
Leica TCS NT/SP confocal microscope for illustration. The number of
varicosities can be counted from the images directly. The Leica
software was used to measure and quantify the terminal length directly
from the images.
Statistical analysis
Numerical data are represented as means ± SD. The one-way ANOVA test was used for comparison of means in the responsiveness to 20-HE, with P < 0.05 chosen as the level of significance. Two-way ANOVA test was used to examine the differences of morphological data. When the basic assumption of parametric ANOVA test was not valid, the nonparametric ANOVA rank test was used.
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RESULTS |
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General morphology and behavior
To determine if the developmental rate is altered for the homozygous ecd1/ecd1 as compared with the control sibling ecd1/TM6B animals, the time it took for 50% of the population to form pupae was monitored. The genotypic identity of these animals was determined based on the dominant phenotypic marker for body size and shape, Tubby (Tb), which is present on the TM6B balancer chromosome. The relative differences shown for the late third instar stadium are prevalent throughout all the instars and pupa. To determine if the developmental rate is altered for the homozygous ecd1/ecd1 as compared with the control sibling ecd1/TM6B animals, the time it took for 50% of the population to form pupa was monitored. The time from the 2-h egg laying period until a pupa formed was considered the time to pupation, and the time taken for all viable larva to become pupae was used as the total time taken. By plotting the commutative sum till total pupae formation occurred, a 50% index value could then be determined and compared between the genotypes (Fig. 1A). The ecd1/ecd1 had a substantial phase lag in developmental timing for the population to form pupae as indicated in the 50-h lag for the 50% index of mean pupation time (Fig. 1B).
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Since it was previously demonstrated that application of 20-HE on
exposed neuromuscular preparations resulted in depression of synaptic
transmission (Ruffner et al. 1999), we compared
locomotive behavior between the wild-type and
ecd1/ecd1
larvae when endogenous 20-HE titers are expected to be different. Body-movement assays consisted of the rate in body-wall contraction and
mouth-hook movements. These behavioral measures are standardized procedures to examine larval function (Neckameyer and Cooper
1998
; Sewall et al. 1975
). The early third
instars corrected for the developmental phase lag were used in the
behavioral tests. The ecd1/ecd1 had a
slower mean rate in body wall contraction and mouth hook movement than
control animals (P < 0.05, t-test; Fig.
1C).
Neuromuscular measures
The muscle m6 is innervated by both Ib and Is motor neurons
(Kurdyak et al. 1994). Both Ib and Is neurons can be
recruited together to produce a composite excitatory junction potential (EJP) as shown in Fig. 2A. The
composite Ib and Is EJP amplitudes are significantly reduced in late
third controls as compared with the
ecd1/ecd1
animals (P < 0.01, n = 6, t-test). There is no difference in the EJP amplitudes
observed between the early third stage of
ecd1/ecd1 and
controls (Fig. 2B). This suggests that the presence of
ecdysone or a secondary effect of ecdysone results in the reduction in the EJP amplitude in late third instar of control sibling
ecd1/TM6B animals. To test the
postulate that a lower titer of ecdysone in
ecd1/ecd1 is
protecting the EJP amplitude in the late third stadium, direct application of 20-HE on exposed neuromuscular junctions was preformed. It was previously demonstrated that early third-staged larvae are
susceptible to exposure to 20-HE in such a manner that the EJP
amplitude would decrease rapidly within minutes (Ruffner et al.
1999
). Such nongenomic actions were also demonstrated in
crustaceans during intermolt (Cooper and Ruffner 1998
).
In both Drosophila and crayfish, the nongenomic actions of
20-HE were shown to be presynaptic, in decreasing the number of
neurotransmitter containing vesicles to be released during nerve
terminal depolarization. Changes in the EJP amplitudes on exposure to
20-HE were examined in the present study within late third instars of
ecd1/ecd1 and
controls. Only the
ecd1/ecd1 flies
showed a substantial decrease in EJP amplitude (P < 0.05, t-test, Fig. 3). This
result suggests that these endogenous production of ecdysone in late
third instar control flies already elicited the nongenomic actions and
further application thus showed little effect.
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Nerve terminal morphology
The two motor axons, Ib and Is, innervating muscle 6 and 7 are
readily distinguishable by the size of the varicosities along the
terminals (Fig. 4). The Ib axon has big
terminal varicosities as compared with the Is axon. The
ecd1/TM6B animals have shorter
terminals than the
ecd1/ecd1
animals; this is readily seen in the overview of the terminals for
ecd1/TM6B (Fig. 4A1)
and ecd1/ecd1
(Fig. 4B1) as well as in the higher magnification (Fig.
