MICROSCOPY RESEARCH AND TECHNIQUE 49:14 –25 (2000)

Drosophila Larval Neuromuscular Junction:

Molecular Components and Mechanisms
Underlying Synaptic Plasticity
YOUNG HO KOH,1,2 L. SIAN GRAMATES,1,3 AND VIVIAN BUDNIK1–3*
1
Biology Department, University of Massachusetts, Amherst, Massachusetts 01003
2
Neuroscience and Behavior Program, University of Massachusetts, Amherst, Massachusetss 01003
3
Program for Molecular, Cellular and Developmental Biology, University of Massachusetts, Amherst, Massachusetts 01003

KEY WORDS: discs large; CAMKII; MAGUKs; in vitro system; synapse assembly; PKA; second
messengers; retrograde signals; postsynaptic protein translation; gene regula-
tion
ABSTRACT Understanding the mechanisms that mediate synaptic plasticity is a primary goal
of molecular neuroscience. The Drosophila larval neuromuscular junction provides a particularly
useful model for investigating the roles of synaptic components in both structural and functional
plasticity. The powerful molecular genetics of this system makes it possible to uncover new synaptic
components and signaling molecules, as well as their function in the intact organism. Together with
the mouse hippocampus and Aplysia dissociated cell culture, the Drosophila larval neuromuscular
junction has been among the most valuable model systems for examining the molecular and cellular
basis of neuronal plasticity. Microsc. Res. Tech. 49:14 –25, 2000. © 2000 Wiley-Liss, Inc.

INTRODUCTION and, therefore, to allow for synaptic plasticity, is not

A chemical synapse is organized into a presynaptic
terminal, containing synaptic vesicles and neurotrans-
mitter release machinery, and a postsynaptic element
that is characterized by the presence of dense clusters
of neurotransmitter receptor and ion-channel com-
plexes. The pre- and postsynaptic elements are sepa-
rated by a synaptic cleft across which neuroactive mol-
ecules and ions can diffuse (reviewed by Garner and
Kindler, 1996). If a signal is to be transmitted effec-
tively across the synaptic cleft, a precise localization of
ion channels and neurotransmitter receptors at both
the pre- and the postsynaptic elements is critical (re-
viewed in Budnik, 1996; Gramates and Budnik,
1999a).
Synaptic plasticity, the process by which connections
between a neuron and its target are modified, is be-
lieved to be the basic mechanism underlying learning
and memory. Studies using mammalian and inverte-
brate models show that there are many common mech-
anisms involved in different categories of learning and
memory, and that these mechanisms are evolutionarily
conserved from lower invertebrate to higher mamma-
lian systems (reviewed by Bailey et al., 1996; Milner et
al., 1998). For example the cAMP and Ca2⫹-dependent
signal transduction pathways appear to be common
elements in every form of learning and memory among
animal species (reviewed by Abel et al., 1998; Dubnau
and Tully, 1998).
Studies of synaptic plasticity in the mammalian hip-
pocampus (Frey and Morris, 1997), Aplysia dissociated
cell cultures (Martin et al., 1997a), and the Drosophila
neuromuscular junction (Davis and Goodman, 1998)
indicate that the minimal unit of synaptic plasticity
may be a single synapse. However, the full array of
components required to make a functional synapse,
fully known.
Most studies aimed at unraveling the mechanisms
underlying synaptic plasticity in the mammalian cen-
tral nervous system (CNS) have been performed using
in vitro systems, in cultured cells, brain slices, or bio-
chemical assays (e.g., Fields and Itoh, 1996; Kryl et al.,
1999; Niethammer et al., 1999). Most in vivo studies
have been limited to immunocytochemical descriptions
of protein localization (e.g., Burgin et al., 1990; Furui-
chi et al., 1993). Therefore, the analysis of plasticity in
the whole organism has been greatly enhanced by the
use of model systems such as the fruit fly and Aplysia.
The Drosophila larval neuromuscular junction
(NMJ) is particularly advantageous to the investiga-
tion of synaptic plasticity. The most favorable features
of this model system include its powerful genetics, an-
atomical and electrophysiological accessibility, and a
well-defined synapse ultrastructure. These features fa-
cilitate the discovery of synaptic constituents and the
functional analysis of these proteins at identified syn-
apses.
Although the Drosophila NMJ is simpler than the
mammalian CNS, both in regard to synaptic connec-
tions and diversity of neurons, it shows striking con-
servation of many key synaptic molecules identified in
mammals. Amongst these conserved components are
Discs-large (DLG), the Drosophila Membrane-Associ-
ated Guanylate Kinase (MAGUK) homologous to PSD-
95/SAP90 family proteins (Budnik et al., 1996; Guan et
Contract grant sponsor: NIH; Contract grant numbers: RO1 NS3007, RO1
NS37061, F31 NS10861-01; Contract grant sponsor: Human Frontiers Grant.
