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The Journal of Neuroscience, August 1, 2002, 22(15):6587-6595
Regulation of Synaptic Connectivity: Levels of Fasciclin II
Influence Synaptic Growth in the Drosophila CNS
Richard A.
Baines1,
Laurent
Seugnet2,
Annemarie
Thompson2,
Paul M.
Salvaterra3, and
Michael
Bate2
1 Department of Biological Sciences, University of
Warwick, Coventry, CV4 7AL, United Kingdom, 2 Department of
Zoology, University of Cambridge, Cambridge, CB2 3EJ, United Kingdom,
and 3 Division of Neurosciences, City of Hope, Beckman
Research Institute, Duarte, California 91010-0269
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ABSTRACT |
Much of our understanding of synaptogenesis comes from studies that
deal with the development of the neuromuscular junction (NMJ). Although
well studied, it is not clear how far the NMJ represents an adequate
model for the formation of synapses within the CNS. Here we investigate
the role of Fasciclin II (Fas II) in the development of synapses
between identified motor neurons and cholinergic interneurons in
the CNS of Drosophila. Fas II is a neural cell adhesion
molecule homolog that is involved in both target selection and
synaptic plasticity at the NMJ in Drosophila. In this
study, we show that levels of Fas II are critical determinants of
synapse formation and growth in the CNS. The initial establishment of
synaptic contacts between these identified neurons is seemingly independent of Fas II. The subsequent proliferation of these synaptic connections that occurs postembryonically is, in contrast,
significantly retarded by the absence of Fas II. Although the initial
formation of synaptic connectivity between these neurons is seemingly
independent of Fas II, we show that their formation is, nevertheless,
significantly affected by manipulations that alter the relative balance
of Fas II in the presynaptic and postsynaptic neurons. Increasing
expression of Fas II in either the presynaptic or postsynaptic neurons,
during embryogenesis, is sufficient to disrupt the normal level of
synaptic connectivity that occurs between these neurons. This effect of Fas II is isoform specific and, moreover, phenocopies the disruption to
synaptic connectivity observed previously after tetanus toxin light
chain-dependent blockade of evoked synaptic vesicle release in these neurons.
Key words:
aCC; neural activity; RP2; synaptic activity; synaptogenesis; tetanus toxin
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INTRODUCTION |
Numerous studies suggest that cues
that guide growing axons to their targets are not in themselves
sufficient to generate the precise patterns of synaptic connectivity
that develop between central neurons. Such studies convincingly
demonstrate that there is an additional phase of activity-dependent
synaptic refinement that is required to either consolidate or eliminate
specific synaptic connections and produce the precision characteristic
of the mature circuitry (Goodman and Shatz, 1993 ; Katz and Shatz,
1996). Studies of this kind have focused primarily on developing
sensory systems, and the contribution of activity to the development of
motor circuitry is less clear (Haverkamp, 1986; Haverkamp and
Oppenheim, 1986; Baines et al., 2001).
In Drosophila, motor neurons establish a stereotyped pattern
of connections with their target muscles, even in the absence of
synaptic transmission (Sweeney et al., 1995). It appears therefore that, in the periphery at least, cues that guide growing axons to their
targets are sufficient to ensure the proper formation of a precise
pattern of connections between presynaptic and postsynaptic cells. One
of the key regulators of growth and guidance of motor axons in
Drosophila is Fasciclin II (Fas II), a homolog of the vertebrate neural cell adhesion molecule (NCAM) (Grenningloh et al.,
1990, 1991). Fas II is expressed in all motor neurons and promotes
adhesion between their axons as they exit the CNS. In addition to axon
guidance, Fas II has also been shown to have important functions at the
neuromuscular junction (NMJ) itself, in which it operates to
stabilize synaptic connections and to mediate activity-dependent
plasticity during postembryonic life (Schuster et al., 1996a,b; Davis
et al., 1997; Davis and Goodman, 1998).
Although the role of Fas II in synaptogenesis and synaptic plasticity
has been intensively studied at the embryonic and larval NMJ, it is not
known whether it has similar functions during the formation and
elaboration of synapses in the CNS (Schuster et al. 1996a). Fas II is
abundantly expressed in the embryonic, larval, and adult CNS in
Drosophila and is, therefore, ideally placed to contribute
to central synapse development (Grenningloh et al., 1991). More
recently, a requirement for Fas II in the formation of odor memory has
been demonstrated, implicating that this CAM is also central to the
mechanisms of synaptic plasticity that underlie learning (Cheng et al.,
2001). Because we developed recently techniques to analyze both the
morphological and functional development of synaptic connections
between cholinergic interneurons and identified postsynaptic motor
neurons in the Drosophila CNS (Baines and Bate, 1998; Baines
et al., 1999, 2001), we are now able to address directly the
contribution of Fas II in the establishment of these connections. In
this study, we find apparent similarities and differences between synaptogenesis centrally and at the NMJ. We show that the embryonic development of these cholinergic central synapses, like the formation of the glutamatergic NMJ, does not require Fas II. However, Fas II is
required for the elaboration of these synapses during postembryonic life. Although Fas II is not required to establish synaptic
connections, the normal pattern of synaptic connectivity is,
nevertheless, sensitive to changes in the relative levels of expression
of Fas II in presynaptic and postsynaptic neurons. In contrast to the NMJ, in which overexpression of Fas II postsynaptically leads to the
stabilization of ectopic connections, in the CNS, overexpression of Fas
II either presynaptic or postsynaptically causes a reduction in
synaptic input to the motor neurons concerned.