4, A2 and B2) images. For both early and late
third instars there are significant differences as indicated between
ecd1/ecd1 and
control sibling ecd1/TM6B
animals (Fig. 5A) although in
both genotypes there is a significant difference between the early
third and late third instars, thus indicating a developmental increase
in the length of both the Is and Ib terminals (P < 0.05, Tukey test; Fig. 5A). The number of varicosities of
the Ib versus Is terminals showed a significant difference between
ecd1/ecd1 and
control sibling ecd1/TM6B
animals for only the late third instar (, P < 0.05, Tukey test; Fig. 5B). No significant differences could be
found between the
ecd1/ecd1 and
controls in early third instars (Fig. 5B). For
ecd1/ecd1 and
controls, there were significant developmental differences between the
early and late third instar stages ( · · · , P < 0.05, Tukey test; Fig. 5B). The developmental differences
for both
ecd1/ecd1 and
controls are that there are more varicosities along Is terminals as
compared with Ib terminals (P < 0.05, Paired
t-test).
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DISCUSSION |
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The ecdysoneless temperature-sensitive
[l(3)ecd1] mutant of Drosophila is a
conditional larval lethal when raised at a restrictive temperature of
29°C (Garen et al. 1977). Ecdysteroid production in
the larval ring gland is reduced as low as 10% of the control level in
this mutant (Garen et al. 1977
; Henrich et al.
1993
), and pleiotropic effects of this mutant strain have also
been examined (Redfern and Bownes 1983
). It appears the
brain, ring gland, and larval salivary glands are smaller in this mutant.
In this study, we investigated if behavior, physiology, sensitivity to 20-HE, and nerve terminal structure showed differences between the reduced ecdysone genotype, ecd1/ecd1, and wild-type control ecd1/TM6B animals during the early and late third instars when raised at 25°C. The ecd1 mutants were able to survive through larval development and form pupae. However, the results demonstrate that the time to pupation is lengthened by about 50 h for the ecd1/ecd1 as compared with the wild-type control siblings. There is variation in both phenotypes within the population, but with the use of the 50% to pupation index, comparisons between strains can be made. In addition to the lengthened larval cycle in the mutants, ecd1/ecd1 animals also display behavioral differences as compared with controls. The rate of body-wall contraction and mouth-hook movements are reduced in the early third instar of ecd1/ecd1 as compared with the wild-type ecd1/TM6B. The physiological measure of EJP amplitude for the combined Is and Ib terminals did not reveal any differences among the two genotypes during the early third instar, but the synaptic strength is reduced in the late third instars for the ecd1/TM6. Since locomotive behavioral differences are observed between the ecd1/ecd1 and the wild-type ecd1/TM6B in early third instars although without measurable differences in the experimentally evoked synaptic responses on m6, one is left to speculate that possible the CNS command of the motor neurons may be different during locomotion. In addition, we have not addressed the function of the other muscles associated with body contractions between these altered genotypes that may in part account for the behavioral observations. It is possible that there maybe pleiotropic effects in the ecd1 mutant that result in a variety of developmental and behavioral problems, such as lower locomotive activities.
Application of exogenous 20-HE is still effective during the late third
instar stadium for the
ecd1/ecd1 but
not the controls. This suggests that endogenous production of ecdysone
has already taken place in the wild-type but not the ecd1/ecd1
larvae, thus the rapid nongenomic responses could still be observed in
the late third
ecd1/ecd1
larvae. It has been shown that motor neurons in the hawkmoth, Manduca sexta, undergo various responses such as apoptosis,
regression, and regrowth during various stages of development where
levels of ecdysteroids are also high (Truman and Reiss
1995). In addition, mushroom bodies isolated from
Drosophila during metamorphosis showed enhanced neurite
outgrowth by direct application of 20-HE in culture (Kraft et
al. 1998
). However, the hormonal control of ecdysone on motor
nerve terminal growth and synaptic strength has not been fully
addressed, thus our interest in the use of the
ecd1/ecd1
mutant for this study.
The mechanisms that govern abrupt behavioral change associated with
molting are also not well understood. In late third instar, the animals
reverse from negative to positive phototactic behavior, and they slow
down their locomotor functions to begin to form pupae. Reduced synaptic
strength may contribute to their lower locomotor activity at this stage
and could possibly be related to the increased ecdysone titers
(Ruffner et al. 1999). The nongenomic action of ecdysone
is not known but since fewer vesicles are released during evoked
stimulation as measured from quantal analysis studies obtained at NMJs
of Drosophila and crayfish there are some likely sites of
action that are feasible (Cooper and Ruffner 1998
;
Ruffner et al. 1999
). The protein-protein interactions
of the SNARE proteins may possible be effected by ecdysone, leading to
fewer vesicles to be docked and released with evoked stimulation. It is
possible that ecdysone could even be disrupting the protein interaction of already docked vesicles. Electron microscopy studies of the presynaptic terminals are needed to address this issue further. In
addition, if steroidal action is affecting evoked calcium entry within
the nerve terminal, this would result in fewer evoked vesicles. The
synthetic ecdysone agonists RH-5849 is known to block a 4-aminopyridine (4-AP)-sensitive voltage-gated K+ channel
(Ortego and Bower 1996
), which could then alter the
entry of calcium through voltage-gated calcium channels. Potentially with the use of calcium-sensitive indicators and confocal microscopy, this issue can be examined (Cooper et al. 1995a
). At
present, we are examining intact vesicle dynamics in the nerve
terminals with the exposure of 20-HE with the use of the dye (FM1-43)
that allows visualization of vesicle populations within nerve
terminals. In addition, there are rapid effects on insect behavior when
20-HE or ecdysone is placed in their diet that have been correlated to
rapid changes in the activity of sensory neurons (Tanaka et al.