*Correspondence to: Vivian Budnik, Biology Department, Morrill (South),
University of Massachusetts, Amherst MA 01003.
E-mail: vbudnik@bio.umass.edu

© 2000 WILEY-LISS, INC.

 REGULATION OF SYNAPTIC COMPONENTS
al., 1996; Lahey et al., 1994; Tejedor et al., 1997;
Thomas et al., 1997); Fasciclin II (FasII), the structural
homologue of neuronal cell adhesion molecules
(NCAMs) (Schuster et al., 1996b; Thomas et al., 1997;
Zito et al., 1997); Drosophila Ca2⫹/calmodulin depen-
dent kinase II (CaMKII) (Griffith et al., 1993; Koh et
al., 1999); and Drosophila cAMP dependent protein
kinase (PKA) (reviewed by Davis et al., 1995, 1998).
Recent reviews (Budnik, 1996; Gramates and Bud-
nik, 1999a; Keshishian et al., 1993) have concentrated
on the assembly and maturation of synapses. In this
review, we will integrate recent findings using the Dro-
sophila NMJ with studies of the mammalian brain and
Aplysia dissociated cell cultures to address the ques-
tion of how synaptic components implicated in devel-
opment and synaptic plasticity are regulated.
Synaptic plasticity is a dynamic process that is reg-
ulated at a number of different levels. Specific synaptic
components are activated or deactivated, assembled in
a spatially restricted cluster or segregated into differ-
ent cell compartments, brought into proximity to or
released from the junctional membrane or cytoskele-
ton, synthesized or degraded. Regulatory signals are
required for the translation of mRNA into newly syn-
thesized proteins, or to signal the genetic machinery in
the nucleus to activate or deactivate transcription of
particular genes.
TAKE ME OUT TO THE BOUTONS: A BRIEF
OVERVIEW OF THE DROSOPHILA LARVAL
NEUROMUSCULAR JUNCTION
The Drosophila larval body wall musculature con-
sists of a segmentally repeated pattern of 30 muscles
innervated by about 40 motorneurons. The axons of
these motorneurons branch onto the surface of the
muscle fibers forming a synaptic arbor composed of a
series of varicosities connected by thin axonal pro-
cesses. These varicosities, or synaptic boutons, fall into
three classes (reviewed in Gramates and Budnik,
1999a; Rheuben, et al., 1999). The major class of bou-
tons (type I) are glutamatergic, and are enveloped by
the subsynaptic reticulum (SSR), an elaborately lay-
ered structure formed from the muscle junctional mem-
brane (Atwood et al., 1993; Jia et al., 1993). All 30 body
wall muscles are innervated by type I glutamatergic
boutons. Subsets of muscles are additionally inner-
vated by type II or type III boutons, which contain
octopamine or peptide neurotransmitters as well as
glutamate (Anderson et al., 1988; Cantera and Nassel,
1992; Gorczyca et al., 1993; Monastriani et al., 1995;
Zhong and Pena, 1995).
Muscles 6 and 7, which have been a particular focus
of NMJ research, are innervated exclusively by type I
boutons. Extensive ultrastructural analyses on these
boutons reveal that they can be subdivided into type I
small (Is) and type I big (Ib) by size, the extent of
folding of the postsynaptic membrane or SSR, and elec-
trophysiological properties (Atwood et al., 1993; Jia et
al., 1993; Kurdyak et al., 1994). These two bouton
subtypes show different functional properties; type Is
boutons have larger stimulation thresholds and excita-
tory junctional currents (EJCs) of larger amplitude,
while more pronounced short-term facilitation is ob-
served at type Ib boutons (Atwood et al., 1997; Budnik
15
et al., 1996; Kurdyak et al., 1994; reviewed by Gra-
mates and Budnik, 1999a).
SHALL WE GATHER AT THE SYNAPSE?
MAGUKS AS ORGANIZERS OF
GLUTAMERGIC SYNAPSES
MAGUKs of the PSD-95 family have been shown to
be important organizers of multiprotein complexes in
both mammalian CNS excitatory synapses (reviewed
by Garner and Kindler, 1996; Kennedy, 1997) and at
the Drosophila larval NMJ (reviewed by Budnik, 1996;
Gramates and Budnik, 1999a). MAGUKs contain three
types of protein-protein interaction modules, one or
more PDZ domains, a Src homology 3 (SH3) domain
(Woods and Bryant, 1993), and a guanylate kinase-like
(GuK) domain (Woods and Bryant, 1991). Some
MAGUKs and MAGUK-related proteins also contain
additional functional regions, such as a calmodulin
binding site and a CaM kinase-like N-terminal in the
cases of CamGUK (Dimitratos et al., 1997), CASK
(Hata et al., 1996) and Lin-2 (Hoskins et al., 1996)
(Fig. 1).