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MATERIALS AND METHODS |
Fly stocks. Flies were fed on apple juice agar
supplemented with yeast. Wild type was Oregon-R. The transgene
e5GAL3M-RRK-GAL4 (RRK-GAL4), which contains regulatory sequences
spanning +7.9 to +9.2 kb of the even-skipped locus, as well as its 3'
untranslated region (Fujioka et al., 1999), was used to selectively
express GAL4-UAS-driven transgenes in aCC and RP2 (this GAL4 insert is identical to, but stronger expressing than, RRC-GAL4 used in previous studies) (Baines et al., 1999). Expression of RRK-GAL4 is first evident in early stage 16 embryos, which precedes the onset of synaptogenesis (Baines and Bate, 1998). GAL4 expression was selectively suppressed in aCC/RP2 using an identical RRK promoter fragment upstream
of GAL80 (M. Fujioka and J. Jaynes, unpublished observation). Cha-Gal4
(19B) contains the entire (7.4 kb) promoter sequence for choline
acetyltransferase (cha), the gene encoding the
synthetic enzyme for ACh and expresses selectively in cholinergic
neurons (Salvaterra and Kitamoto, 2001). 1407-GAL4 was used to express UAS transgenes throughout the entire CNS. Expression of tetanus toxin
light chain (TeTxLC) (UAS-TNTG and the inactive variant TNT-VIF) and
Fas II were confirmed by antibody labeling (Sweeney et al., 1995;
Schuster et al., 1996a). UAS-Fas II (PEST+),
fasIIeB112,
fasIIe76,
fasIIe86,
fasIIe93, and anti-Fas II were kindly
provided by C. Goodman. Fas II null (fasIIeB112) larvae were identified
based on their failure to stain with Fas II antibody. UAS-Fas
[glycosylphosphatidylinositol (GPI)] was provided by G. Davis
(University of California, San Francisco, San Francisco, CA),
UAS-connectin by A. Chiba (University of Illinois, Urbana, IL), and
UAS-Fas I by M. Hiramoto (University of Tokyo, Bunkyo, Japan).
Embryo dissection. Larvae were dissected, and central
neurons were accessed as described by Baines and Bate (1998). The
embryo was viewed using a 63× water immersion lens combined with
Nomarski optics (Olympus BX50WI microscope; Olympus Optical, Tokyo, Japan).
DiI labeling and electron microscopy. Larvae were dissected,
fixed in formaldehyde [8% in 75 mM phosphate
buffer (PB), pH 7.2, for 1 hr], washed in PB, and aCC labeled by
applying a droplet of DiI (Molecular Probes, Eugene, OR) to its NMJ on
muscle DA1. After overnight incubation at 4°C, embryos were examined
with epifluorescence, and those preparations in which only aCC was labeled were prepared for electron microscopy. Suitable embryos were
fixed again in formaldehyde (8% and 75 mM PB, pH
7.2, for 8 min), washed, and transferred into Tris buffer (0.1 M, pH. 7.5) before photoconversion using
3,3'-diaminobenzidine tetrachloride (3 mg/ml in Tris buffer; Fisher,
Loughborough, UK). After washing in Tris buffer followed by
H20, embryos were postfixed in osmium (1% in
dH2O, for 1 hr), stained with aqueous uranyl
acetate (2%, for 30 min), dehydrated, and embedded in Araldite resin.
Embryos were sectioned at 2 µm thickness until labeled profiles were
encountered, at which point a series of ultrathin sections (30-50 nm,
silver-gray) were taken. Labeled neurons were not serially sectioned in
their entirety but instead were sampled at 2 µm intervals, with
20-30 consecutive ultrathin sections taken at each successive level. For each genotype, sections were taken from at least five neurons sectioned from at least four animals. Sections were stained with lead
citrate (5 min) and analyzed on a Philips EM 300.
Electrophysiology. The procedure for whole-cell recordings
and composition of salines used are described by Baines and Bate (1998), with the exception that potassium methylsulfonate was substituted for KCl in the patch saline. This substitution greatly extends the maximum recording time possible for these neurons (up to a
maximum of ~20 min). Only cells with an input resistance >1 G
(average of 4.05 ± 0.46 G; n = 100; mean ± SE) were accepted for analysis. Current traces were sampled at 20 kHz and were filtered at 2 kHz. All recordings were made at room
temperature (22-24°C). Cells were unequivocally identified by
labeling with Lucifer yellow (K salt, 0.1%; Molecular Probes), which
was included in the patch saline. Spontaneous currents were recorded in
the presence of TTX (100 nM; Alomone Labs,
Jerusalem, Israel).
Statistics. Data were compared using the nonparametric
Mann-Whitney U test, unless stated otherwise. Results were
deemed significant at p  0.05. All values shown are
mean ± SE
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RESULTS |
Motor neurons receive rhythmic synaptic input from cholinergic
interneurons during embryonic and larval development
Whole-cell current-clamp recordings from two identified
motor neurons, aCC and RP2, in young first instar larvae (L1, ~4 hr after hatching at 25°C) reveal depolarizations that are long-lived (lasting for up to 2 sec), rhythmic (23.4 ± 1.9 events per min; n = 24), and sufficient to evoke action potentials in
the motor neurons (Fig. 1). No
differences were seen between these two motor neurons, and this
reflects their almost identical electrophysiological properties (Baines
et al., 2001). The frequency of these rhythmic depolarizations, which
we term suprathreshold throughout this study to denote the fact that
they elicit spikes, increases during larval development such that, in
young second instar larvae (L2, ~28 hr after hatching), the average
frequency observed is 54 ± 5 per min (n = 7)
(Fig. 1C). These depolarizations, which are first evident,
although at a much lower frequency, in late stage 17 embryos (~18-19
hr after egg laying) (Fig. 1C) (Baines et al., 1999),
probably reflect the synaptic drive from presynaptic interneurons that
form part of the motor pattern generator. Although we are unable to
identify any individual interneurons that make synaptic contact with
aCC/RP2, we established that these suprathreshold inputs are
cholinergic. This conclusion is based on a number of observations.
First, the presence of cholinergic antagonists blocks the synaptic
inputs to aCC/RP2 (Baines et al., 1999). Second, blocking
neurotransmitter release in all cholinergic neurons by expression of
TeTxLC (Sweeney et al., 1995) causes embryonic paralysis and a total
absence of synaptic inputs to motor neurons (R. A. Baines,
unpublished data). Third, these currents are absent in cha
mutants that lack acetylcholine (Baines et al., 2001). Fourth, the
reversal potential of these depolarizations is ~0 mV, which is
identical to currents produced by iontophoretic application of ACh to
these neurons (Baines, unpublished data).
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Figure 1.
Synaptic drive to the aCC/RP2 motor neurons.
A, B, Whole-cell current-clamp recordings
from either aCC or RP2 (aCC shown) in wild-type young first instar
larvae show rhythmic depolarizations that are sufficient to evoke
action potentials, which are more clearly visible in trace
B. C, Synaptic depolarizations are first evident
in late stage 17 embryos (labeled E) and increase in
frequency during larval development (L1, first instar;
L2, second instar). Values are mean ± SE;
n  10.