1994
).
Structurally the number of varicosities and the terminal length showed
significant differences between
ecd1/ecd1 and
the wild-type ecd1/TM6B genotype
in the late third instars. The relationship between terminal morphology
and synaptic strength in Drosophila NMJs has been examined
previously. For instance, a larger ratio of terminal size per muscle
size gives rise to larger EJP amplitudes (Lnenicka and
Keshishian 2000). It is also suggested that the cell-adhesion molecule Fasciclin II plays a role in controlling synaptic
stabilization and growth in Drosophila NMJs. In e76 mutant
flies that possess a hypomorphic allele of the Fas II gene, there are
fewer synapse-bearing nerve terminal varicosities (Stewart et
al. 1996
). Schuster et al. (1996a
,b
)
demonstrated that the increase or decrease of axon sprouting in
Drosophila NMJs depends on the expression level of Fas II.
In these mentioned cases, the synaptic strength is maintained at a
normal level for the muscle cell as a whole in spite of differences in
the length and number of varicosities of the nerve terminals. Our
results indicate that the
ecd1/ecd1 late
third instar larvae have longer nerve terminals with more varicosities
than the ecd1/TM6B larvae. These
morphological differences in the motor nerve terminals could account
for the larger EJPs measured in the
ecd1/ecd1 late
third instars, although there are several other factors that need to be
considered as well that will require further investigation to determine
the true nature of why the EJPs are more pronounced in the
ecd1/ecd1 late
third instars. Such scenarios other than nerve terminal morphological
differences are that the ecdysteroid titers are higher in
the ecd1/TM6B larvae than the
ecd1/ecd1
larvae and therefore a greater suppression in transmitter release is
measured. In addition, the background strain
ecd1/TM6B larvae are of the
Tubby (Tb) fly genotype, which is observed by the
larvae being shorter yet wider than the
ecd1/ecd1
strain. This difference in body morphology is also noted among the
longitudinal muscles (e.g., m6), meaning that m6 is shorter and wider
in ecd1/TM6B larvae than in
ecd1/ecd1
larvae; this may also account for differences in total surface area of
the muscle. This morphological difference of the muscle can lead to a
difference in input resistance of the muscle fiber, thus altering the
amplitude of the EJP given the same synaptic current. Studies are
currently underway in our laboratories in examining such differences as
synaptic physiology, nerve terminal development, and structure using
these ecdysoneless strains with other background strains.
With several of the mechanisms underlying maintenance and modulation of
synaptic strength during development and maturation being elucidated in
various experimental systems, much still remains to be uncovered. In
particular, little is know about the nongenomic actions of steroids on
synaptic efficacy. One model system for studying fundamental questions
about steroid action is the Drosophila neuromuscular
junction. In Drosophila, the advantage of known identifiable
cells with the powerful techniques of molecular genetics and the
ability to perform anatomical analysis as well as electrophysiological measures allow experimental insights that are not possible, at present,
in other systems. Taking advantage of mutations that results in lower
ecdysone titers allows further investigations into the steroid actions
of ecdysone. There are well-documented genomic effects of steroids such
as estradiol, aldosterone, vitamin D3, and cortisol. Processes such as
activation of the IP3-, cGMP-, and cAMP-signaling
pathways and increased release of internal calcium are future avenues
to be investigated (Orchink et al. 1991; Thummel
1996
; Wehling 1995
). Since this model system has played, and continues to play, important roles in answering questions of regulation of chemical synaptic transmission, we feel that investigations of steroid action will be fruitful in providing answers
to the mechanistic actions of a variety of steroids.
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ACKNOWLEDGMENTS |
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We thank Dr. Vincent C. Henrich, University of North Carolina at Charlotte, for supplying the flies.
Funding was provided by National Science Foundation Grants IBN-9808631 and NSF-ILI-DUE 9850907 to R. L. Cooper.
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FOOTNOTES |
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Address for reprint requests: R. L. Cooper, 101 T.H. Morgan School of Biological Sciences, University of Kentucky, Lexington, KY 40506-0225 (E-mail: RLCOOP1{at}pop.uky.edu).
Received 5 June 2000; accepted in final form 22 September 2000.
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REFERENCES |
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