An ever-growing array of neuronal MAGUK binding
partners have been identified, as summarized in Fig-
ure 1. Recent findings implicate PSD-95 family
MAGUKs in the structural and functional regulation of
synaptic plasticity via interaction with receptors (re-
viewed by Sheng, 1996), ion channels (reviewed by
Garcia et al., 1998; Sheng, 1996), cell adhesion mole-
cules (Irie et al., 1997; Thomas et al., 1997; Zito et al.,
1997), protein kinases (Koh et al., 1999; Tezuka et al.,
1999), or via regulation of retrograde signaling path-
ways (Brenman et al., 1996; Jaffrey et al., 1998) or
changes in GTPase activity (Chen at al., 1998; Kim et
al., 1998). The existence of at least three different pro-
tein-protein interaction motifs in MAGUKs and the
identification of many of their binding partners suggest
that MAGUKs may serve to assemble and to anchor
specific synaptic components to allow for efficient reg-
ulation of signal transduction pathways.
The first MAGUK binding partners identified were
Shaker-type K⫹ channels (Kim et al., 1995) and
NMDA-type glutamate receptor subunits (Kim et al.,
1996; Kornau et al., 1995; Muller, et al., 1996). These
transmembrane proteins were shown to become clus-
tered in heterologous cells upon transfection with
mammalian MAGUKs PSD-95 and chapsyn-110 (Kim
et al., 1995, 1996; Niethammer et al., 1998). These
studies demonstrated that the first and second PDZ
domains of the MAGUK bind the cytoplasmic C-termi-
nal S/TXV motifs of the binding partners (Doyle et al.,
1996; Kim et al., 1995; Kornau et al., 1995; reviewed by
Budnik, 1996; Gramates and Budnik, 1999a; Sheng,
1996). Similarly, the third PDZ domain of PSD-95 was
found to interact with the cytoplasmic C-terminal
S/TXV motif of neuroligin (Irie et al., 1997). Neuroligin
is a postsynaptic transmembrane protein that func-
tions as a heterophilic cell adhesion molecule, binding
tightly to the extracellular domain of presynaptic-neur-
exin in a Ca2⫹-dependent reaction (Nguyen and Sud-
hof, 1997). The intracellular C-terminal of ␤-neurexin
binds to the PDZ domain of CASK, a MAGUK-related
protein containing an N-terminal similar to CaM ki-
nase, in addition to PDZ, SH3, and GuK domains (Hata
et al., 1996). This intricate multiprotein complex span-
16 Y.H. KOH ET AL.
Fig. 1. Top: Protein-protein interaction modules of PSD-95 family
MAGUKs. Examples include DLG (Woods and Bryant, 1991), SAP97
(Muller et al., 1995), PSD-95 (Cho et al, 1992; Kistner et al., 1993),
SAP102 (Muller et al., 1996), and Chapsyn-110 (Kim et al., 1996).
Middle: Schematic diagram of MAGUK-related proteins containing a
CaMKII-like domain, including CASK (Hata et al., 1996), CamGUK
(Dimitratos et al., 1997), and Lin-2 (Hoskins et al., 1996). Bottom:
Selected binding partners of PSD-95 family MAGUKs, including
Shaker-type K⫹ channels (Kim et al., 1995), Fasciclin II (Thomas et
al., 1997; Zito et al., 1997), NMDA-type glutamate receptor subunit
(Kornau et al., 1995; Kim, et al., 1996; Muller, et al., 1996), nNOS
(Brenman et al., 1996), SynGAP (Kim et al., 1998; Chen et al., 1998),
CRIPT (Niethammer et al. 1998), Citron (Furuyashiki et al., 1999;
Zhang et al., 1998), neuroligin (Irie et al., 1997), kainate receptor
(Garcia et al., 1998), band 4.1 (Lue et al., 1996), GKAP (Kim et al.,
1997; Naisbett, et al., 1997; Takeuchi, et al., 1997) and GUKHolder
(Gramates and Budnik, 1999b).
ning the cell membranes of both the pre-and postsyn-
aptic cells provides an insight into how MAGUKs may
serve to organize pre- and postsynaptic elements into
the precise arrangement required for efficient trans-
mission of signals across the synaptic cleft, clustering
receptors and ion channels, and the association of these
clusters with the synaptic cytoskeleton.
MAGUKs have also been implicated in an important
reversible regulatory mechanism of synaptic transmis-
sion, the phosphorylation of ion-channels or receptors.