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Fas II is expressed in a subpopulation of
cholinergic interneurons
Motor neurons, including aCC, express Fas II (Grenningloh et al.,
1991; Van Vactor et al., 1993), but the extent of Fas II expression in
cholinergic interneurons is not known. To determine this, we crossed a
 -galactosidase enhancer trap line inserted in
fasII (A31) (Ghysen and O'Kane, 1989) to flies carrying a
single copy of both B19-GAL4 (which is expressed in all cholinergic
neurons) (Salvaterra and Kitamoto, 2001) and UAS-GFP. The neuronal
expression pattern of A31 mirrors that of fasII mRNA, and
the P element does not alter Fas II protein expression or induce a
mutant phenotype (Grenningloh et al., 1991). Confocal analysis of
CNS from both early (at the initiation of synaptogenesis) and late
stage 17 (mature synapses; Baines and Bate, 1998) embryos shows that a proportion of cholinergic neurons (labeled green) also
express -galactosidase (Fig.
2, red). The number of
coexpressing neurons is difficult to determine precisely but is
relatively small. There are between 5 and 10 such neurons per segment
in the ventral nerve cord (Fig. 2A) and between 20 and 30 in each brain lobe (Fig. 2B). Thus, in
addition to motor neurons, a subpopulation of cholinergic interneurons
appear to express fasII, although we cannot, of course, infer from this that these particular interneurons are presynaptic to
aCC/RP2.
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Figure 2.
A subpopulation of cholinergic interneurons
coexpresses Fas II. Confocal sections (2 µm) through the ventral
nerve cord (VNC) and brain lobe (Brain)
in a late stage 17 embryo. Cholinergic neurons are visualized by
anti-GFP (cha B19-GAL4 driving UAS-GFP;
green), and Fas II expression is reported by a P element
driving a nuclear -galactosidase
(-gal antibody labeled red). A number
of cholinergic cells coexpress Fas II in both the ventral nerve cord
and brain lobe (merged image on right,
arrowheads).
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Fas II is not required for central synaptogenesis
Fas II is not required for the formation of the embryonic NMJ,
although it is required later for the maintenance and proliferation of
these synaptic contacts (Schuster et al., 1996a,b). The requirement for
Fas II for plastic changes at the NMJ was established, in part, through
the use of a series of fasII alleles. These alleles, which
affect only the level of Fas II expression but not protein structure or
tissue distribution, include fasIIeB112 (a
protein null), fasIIe76 (5-10% of normal
levels), fasIIe86 (~50%), and
fasIIe93 (100% and a control for genetic
background) (Grenningloh et al., 1991; Schuster et al., 1996a). To show
whether Fas II is required for the formation of a normal pattern of
presynaptic inputs onto embryonic motor neurons, we studied synaptic
communication in these differing fasII alleles. We analyzed
synaptic drive to aCC/RP2 and made an ultrastructural survey of
connections between aCC and its presynaptic partner neurons.
We find that the synaptic drive to aCC/RP2 in L1 is not
significantly different in these alleles (Fig.
3A). The frequency of
suprathreshold synaptic inputs recorded was 17.4 ± 4.6 (fasIIeB112), 22 ± 2.8 (fasIIe76), 16.3 ± 3.2 (fasIIe86), and 19 ± 2 (fasIIe93) (n 8; p > 0.05). Dendritic processes in the
neuropil belonging to aCC were revealed by photoconversion of
DiI-labeled neurons to produce an electron-dense precipitate. Sites of
presynaptic input were identified by the presence of a cluster of clear
synaptic vesicles, with a requirement that some vesicles be docked to
the presynaptic membrane immediately adjacent to the labeled profile (Fig. 3C,D) (Baines et al., 1999, 2001). There
was no significant difference in the number of presynaptic terminals
that contact aCC in L1 in the presence or absence of Fas II (12.4%, 32 of 258 labeled profiles examined exhibited a presynaptic element,
fasIIe93 vs 11.6%, 32 of 276, fasIIeB112; p > 0.05;
 2 test) (Fig. 3B). We
conclude that Fas II is not required for the establishment of synaptic
connections between embryonic motor neurons and their presynaptic
partners.
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Figure 3.
Synaptic proliferation during larval development
requires Fas II. A, During the first 28 hr of larval
life, the frequency of suprathreshold synaptic inputs recorded in
aCC/RP2 increases approximately twofold (control line,
fase93). In the absence of Fas II
(faseB112), this developmental
increase is significantly reduced (p 0.01). The increase remains normal, however, in Fas II hypomorphs
(lines fase86 and
fase76, respectively). Values are
mean ± SE; n 8. B,
Ultrastructural analysis reveals that the number of presynaptic
terminals that contact aCC (see Materials and Methods) increases during
the same period (fase93). In
the absence of Fas II
(faseB112), however, this
increase fails to occur. C, D, In 28 hr
larvae, the presynaptic terminals (arrows) seen to
contact aCC are qualitatively similar, regardless of the presence
(C) or absence (D) of Fas
II. E, An example of a labeled profile of aCC that is
not associated with a presynaptic input.
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Fas II is required for postembryonic synaptic proliferation
During the first 28 hr of larval life, the frequency of
suprathreshold synaptic drive to aCC/RP2 increases considerably
(19 ± 2.0 to 54 ± 5 events per min,
fasIIe93) (Fig. 3A). This
change is associated with a similarly large increase in the number of
presynaptic terminals that contact aCC (23.2%, 63 of 271, fasIIe93) (Fig. 3B). This
suggests that, in addition to a proliferation of synaptic connections
between presynaptic neurons and aCC/RP2, the probability of firing
activity in the presynaptic neurons also increases during larval
development. In the absence of Fas II, the frequency of synaptic drive
to aCC/RP2 still increases during this period (increasing to 32.5 ± 4 events per min; n = 8;
fasIIeB112), although this increase is
significantly less than when Fas II is present
(p 0.01) (Fig. 3A). Strikingly,
however, in the absence of Fas II, the number of presynaptic terminals
that contact aCC remains unchanged (12.5%, 34 of 272) (Fig.
3B). The ultrastructure of the presynaptic terminals at this
stage is qualitatively indistinguishable from those that develop in WT
(Fig. 3C,D). Thus, although there is no absolute
requirement for Fas II to support either initial synapse formation or
the consolidation of these early connections, it is required for the
proliferation of these contacts postembryonically. The increase in the
frequency of synaptic drive to aCC/RP2 is not affected in the
hypomorphic alleles examined (52 ± 7.6, fasIIe76 and 46 ± 4.2, fasIIe86; n 8) (Fig.