Fyn, a member of the Src family of protein tyrosine
kinases, phosphorylates the NR2A subunit of NMDA
receptors in heterologously transfected cells. fyn mu-
tant mice exhibit a reduced level of NR2A phosphory-
lation, indicating that Fyn is likely to have an in vivo
regulatory role (Tezuka et al., 1999). Deletion analysis
indicates that the SH2 domain of Fyn binds to the third
PDZ domain of PSD-95, and that this association is
independent of tyrosine phosphorylation. A number of
Src family protein tyrosine kinases, including Src, Yes,
Lyn, and Fyn, are coimmunoprecipitated with PSD-95,
suggesting that they are complexed with PSD-95 (Te-
zuka et al., 1999). These data indicate that PSD-95
brings receptors and kinases together for proper signal
transmission.
Changes in synaptic plasticity are accompanied by
changes in synapse morphology; axons form new
branches, new boutons are formed and stabilized or
retracted. Such alterations in cell morphology are nec-
essarily accompanied by cytoskeletal rearrangements.
In the mammalian synapse, the postsynaptic density
(PSD) is heavily enriched in cytoskeletal elements. Re-
cent experiments have raised the intriguing possibility
that PSD-95 may function in the rearrangement of the
PSD cytoskeleton, acting indirectly through its binding
partners. For example, the microtubule binding protein
CRIPT (Cysteine-rich interactor of PDZ three) also
binds to the third PDZ domain of PSD-95 (Niethammer
et al., 1998). CRIPT colocalizes with PSD-95 in the
postsynaptic density of brain synapses, and can be
coimmunoprecipitated with antisera to PSD-95 and tu-
bulin. If both proteins are co-transfected into heterolo-
gous cells, CRIPT redistributes PSD-95 to the microtu-
bule-based reticular network (Niethammer et al.,
1998).
PSD-95 also forms a complex with the Rho effector
Citron in hippocampal GABAergic neurons (Zhang et
al., 1999) and thalamic excitatory neurons (Furu-
yashiki et al., 1999); as with CRIPT, this interaction is
mediated by the third PDZ domain (Zhang et al., 1999).
The Rho family of small GTPases is known to act on
rearrangements of the actin-based cytoskeleton, e.g.,
during cytokinesis (Madaule et al., 1998), axonal out-
growth, and cell migration (Furuyashiki et al., 1999;
Zipkin et al., 1997). Citron interacts with the GTP-
bound form of Rho (Madaule et al., 1995). The interac-
tions of PSD-95 with proteins known to be involved in
cytoskeleton dynamics suggest that synaptic MAGUKs
could have a regulatory relationship with cytoskeletal
elements of the postsynaptic density.
Nearly all of these observations have been made
using biochemical assays, cell culture, or other in vitro
methods, with in vivo studies largely limited to the
determination of patterns of protein distribution via
immunocytochemistry. Very few mammalian MAGUK
mutants exist to assess their in vivo function. So far,
only the generation of PSD-95 knockout mice, which
express a truncated form of the protein, has been re-
ported (Migaud et al., 1999). These mice exhibit en-
hanced long-term potentiation and severe impairment
of spatial learning and memory (Migaud et al., 1999).
However, there are no significant changes in synapse
number or in clustering of NMDA NR1 subunits in the
hippocampus, possibly due to genetic redundancy of
MAGUKs in the mammalian CNS (Migaud et al.,
1999).
In the fly, however, direct demonstrations of the clus-
tering function of MAGUKs have proved feasible. The
Drosophila MAGUK Discs-large (DLG) is required both
pre- and postsynaptically for synaptic targeting and
clustering of Shaker-type K⫹ channels and the cell
adhesion molecule FasII (Tejedor et al., 1997; Thomas
et al., 1997; reviewed by Budnik, 1996; Gramates and
Budnik, 1999a). Mutations deleting all but the first two
PDZ domains of DLG induce the ectopic clustering of
Shaker-type K⫹ channels (Tejedor et al., 1997). Muta-
 REGULATION OF SYNAPTIC COMPONENTS
tions with low levels of DLG result in mislocalization of
both Shaker-type K⫹ channels (Tejedor et al., 1997)
and FasII (Thomas et al., 1997) over muscles. Ultra-
structural changes in pre- and postsynptic elements of
dlg mutants are also observed at the EM level. In
hypomorphic dlg alleles, the number of active zones
within presynaptic terminals is significantly increased
(Thomas et al., 1997), while the elaborate membrane
folding of the postsynaptic SSR is substantially re-
duced (Budnik et al., 1996; Koh et al., 1999; Lahey et
al., 1994). The increased number of active zones, as
well as an increase in bouton size, seen in severe dlg
hypomorphic alleles, is similar to the phenotype seen in
hyperexcitability mutants affecting K⫹ channels, such
as Shaker and ether á gógo (Jia et al., 1993), and is
accompanied by an increase in synaptic efficacy (Bud-
nik et al., 1996). Both increased activity and decreased
DLG expression result in decreased levels of FasII at
the synapse, which may explain this phenotype (Schus-
ter et al., 1996b; Thomas et al., 1997). Evidence sugg-
gests that decreased levels of FasII, or of the Aplysia
FasII homolog ApCAM, result in synapse growth (see
Ca2⫹/Calmodulin Kinase Dependent Phosphorylation
of Synaptic Components) (Abel et al., 1998; Bailey and
Kandel, 1993; Koh et al., 1999; Schuster et al., 1996b;
Thomas et al., 1997).
RETURN TO SENDER: SECOND MESSENGER
PATHWAYS AND RETROGRADE SIGNALS AT
THE SYNAPSE
cAMP-Dependent Pathway
The cAMP-dependent second messenger pathway is
believed to be intimately involved in activity-depen-
dent changes in synaptic plasticity. Mutations of com-
ponents of this pathway have a profound effect on
functional and structural synaptic plasticity of the lar-
val NMJ, affecting the degree of branching of axon
terminals, bouton number, number of active zones per
bouton, and number of vesicles released per nerve im-
pulse (Davis et al., 1995; Griffith and Greenspan, 1993;
Malenka et al., 1989; Wang et al., 1994; Zhong and Wu,
1991; Zhong et al., 1992; reviewed in Budnik, 1996;
Gramates and Budnik, 1999a; Hannan and Zhong,
1999).
cAMP-dependent protein kinase A (PKA) is one of
the multitudinous proteins regulated by cAMP. Inac-
tive PKA exists as a tetramer, made up of two catalytic
and two regulatory subunits. When the regulatory sub-
units are bound by cAMP, the catalytic subunits are
released as active monomers (Coffino et al., 1976).
There is some evidence that PKA phosphorylation of
postsynaptic glutamate receptors has a regulatory ef-
fect on functional synaptic plasticity. Overexpression of
wild type PKA catalytic or regulatory subunits in Dro-
sophila larval muscles decreases quantal size, as mea-
sured by the amplitude of miniature excitatory junc-
tional currents (mEJCs) or miniature excitatory junc-
tional potentials (mEJPs). Conversely, muscle-specific
transgenic expression of a defective regulatory subunit
in which the cAMP binding site is mutated has the
opposite effect; quantal size is increased (Davis et al.,
1998). These effects are not seen in mutants that lack
muscle-specific glutamate receptor IIA (DGluRIIA)
(Davis et al., 1998). DGluRIIA (Schuster et al., 1991) is
one of two glutamate receptors expressed in the mus-
17
cles of Drosophila larvae; mutations that delete one of
the receptors are homozygous viable (Petersen et al.,
1997). DGluRIIA, but not DGluRIIB, has an optimal
consensus PKA phosphorylation site, thus making it a
possible substrate for PKA regulation (Petersen et al.,
1997). Unlike wild type larvae, mutants lacking DGlu-
RIIA do not show decreased quantal size in response to
bath application of Sp-cAMPS, a nonhydrolyzable
cAMP analog known to increase PKA activity (Davis et
al., 1998). These data strongly suggest that PKA may
regulate the activity of DGluRIIA. The PKA-dependent
reduction in quantal size is accompanied developmen-
tally by an increase in quantal content, indicating the
presence of a retrograde signal regulating presynaptic
release of glutamate (Davis et al., 1998).
NO as a Potential Retrograde Signal at the
Neuromuscular Junction
The change in axon arborization induced by selective
overexpression of the cell adhesion molecule FasII also
points to the existence of a retrograde signal from
postsynaptic muscle to presynaptic motorneuron
(Davis and Goodman, 1998). When Fas II is overex-
pressed in body wall muscles 6 and 13, they become
hyperinnervated by RP3 or RP1 and RP5 motor neu-
rons, while their companion muscles 7 and 12, which
normally share innervation by the same motor neuron,
have reduced innervation. The dramatic increase in
bouton number on muscles 6 and 13 is compensated by
a decrease in the amount of neurotransmitter released
per bouton. Likewise, quantal size undergoes a com-
pensatory increase at the muscles with reduced inner-
vation (Davis and Goodman, 1998). These differential
changes in synaptic efficacy between hyper- and hypo-
innervated muscle indicate a muscle-to-motor neuron
retrograde regulation at specific presynaptic terminals.
Several candidates have been suggested to poten-
tially act as retrograde signalling molecules (reviewed
in Harish and Poo, 1992; Nguyen and Lichtman, 1996).