3A), suggesting that even severely reduced levels of Fas II
are still sufficient to allow normal postembryonic maturation of the
motor network.
Genetic manipulations that increase the levels of Fas II in motor
neurons reduce synaptic input
Although the embryonic NMJ forms normally in the absence of Fas
II, an increase in Fas II (PEST+) expression in the postsynaptic muscle
cell during synaptogenesis is sufficient to alter the pattern of
synaptic connectivity by driving the formation of additional, ectopic
synaptic contacts by innervating motor neurons (Davis et al., 1997). We
decided to investigate whether increased levels of Fas II expressed
postsynaptically in motor neurons have a similar effect on synapse
formation in the CNS. Three major isoforms of Fas II have been reported
in Drosophila (Lin et al., 1994; Goodman et al., 1997). Two
of these contain a single transmembrane (TM) domain and are
distinguishable by the presence of a PEST degradation sequence (PEST+)
(Rechsteiner, 1988) in the cytoplasmic domain of one but not in the
other (PEST ). The third isoform is a GPI-linked form that lacks a TM
domain. Using current clamp, we measured the effect of increasing both
Fas II (PEST+) and Fas II (GPI-linked) in aCC/RP2 by recording the
frequency of suprathreshold synaptic inputs observed in these neurons.
Expressing Fas II (PEST+) in aCC/RP2 (using an RRK-GAL4 driver)
significantly reduced the frequency of suprathreshold inputs recorded
in L1 (7.2 ± 1.8 vs 26 ± 3.3 events per min;
n = 10; p 0.001) (Fig.
4A). The effect of
expressing Fas II in aCC/RP2 was similar for a second independent UAS
insert (third chromosome, 10.5 ± 3.2 vs 24 ± 3.0 events per
min; n = 9; p 0.01) and was rescued
by the simultaneous coexpression of a GAL4 inhibitor (RRK-GAL80) in
aCC/RP2 (20 ± 4 events per min; n = 8;
p > 0.05) (Fig. 4A). Together, these
controls rule out any contribution of genetic background. In addition
to recording from aCC/RP2, we recorded the synaptic drive to a third,
control motor neuron (RP3) that does not express GAL4 in these embryos
and larvae. The frequency of suprathreshold inputs in RP3 was
unaffected by expression of Fas II in aCC/RP2 (15 ± 2.9 vs
13.8 ± 0.9 events per min, experimental vs control;
n = 5; p > 0.05) (Fig.
4B). Expressing the GPI-linked isoform of Fas II in
aCC/RP2 did not significantly reduce the frequency of
suprathreshold synaptic drive (19.1 ± 3.9 events per min;
n = 5; p > 0.05). Expression of Fas I
or connectin, two other Drosophila CAMs (Chiba, 1999), in
aCC/RP2 also failed to affect the frequency of synaptic input
(23.2 ± 3.5 and 23 ± 3.6 events per min, respectively;
n = 5; p > 0.05). We conclude that the observed
reduction in suprathreshold inputs from presynaptic partner neurons is
a consequence specifically of enhancing Fas II (PEST+) expression in
aCC/RP2.
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Figure 4.
Increased levels of Fas II in either the
presynaptic or postsynaptic neurons disrupt synaptogenesis.
A shows the frequency of suprathreshold synaptic inputs
recorded in aCC/RP2 in L1. control represents the
average frequency seen in parental GAL4s (1407, RRK, and B19) and UAS
transgenic lines (no individual line was significantly different from
any other). Expression of Fas II (PEST+) in all neurons of the CNS
(1407-GAL4) does not influence the frequency of synaptic inputs.
However, selective expression of Fas II in either aCC/RP2 (RRK-GAL4) or
presynaptic cholinergic (B19-GAL4) neurons significantly reduces input
frequency (p 0.001 for both treatments).
Simultaneous expression of Fas II in both presynaptic cholinergic
neurons and aCC/RP2 (B19/RRK-GAL4s) does not reduce the frequency of
synaptic drive. The effect of expressing Fas II in aCC/RP2 is rescued
by the presence of GAL80 in these motor neurons (RRK-GAL4/GAL80). For
all values, n 8; mean ± SE. B,
Expression of Fas II (PEST+) in aCC/RP2 (RRK-GAL4) does not influence
the frequency of suprathreshold synaptic drive recorded in the RP3
motor neuron (which does not express GAL4 in these larvae). However,
combined expression in both aCC/RP2 and cholinergic interneurons
(B19/RRK-GAL4s) results in a significant decrease
(p 0.05; for explanation, see
Results). C, Ultrastructural analysis reveals
that expression of Fas II (PEST+) in aCC (RRK-GAL4) significantly
reduces the number of presynaptic terminals observed to contact this
neuron (p 0.05; 2 test).
Simultaneous expression of Fas II in both aCC and cholinergic neurons
(B19/RRK) does not, however, affect the number of presynaptic terminals
that contact aCC. D, Expression of Fas II (PEST+) in aCC
does not reduce the number of presynaptic terminals that contact RP3.
Presynaptic terminals contacting this neuron (which does not express
GAL4 in these larvae) are significantly reduced in number after the
combined expression of Fas II in both aCC/RP2 and cholinergic neurons
(p 0.05; 2 test).
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To analyze more rigorously the effect that increasing expression of Fas
II (PEST+, hereinafter termed Fas II) in aCC/RP2 has on the strength of
synaptic currents, we repeated the recordings in voltage clamp. Voltage
clamp is advantageous because it removes any contributions of
voltage-gated ion channels in the motor neuron membrane to the synaptic
currents observed. This is of particular relevance because our previous
work shows that, when deprived of excitatory synaptic input, aCC/RP2
compensate by upregulating the relative strength of specific
voltage-gated channels (Baines et al., 2001). Recordings of synaptic
transmission, under voltage clamp (Vh
60 mV), show clearly that, when Fas II is expressed in aCC/RP2, the
amplitude distribution of evoked synaptic currents is significantly
reduced compared with controls (Fig.
5Ai,Aii). Thus, the
underlying effect of overexpressing Fas II in aCC/RP2 appears to be one
of reduced synaptic function, which results in a significant reduction
of the suprathreshold synaptic drive that these neurons receive.