Of these, an attractive candidate for this retrograde
signal is nitric oxide (NO), which has been implicated
in retrograde signaling in the mammalian hippocam-
pus (O’Dell, et al., 1991; reviewed in Hawkins et al.,
1994, 1998) as well as in snail motor neurons (Park et
al., 1998). NO, synthesized by neuronal NO synthase
(nNOS), has been shown to activate soluble guanylyl
cyclase and lead to formation of cyclic GMP (reviewed
in Bredt and Snyder, 1992). Mammalian nNOS is en-
riched postsynaptically, where its activation is tightly
coupled to NMDA receptor-mediated Ca2⫹ influx.
nNOS contains a PDZ domain, and interacts with the
MAGUKs PSD-95 or PSD-93 through a PDZ-PDZ in-
teraction (Brenman et al., 1996), thus physically as
well as functionally linking nNOS to the NMDA recep-
tor. If expression of PSD-95 is suppressed in cultured
cortical neurons, nNOS expression and function is un-
affected, but Ca2⫹-activated NO production is blocked,
confirming that the physical association of NMDA re-
ceptors with nNOS is critical to the functional associ-
ation (Sattler et al., 1999). This association appears to
be regulated by CAPON (carboxy terminal PDZ ligand
of nNOS), which interacts with the nNOS PDZ domain
via a C-terminal S/TXV–like motif (Jaffrey et al., 1998).
PSD-95/nNOS clusters are lost in transfected cultured
cells overexpressing transgenic CAPON, which directly
18 Y.H. KOH ET AL.
competes with PSD-95 for binding of the nNOS PDZ
domain (Jaffrey et al., 1998).
There is evidence that NO may also function at the
Drosophila NMJ. An NO-sensitive guanylyl cyclase is
expressed in a stage-specific manner in the RP3 motor
neuron, which innervates larval muscles 6 and 7, sug-
gesting that an NO/cGMP signalling system could exist
(Wildemann and Bicker, 1999a). Application of the NO
donor sodium nitroprusside (SNP) to the larval NMJ
induces an increase in cGMP immunoreactivity in the
boutons and neuronal cell bodies of presynaptic nerve
terminals, but not in the postsynaptic muscle. This can
be blocked by specific inhibitors of soluble guanylyl
cyclase (Wildemann and Bicker, 1999b). Furthermore,
NO donors and membrane permeant cGMP analogues
cause vesicle release at the NMJ (Wildeman and
Bicker, 1999b). However, there is no direct evidence
that larval muscles are the source of NO release. Mus-
cle-specific expression of Drosophila NOS has not yet
been detected by immunocytochemistry (Wildemann
and Bicker, 1999b). dNOS, unlike mammalian nNOS,
does not have a PDZ domain (Regulski and Tully,
1995), and hence may not be able to interact directly
with DLG as nNOS interacts with PSD-95 (Brenman et
al., 1996).
Ca2ⴙ/Calmodulin Kinase Dependent
Phosphorylation of Synaptic Components
Ca2⫹/calmodulin-dependent protein kinase II (CaM
kinase II) is one of the most abundant proteins in the
postsynaptic density of the mammalian central syn-
apse, although it is also expressed presynaptically. It
has been suggested that it may play critical roles in
long-term potentiation (Mayford et al., 1996). In cul-
tured hippocampal neurons, transgenic CaMKII be-
comes dissociated from filamentous actin in response to
NMDA receptor activation by glutamate. Unbound
CaMKII is then translocated to the postsynaptic den-
sity of dendritic terminals, a process that requires the
binding of calmodulin. Transgenic CaMKII is colocal-
ized with PSD-95 immunoreactivity at the postsynap-
tic density, suggesting a possible functional or struc-
tural interaction between MAGUKs and CaMKII
(Shen and Meyer, 1999). Interestingly, calmodulin
binds near the SH3 domain of the human MAGUK
NE-dlg/SAP-102 in a Ca2⫹-dependent manner, and
NE-dlg/SAP 102 interacts in vitro with the GuK do-
main of PSD-95 in the presence of Ca2⫹ and calmodulin
(Masuko et al., 1999). Thus, the interaction of neuronal
MAGUKs may be modulated by Ca2⫹/calmodulin.
A likely CaMKII substrate is SynGAP, another com-
ponent of the postsynaptic density. p135 SynGAP is a
synaptic Ras-GTPase activating protein. It interacts in
vitro with all three PDZ domains of PSD-95 and SAP-
102 by its C-terminal S/TXV motif. Moreover, SynGAP
colocalizes with PSD-95 at excitatory synapses (Chen
et al., 1998; Kim et al., 1998). CaMKII-dependent phos-
phorylation of p135 SynGAP inhibits its Ras-GTPase
activating activity (Chen et al., 1998). The GTP-bound
form of Ras recruits Raf kinase to the cell membrane,
where it can then initiate a phosphorylation cascade
leading to activation of mitogen-activated protein
(MAP) kinase (Cobb and Goldsmith, 1995). MAP ki-
nase has been suggested to be intimately involved in
the induction of long-term potentiation (Bading and
Greenberg, 1991; English and Sweatt, 1996, 1997).
Ras-GTPases block this cascade by hydrolyzing the
Ras-bound GTP (Downward et al., 1990; Trahey et al.,
1988; Vogel et al., 1988; Wigler, 1990). Activation of
NMDA receptors is known to activate the MAP kinase
signal transduction pathway (Bading and Greenberg,
1991; English and Sweatt, 1996, 1997). The PSD-95-
mediated association of CaMKII and SynGAP with
NMDA receptors could establish a link between the
NMDA receptor-mediated rise in Ca2⫹ and the activa-
tion of the MAP kinase pathway (Chen et al., 1998).
CaMKII is equally important at fly central and NMJ
synapses where, much as in the mammalian central
synapse, it is believed to be a major factor in synaptic
plasticity (Griffith et al., 1994; Koh et al., 1999). The
Drosophila CaMKII (DCaMKII) gene shows highest
homology to the mammalian-CaMKII subunit (Cho et
al., 1992), which is exclusively expressed in the mam-
malian hippocampus (Mayford et al., 1996). It has been
proposed that CaMKII can transduce transient synap-
tic activity to persistent synaptic structural and func-
tional changes via phosphorylation of its substrates
(Lisman, 1994). The activities of the mammalian-
CaMKII and DCaMKII are similarly regulated in a
complex manner by Ca2⫹/calmodulin and by autophos-
phorylation. This process has been demonstrated in
molecular-genetic experiments, in both mammals and
flies, in which mutant forms of the molecules are over-
expressed in specific tissues. In the presence of Ca2⫹/
calmodulin CaMKII autophosphorylates serine 287
(286 in ␣-CaMKII) to produce a Ca2⫹/calmodulin-inde-
pendent kinase. Mutation of serine 287 to alanine
(CaMKIIT287A) prevents autophosphorylation and re-
sults in kinase activity that is exclusively dependent on
Ca2⫹/calmodulin (Mayford et al., 1996; Meyer et al.,
1992; Wang et al., 1998). In contrast, changing serine
287 to aspartic acid (CaMKIIT287D) mimics autophos-
phorylation, so that the kinase becomes constitutively
active, even in the absence of Ca2⫹/calmodulin (May-
ford et al., 1996; Wang et al., 1998). It is possible to
inhibit CaMKII activity by overexpressing alanine
CaM kinase II inhibitory peptide (ala), which competes
for its catalytic site, thus reducing kinase activity (Grif-
fith and Greenspan, 1993; Wang et al., 1994).
Studies at the Drosophila larval NMJ show that
DCaMKII has an important role in the regulation of
synapse structure and function via its interaction with
DLG. DCaMKII appears to be expressed both pre- and
postsynaptically at the larval NMJ where it partially
co-localizes with DLG (Koh et al., 1999). Antibodies to
DCaMKII coimmunoprecipitate DLG from larval body
wall muscle extracts (Koh et al., 1999). Persistent
DCaMKII activation via overexpression of the consti-
tutively active form at the NMJ induces abnormal
NMJ morphology that is similar to the phenotype seen
in dlg mutant alleles. These abnormal phenotypes in-
clude increased bouton size and abnormal NMJ ar-
borization pattern as well as mislocalization of FasII
(Koh et al., 1999). The striking similarity in NMJ mu-
tant phenotypes is also seen at the ultrastructural
level. EM analysis reveals that the length of the SSR,
which is dependent on DLG levels (Budnik et al., 1996),
is significantly decreased compared to wild type con-
trols. Further, the number of active zones and size of
boutons is increased, with values similar to those seen
 REGULATION OF SYNAPTIC COMPONENTS
Fig. 2. Dynamic regulation of DLG at synapses by CaMKII depen-
dent phosphorylation. Increased Ca2⫹ concentration at the postsyn-
aptic cell activates CaMKII. Activated CaMKII then phosphorylates
serine 48 of PDZ1 of DLG. Phosphorylated DLG dissociates from the
synaptic membrane. DLG-binding synaptic components, such as
FasII and Shaker, could subsequently be free to move away from
synaptic complexes. Reproduced from Koh et al. (1999) with permis-
sion of the publisher.
in dlg mutant alleles. Interestingly, when DCaMKII
activity is reduced by expression of ala, an opposite
phenotype is observed—the length of the SSR increases
compared to wild type control. Consistent with these
EM results, DLG immunoreactivity at NMJs also de-
creases by constitutive DCaMKII activation and in-
creased by partial inhibition of DCaMKII activity (Koh
et al., 1999). These observations suggest that CaMKII
and DLG are involved in similar aspects of synapse
development, and that CaMKII may regulate DLG
function.