Such a shift in distribution could result from either a reduction in
the total number of presynaptic terminals that normally excite these
neurons or, alternatively, from a more general weakening of the
synaptic efficacy of the normal complement of synapses present. Because
we do not know whether evoked currents in these neurons result from
activity at just one synapse or require the combined activity of many
synapses, we cannot readily distinguish between these different
mechanisms. However, an analysis of spontaneous currents in aCC/RP2
shows no such reduction in amplitude distribution after expression of Fas II in these neurons (Fig. 5Bi,Bii). Moreover,
the frequency of spontaneous events is significantly reduced after
expression of Fas II (11.1 ± 2.7 vs 4.75 ± 0.6 events per
min, control vs Fas II expression; p < 0.05). A
reduction in spontaneous current frequency, without an associated
change in current amplitude, is consistent with a reduction in the
number of presynaptic input sites to aCC/RP2 rather than a more general
weakening of the synapses normally present (see below).
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Figure 5.
Fas II expression in aCC/RP2 reduces the strength
of synaptic inputs. Ai, Amplitude distribution of evoked
synaptic currents recorded in voltage clamp
(Vh 60 mV) from aCC/RP2 in control L1
(GAL4 and UAS parental lines). Current amplitude is normally (Gaussian)
distributed with a peak amplitude of 102 ± 1.5 pA
(n = 542). Aii, The amplitude
distribution of synaptic currents recorded in aCC/RP2 is significantly
reduced after expression of Fas II (PEST+) in just these neurons
(p < 0.001). Under these conditions, the
distribution is centered around a mean amplitude of 48 ± 1.3 pA
(n = 487). B, Spontaneous currents
(those which persist in the presence of TTX) show no significant
difference in amplitude distribution attributable to expression of Fas
II in aCC/RP2. In control L1 (Bi; GAL4 and UAS parental
lines), currents are normally distributed around a mean of 5.2 ± 0.3 pA (n = 398), whereas after expression of
Fas II in aCC/RP2 (Bii), the distribution mean is
6.3 ± 0.2 pA (n = 190). Individual currents
were obtained from at least six neurons for each analysis.
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Synaptic transmission is influenced by the relative levels of Fas
II expression across the synapse
The fact that genetic manipulations that increase the levels of
Fas II expression in motor neurons are sufficient to alter the synaptic
drive that they receive suggests that synaptogenesis might be sensitive
to the relative levels of Fas II expression in the presynaptic and
postsynaptic neurons. To test this idea, we used a GAL4 construct
driven by the Drosophila cha promoter that is
expressed in all cholinergic neurons (B19-GAL4) (Salvaterra and
Kitamoto, 2001). Using this construct to drive Fas II expression in
cholinergic neurons, we found a significant reduction in the frequency
of suprathreshold synaptic drive recorded in aCC/RP2 (8 ± 1.2 vs
24 ± 3 events per min; n = 10; p 0.001) (Fig. 4A). Thus, increased Fas II in either
the presynaptic or postsynaptic neurons is seemingly sufficient to
disrupt synaptogenesis.
It might be argued that misexpressing Fas II interferes with
synaptogenesis by altering axon guidance before synapse formation. However, this was a possibility that we tested by an additional experiment in which we expressed Fas II simultaneously in both cholinergic neurons and in aCC/RP2 (RRK/B19-GAL4). Recordings from
aCC/RP2 in L1 of this genotype show no significant reduction in the
frequency of suprathreshold synaptic inputs (24.7 ± 7.9 events
per min; n = 5) (Fig. 4A). In this
experiment, however, recordings from the RP3 motor neuron showed a
marked reduction in synaptic drive (6 ± 1.5 vs 15 ± 2.9 events per min; n = 5; p 0.01) (Fig.
4B). Thus, although we have not shown directly that
this manipulation does not affect axon guidance, the experimental outcome reinforces our view that an imbalance in presynaptic and postsynaptic levels of Fas II expression interferes with
synaptogenesis, because in this experiment, RP3, which also receives
input from cholinergic presynaptic neurons, does not itself express
GAL4. Thus, in the case of RP3, elevated expression of Fas II is only presynaptic. We also expressed Fas II in all neurons of the embryo (1407-GAL4) but, again, did not observe any alteration in the frequency
of suprathreshold synaptic currents recorded in aCC/RP2 in L1 (20 ± 2.5 vs 24.7 ± 8 events per min, respectively, experimental vs
control; p > 0.05) (Fig. 4A). We
conclude from these electrophysiological data that the normal
development of synaptic communication between embryonic motor neurons
and their presynaptic partners is sensitive to the relative amount of
Fas II expressed on either side of the synapse but is unaffected by the
absolute level of Fas II present.
Synaptogenesis is disrupted by a forced imbalance of Fas II between
synaptic partner neurons
We showed that experimentally induced increases in
levels of Fas II, in either presynaptic or postsynaptic neurons, are
sufficient to reduce synaptic efficacy. However, the mechanism that
underlies this effect is unknown. Our electrophysiological analysis
(see above) is consistent with overexpression of Fas II resulting in a
reduction in the number of synapses that normally form between these
populations of neurons. Because we are able to quantify the number and
appearance of presynaptic terminals that contact aCC/RP2, this
hypothesis is testable.
An ultrastructural analysis reveals that increased expression of Fas II
in aCC (RRK-GAL4) is accompanied by a significant reduction in the
number of presynaptic terminals that contact this neuron in L1 (5.2%,
32 of 258 vs 12.4%, 17 of 324; p 0.05; 2 test) (Fig. 4C, RP2 was
not examined). The expression of Fas II in these larvae does not,
however, affect the number of presynaptic terminals that contact RP3,
the control neuron that does not express GAL4 (12.7%, 40 of 314 vs
13.6%, 41 of 302 profiles, control vs experimental; p > 0.05) (Fig. 4D). Expressing Fas II simultaneously in both cholinergic neurons and aCC/RP2 (RRK/B19-GAL4s), which does not
affect synaptic drive (see above), also has no affect on the number of
presynaptic terminals that contact aCC (12.4%, 32 of 258 vs 12.2%, 33 of 270, control vs experimental) (Fig. 4C). In these same
larvae, however, the number of presynaptic terminals that contact RP3
(which has a reduced frequency of synaptic input; see above), is
significantly reduced (5.4%, 15 of 277 vs 12.7%, 40 of 314;
p 0.05; 2 test)
(Fig. 4D). The most parsimonious explanation for
these observations is that synaptogenesis between motor neurons and their presynaptic partners is decisively influenced by the relative levels of Fas II expression between the two types of neurons.
TeTxLC-dependent disruption of synaptogenesis is partly dependent
on Fas II
There is a striking similarity between the results of these
experiments and the effects we reported previously of misexpressing TeTxLC in the developing CNS (Baines et al., 1999). In those
experiments, we showed that the selective expression of TeTxLC in
aCC/RP2 results in a reduction in synaptic input to these neurons that
is comparable with the effects we describe here. The underlying cause
of the reduction is, again, a failure of synapse formation between
presynaptic elements and the affected motor neurons. The similarity
between the results of the two experiments suggests that Fas II might be a downstream effector of TeTxLC expression. Indeed, evidence for
such a link has been reported recently in the Drosophila eye in which expression of TeTxLC in photoreceptors results in enhanced levels of Fas II in these cells (Hiesinger et al., 1999).
To establish a link between the two sets of experiments, we examined
the effect of TeTxLC expression in a Fas II null background (fasIIeB112). In the presence of
Fas II, expression of TeTxLC in aCC/RP2 severely reduces the frequency
of suprathreshold inputs to these neurons when compared with
controls (4.4 ± 1.7 vs 17.4 ± 4.4 inputs per min; mean ± SE; n = 7; p 0.01) (Baines et
al., 1999). In the absence of Fas II
(fasIIeB112), the effect of TeTxLC
expression on the frequency of such synaptic inputs is significantly
diminished (10.9 ± 2.4 inputs per minute; mean ± SE;
n 8; p 0.05 compared with TeTxLC in
the presence of Fas II) (Fig. 6). This
suggests that part of the effect of misexpressing TeTxLC is likely to
be caused by altered levels of Fas II expression. However, the fact
that the rescue of the toxin effect is incomplete in the absence of Fas
II (10.9 ± 2.4 vs 17.4 ± 4.4) implies that TeTxLC also acts
to alter the expression levels of other proteins that are involved in
the formation of appropriate synaptic connectivity (adult
photoreceptors; cf. Hiesinger et al., 1999).
View larger version (11K):
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Figure 6.
Fas II is required for TeTxLC-induced reduction in
synaptic inputs. Expression of TeTxLC in aCC/RP2 (RRK-GAL4) is
sufficient to reduce the frequency of suprathreshold synaptic inputs to
these neurons (Baines et al., 1999). However, in the absence of Fas II
(fasIIeB112), the
TeTxLC-induced reduction of input is diminished. For all values,
n 7; mean ± SE. Similarity of
letters denotes statistical significance at
p 0.05. Control larvae contained either the GAL4
or UAS transgenes (but not both) and normal Fas II levels.
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DISCUSSION |
Previous studies of Fas II and synaptogenesis have focused on the
accessible synapse formed at the larval NMJ in Drosophila. In this paper, we extend these studies to the more complex issue of the
formation of central synapses, using a relatively well defined set of
synaptic contacts that form during embryogenesis between cholinergic
interneurons and identified motor neurons. The analysis at the NMJ
shows that Fas II is expressed both presynaptic and postsynaptically
but that it is not required for the formation of synaptic connections
between motor neurons and their target muscles (Schuster et al.,
1996a,b). However, if Fas II is overexpressed in muscles during a
critical period of embryogenesis, it allows additional, ectopic
synapses to form and become stabilized on the muscles concerned (Davis
and Goodman, 1998). These findings together with immunocytochemical
studies of Fas II expression have suggested that, during the initial
phase of synaptogenesis, Fas II is present in limiting amounts on the
postsynaptic cell and that the protein then becomes aggregated under
contacts formed by the innervating motor neuron, so inhibiting the
formation of stable, ectopic contacts by other neurons (Davis et al.,
1997). These observations imply that, although not essential to
synaptogenesis, Fas II can act as a powerful determinant of the
distribution and number of contacts on the postsynaptic cell.
Our first aim was to show whether or not Fas II can act in a similar
manner during the embryonic formation of central synapses. We know that
Fas II is expressed in motor neurons (Grenningloh et al., 1991; Van
Vactor et al., 1993), and our studies show that it is also expressed in
a subset of cholinergic interneurons, although we are unable to say
whether these are immediately presynaptic to the motor neurons that we
are studying. Moreover, we do not know the precise distribution of Fas
II protein in either the presynaptic or postsynaptic neurons, nor do we
know anything about the relative expression of Fas II in different
neurons. However, the results of both our physiological and
ultrastructural analyses show that an apparently normal pattern of
interneuron to motor neuron synapses develops in the absence of Fas II.
In the continued absence of Fas II, however, these synapses clearly
fail to proliferate, and, as a consequence, the synaptic drive to motor
neurons is reduced. On the other hand, increased Fas II expression in
either the presynaptic or postsynaptic cells is sufficient to reduce synaptic inputs to the motor neurons as judged physiologically or
ultrastructurally. The puzzling aspect of this latter result is that,
although it suggests that, as at the NMJ, Fas II can act centrally to
influence the pattern of synaptic contacts, it appears to do so in
completely the opposite sense: additional Fas II reduces the number of
synapses rather than promoting the formation of additional, ectopic
contacts. Our findings also differ from the observed consequence of
disproportionately increasing the mammalian homolog of Fas II, NCAM, in
postsynaptic hippocampal neurons maintained in culture, which, similar
to the NMJ, is also sufficient to strengthen synaptic connectivity
(Dityatev et al., 2000). We cannot discount the possibility that
increased expression of Fas II in aCC/RP2 results in the additional
formation of inappropriate synaptic connections to these neurons, which
may be sufficient to weaken, structurally or functionally, the
connections that normally form between these neurons and their normal
presynaptic partners. However, because we see a clear reduction in the
number of presynaptic input sites in young first instar larvae, under these conditions, it would suggest that any such inappropriate connections are likely to have retracted by this stage. Simply interpreted, the effects that we report suggest that, although Fas II
is required for postembryonic synapse proliferation, disproportionate increases in levels of Fas II in central neurons has a potentially repressive effect on the formation of synapses between the cell concerned and its putative synaptic partners, regardless of its site of
expression. We should be cautious, however, in finally adopting this
conclusion, because the environment of dendritic arborizations in the
embryonic neuropil is likely to be complex, and we do not understand
the contribution of Fas II to dendritic patterning. Thus, although
increased levels of Fas II expression, or absence of this CAM, does not
alter the gross morphology of aCC based on an analysis of DiI-labeled
cells (Baines, personal observation), such manipulations could
conceivably alter more subtle aspects of dendritic morphology and
disrupt the normal pattern of synaptic connectivity. A detailed
analysis of dendritic patterning in these neurons is reliant on, and
must wait until, we are able to visualize individual presynaptic
partner neurons.
Our experiments concentrate on two motor neurons, aCC and RP2, that
innervate dorsal muscles. These neurons are identifiable in the
embryonic and larval CNS and are relatively accessible to patch-clamp
electrodes. In addition, the RRK-Gal4 line allows us to misexpress
proteins such as Fas II selectively in these cells (Baines et al.,
1999). We also monitor the results of our experiments in a third,
control motor neuron, RP3, that innervates ventral longitudinal
muscles. The effects of our experiments on RP3 are interesting and
revealing. First, under control conditions, the frequency of
suprathreshold synaptic input to RP3 is approximately one-half that
seen in aCC/RP2. This suggests that, under the conditions of our
experiments, RP3 (ventral muscles) and aCC/RP2 (dorsal muscles) receive
distinct inputs from interneurons involved in generating rhythmic motor
outputs. Second, the frequency of input to RP3 remains unchanged when
the level of Fas II is increased in aCC/RP2, and the frequency of
synaptic input declines in these neurons. This result suggests that the
alterations in synaptic communication that we detect are the result of
local events in the neurons concerned. The third observation is the
most significant and reinforces our interpretation that the effects we
describe depend on the relative levels of Fas II expressed in
presynaptic and postsynaptic neurons. Thus, in experiments in which Fas
II is simultaneously expressed in cholinergic interneurons and aCC/RP2, the decline in input frequency to aCC/RP2, seen when Fas II is expressed in either of these sets of neurons alone, fails to occur. This result implies that it is the balance of Fas II in presynaptic and
postsynaptic cells that is decisive for the formation of a normal
pattern of synaptic inputs. Significantly, in this experiment, the
control neuron RP3, with normal levels of Fas II, is innervated by
interneurons whose level of Fas II has been increased: we predict that
synaptic communication should be weakened, and this is indeed the
effect that we observe. Thus, alterations in the relative levels of Fas
II in presynaptic and postsynaptic cells have local effects that are
selective and predictable for individual neurons. This strongly
suggests that, during synaptogenesis, the balance of Fas II in
presynaptic and postsynaptic cells can influence the formation of a
normal pattern of synaptic contacts.
The strikingly similar results of misexpressing Fas II or TeTxLC in
aCC/RP2 suggested to us that the effects of TeTxLC might be caused at
least in part by elevated levels of Fas II in the neurons in which it
is expressed. Indeed, we find that the toxin effects are partially
rescued by the complete loss of Fas II function. This, together with
our observation that an imbalance in presynaptic and postsynaptic
levels of Fas II expression is sufficient to interfere with normal
synaptogenesis, offers an explanation for our previously puzzling
finding that blocking vesicle release from the postsynaptic neuron
leads to a reduction in presynaptic input to that cell (Baines et al.,
1999). If, as in photoreceptor cells, expression of TeTxLC leads to an
overall increase in levels of Fas II in the affected cells (Hiesinger
et al., 1999), then we would expect synaptic inputs to those cells to
be disturbed. The finding that the local balance of Fas II influences
the formation of central synapses, together with the strong implication
that alterations in vesicle trafficking can interfere with this
balance, is important for our understanding of normal synaptogenesis
and its control. We would predict that, although synaptogenesis can proceed successfully in the absence of Fas II, any, possibly
activity-dependent, modulation of Fas II levels in presynaptic or
postsynaptic cells has the potential to influence the number and
pattern of connections formed in a normal embryo. How activity might
regulate levels of Fas II in synaptic terminals remains to be
determined. Synaptically targeted membrane proteins, including
neurotransmitter receptors, are thought to be constantly moving in to
and out of the synaptic membrane, this movement being dependent on
successive rounds of vesicular endocytosis and exocytosis (Turrigiano,
2000). A perturbation at any point in this cycle has the potential to
result in an inappropriate surface expression of these proteins and
perhaps provide a viable route to influence synaptic plasticity. An
example of such a mechanism is long-term sensitization in
Aplysia, which involves an activity-dependent downregulation
of apCAM (a homolog of Fas II) in the presynaptic sensory neuron
(Mayford et al., 1992; Zhu et al., 1995). This downregulation appears
to be attributable to a cAMP-dependent reduction in gene
expression and a simultaneous increase in the rate of endocytotic
internalization of preexisting protein from the presynaptic membrane
(Bailey et al., 1992; Mayford et al., 1992).
Although Fas II is not required for the initial formation of an
appropriate pattern of synaptic contacts either peripherally (at the
NMJ) or centrally, our experiments show that, as at the NMJ, Fas II is
essential for the further growth and elaboration of synaptic contacts
during postembryonic life. During this larval phase of active feeding
and growth, the increasing size of the muscles is matched by an
increase in the size and complexity of motor neuron dendritic arbors
(Uhler, 2001). Our ultrastructural analysis of synaptic inputs to motor
neuron arbors during early larval life shows that, at least in these
early phases, there is a corresponding increase in the number of
presynaptic contacts on the dendrites of aCC/RP2. Strikingly, this
increase fails completely in the Fas II null larvae. It is likely that
this, together with the previously documented reduced innervation at
the NMJ, contributes to the increasing sluggishness and ultimate death
of these mutant larvae. We have not surveyed the presynaptic contacts
formed postembryonically in the series of hypomorphic Fas II alleles,
but, given the relatively normal maturation of the synaptic drive that
we detect in these animals, it seems likely that there is an essential
but low level of Fas II that is required for the proper growth and
elaboration of presynaptic endings on the motor neuron dendritic
arbors. It may well be that, as at the larval NMJ, this critical level
of Fas II is a significant determinant of plasticity at central
synapses in the fly.
| |
FOOTNOTES |
Received March 4, 2002; revised May 3, 2002; accepted May 9, 2002.
This study was funded by the Wellcome Trust. M.B. is a Royal Society
Research Professor. We are grateful to G. Davies, C. Goodman, J. Jaynes, M. Fujioka, A. Chiba, M. Hiramoto, and H. Skaer for flies
and/or antibodies. We also thank M. Day for help with electron
microscopy and M. Landgraf for comments and discussion.
Correspondence should be addressed to Dr. R. A. Baines, Department
of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK.
E-mail: rbaines{at}bio.warwick.ac.uk.
| |
REFERENCES |
-
Bailey CH,
Chen M,
Keller F,
Kandel ER
(1992)
Serotonin-mediated endocytosis of apCAM: an early step of learning-related synaptic growth in Aplysia.
Science
256:645-649[Abstract/Free Full Text].
-
Baines RA,
Bate M
(1998)
Electrophysiological development of central neurons in the Drosophila embryo.
J Neurosci
18:4673-4683[Abstract/Free Full Text].
-
Baines RA,
Robinson SG,
Fujioka M,
Jaynes JB,
Bate M
(1999)
Postsynaptic vesicle release is essential for synaptogenesis in Drosophila.
Curr Biol
9:1267-1270[Web of Science][Medline].
-
Baines RA,
Uhler JP,
Thompson A,
Sweeney ST,
Bate M
(2001)
Altered electrical properties in Drosophila neurons developing without synaptic transmission.
J Neurosci
21:1523-1531[Abstract/Free Full Text].
-
Cheng Y,
Endo K,
Wu K,
Rodan AR,
Heberlein U,
Davis RL
(2001)
Drosophila fasciclinII is required for the formation of odor memories and for normal sensitivity to alcohol.
Cell
105:757-768[Web of Science][Medline].
-
Chiba A
(1999)
Early development of the Drosophila neuromuscular junction: a model for studying neuronal networks in development.
Int Rev Neurobiol
43:1-24[Medline].
-
Davis GW,
Goodman CS
(1998)
Synapse-specific control of synaptic efficacy at the terminals of a single neuron.
Nature
392:82-86[Medline].
-
Davis GW,
Schuster CM,
Goodman CS
(1997)
Genetic analysis of the mechanisms controlling target selection: target-derived Fasciclin II regulates the pattern of synapse formation.
Neuron
19:561-573[Web of Science][Medline].
-
Dityatev A,
Dityateva G,
Schachner M
(2000)
Synaptic strength as a function of post- versus presynaptic expression of the neural cell adhesion molecule NCAM.
Neuron
26:207-217[Web of Science][Medline].
-
Fujioka M,
Emi-Sarker Y,
Yusibova GL,
Goto T,
Jaynes JB
(1999)
Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients.
Development
126:2527-2538[Abstract].
-
Ghysen A,
O'Kane C
(1989)
Neural enhancer-like elements as specific cell markers in Drosophila.
Development
105:35-52[Abstract].
-
Goodman CS,
Shatz CJ
(1993)
Developmental mechanisms that generate precise patterns of neuronal connectivity.
Cell
72:77-98.
-
Goodman CS,
Davis GW,
Zito K
(1997)
The many faces of Fasciclin II: genetic analysis reveals multiple roles for a cell adhesion molecule during the generation of neuronal specificity.
Cold Spring Harb Symp Quant Biol
62:479-491[Abstract/Free Full Text].
-
Grenningloh G,
Bieber A,
Rehm J,
Snow PM,
Traquina Z,
Hortsch M,
Patel NH,
Goodman CS
(1990)
Molecular genetics of neuronal recognition in Drosophila: evolution and function of immunoglobulin superfamily cell adhesion molecules.
Cold Spring Harbor Symp Quant Biol
55:327-340[Abstract/Free Full Text].
-
Grenningloh G,
Rehm EJ,
Goodman CS
(1991)
Genetic analysis of growth cone guidance in Drosophila: Fasciclin II functions as a neuronal recognition molecule.
Cell
67:45-57[Web of Science][Medline].
-
Haverkamp LJ
(1986)
Anatomical and physiological development of the Xenopus embryonic motor system in the absence of neural activity.
J Neurosci
6:1338-1348[Abstract].
-
Haverkamp LJ,
Oppenheim RW
(1986)
Behavioral development in the absence of neural activity: effects of chronic immobilization on amphibian embryos.
J Neurosci
6:1332-1337[Abstract].
-
Hiesinger PR,
Reiter C,
Schau H,
Fischbach K-F
(1999)
Neuropil pattern formation and regulation of cell adhesion molecules in Drosophila optic lobe development depend on synaptobrevin.
J Neurosci
19:7548-7556[Abstract/Free Full Text].
-
Katz LC,
Shatz CJ
(1996)
Synaptic activity and the construction of cortical circuits.
Science
274:1133-1138[Abstract/Free Full Text].
-
Lin DM,
Fetter RD,
Kopczynski C,
Grenningloh G,
Goodman CS
(1994)
Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation, and altered fasciculation.
Neuron
13:1055-1069[Web of Science][Medline].
-
Mayford M,
Barzilai A,
Keller F,
Schacher S,
Kandel ER
(1992)
Modulation of an NCAM-related adhesion molecule with long-term synaptic plasticity in Aplysia.
Science
256:638-643[Abstract/Free Full Text].
-
Rechsteiner M
(1988)
Regulation of enzyme levels by proteolysis: the role of PEST regions.
Adv Enzyme Regul
27:135-151[Web of Science][Medline].
-
Salvaterra PM,
Kitamoto T
(2001)
Drosophila cholinergic neurons and processes visualized with Gal4/UAS-GFP.
Gene Expr Patterns
1:73-82[Medline].
-
Schuster CM,
Davis GW,
Fetter RD,
Goodman CS
(1996a)
Genetic dissection and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilisation and growth.
Neuron
17:641-654[Web of Science][Medline].
-
Schuster CM,
Davis GW,
Fetter RD,
Goodman CS
(1996b)
Genetic dissection and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity.
Neuron
17:655-667[Web of Science][Medline].
-
Sweeney ST,
Broadie K,
Keane J,
Niemann H,
O'Kane J
(1995)
Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.
Neuron
14:341-351[Web of Science][Medline].
-
Turrigiano GG
(2000)
AMPA receptors unbound: membrane cycling and synaptic plasticity.
Neuron
26:5-8[Web of Science][Medline].
-
Uhler JP
(2001)
The development of dendritic arbors in Drosophila motoneurons,
In: PhD thesis University of Cambridge.
-
Van Vactor D,
Sink H,
Famborough D,
Tsoo R,
Goodman CS
(1993)
Genes that control neuromuscular specificity in Drosophila.
Cell
73:1137-1153[Web of Science][Medline].
-
Zhu H,
Wu F,
Schacher S
(1995)
Changes in expression and distribution of Aplysia cell adhesion molecules can influence synapse formation and elimination in vitro.
J Neurosci
15:4173-4183[Abstract].
Copyright © 2002 Society for Neuroscience 0270-6474/02/22156587-09$05.00/0
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ABSTRACT
INTRODUCTION
Gene