A possible mechanism for this regulation is that
CaMKII may affect the degree of association of DLG
with the synaptic membrane/cytoskeleton (Fig. 2). The
first PDZ domain of all MAGUKs contains a conserved
CaMKII phosphorylation consensus site that appears
to be a target for CaMKII. In vitro, this site (serine 48)
is phosphorylated by CaMKII, and this phosphoryla-
tion can be prevented by substituting serine 48 to ala-
nine or aspartic acid (Koh et al., 1999). A demonstra-
tion that phosphorylation of serine 48 is required in
vivo for regulation of DLG localization at the NMJ was
obtained by using three different enhanced green fluo-
rescence protein (eGFP)-tagged DLG constructs, with
or without point mutations of serine 48. These trans-
genic DLG forms were expressed in both the pre- and
postsynaptic cells in dlg mutant larvae (Koh et al.,
1999). In one mutant construct (eGFP-DLGS48D)
serine 48 was substituted by aspartic acid, mimicking
CaMKII phosphorylation; in the other (eGFP-
DLGS48A) serine 48 was substituted by alanine, pre-
venting CaMKII-dependent phosphorylation. When
wild type eGFP-DLG was expressed, eGFP signal was
concentrated at the NMJ, and a low level of fluores-
cence was observed at extrasynaptic regions. In con-
trast, when eGFP-DLGS48A was expressed, GFP fluo-
rescence was restricted to the NMJ. However, expres-
sion of eGFP-DLGS48D resulted in GFP signal
throughout muscle cytoplasm and membrane, and rel-
19
atively low levels of signal at the NMJ (Koh et al.,
1999). Thus, CaMKII appears to regulate DLG function
by affecting its synaptic localization through phosphor-
ylation (Koh et al., 1999). As phosphorylated DLG is
dissociated from the synaptic terminals, FasII and
other DLG binding partners could then be free to move
away from the NMJ. Intriguingly, X-ray crystallogra-
phy of the third PDZ domain of DLG shows that a 7
amino acid stretch contains both the sequence RGNS,
the CaMKII phosphorylation consensus site, and the
sequence GLG, a motif conserved in all MAGUK PDZ
domains, which is the site to which the C-terminal
S/TXV COOH motifs of MAGUK binding partners bind
(Doyle et al., 1996). Thus, phosphorylation of serine 48
by DCaMKII may alter binding affinities between the
first PDZ domain and the C-terminal S/TXV motifs of
FasII or Shaker-type K⫹ channels.
During larval development, the muscle fibers of
growing fly larvae increase exponentially in size; syn-
aptic arbors continuously expand to keep pace (Gorc-
zyca et al., 1993; Guan et al., 1996; Keshishian et al.,
1993). Structural plasticity is affected by neuronal ac-
tivity levels and by cell adhesion (Budnik et al., 1990;
Schuster et al., 1996a,b). Hyperexcitable mutants that
have increased electrical activity levels have larger and
more elaborately branched NMJs with more boutons
than do wild type larvae (Budnik et al, 1990). A similar
phenotype has been shown to be mediated by the level
of FasII present at the synapse (Schuster et al., 1996b;
Thomas et al., 1997; Zito et al., 1997). FasII mutants
that have a very low level of FasII expression exhibit
poorly elaborated NMJs with few boutons, while mu-
tants with a moderate reduction of FasII expression
show an expansion of the axonal arbor (Schuster et al.,
1996a). These observations suggest a model in which
synapses are stabilized by the presence of high levels of
cell adhesion molecules, but can expand when this
restriction is attenuated; partial disassembly of exist-
ing stable synapses permits the assembly of new syn-
aptic connections.
The regulation of DLG function by CaMKII provides
a potential mechanism by which synaptic activity af-
fects synaptic plasticity. The dissociation of CaMKII-
phosphorylated DLG from the synaptic complex allows
FasII to diffuse away from the synapse, thus permit-
ting the activity-dependent expansion of the NMJ (Koh
et al., 1999).
There are several alternative ways by which activity
may regulate NMJ development. For example overex-
pression of frequenin, a member of the Neuronal Cal-
cium Sensor family, also alters NMJ terminal morphol-
ogy (Angaut-Petit et al., 1998). Whether this effect is
mediated through the pathways described above is cur-
rently not known.
CaMKII appears to be expressed at both pre-and
postsynaptic sites, in vertebrates (reviewed in Green-
gard et al., 1993; Turner et al., 1999) as well as in flies
(Koh et al., 1999), where it interacts with a number of
molecules. In flies, one such substrate is Slowpoke
binding protein (Slob). Drosophila Slowpoke (dSlo) is a
Ca2⫹-dependent potassium channel with a consider-
ably extended carboxy terminal domain (Adelman et
al., 1992; Atkinson et al., 1991). Slob was identified in
a yeast two-hybrid screen using this long carboxy ter-
minal as bait (Schopperle et al., 1998). Using Slob as
20 Y.H. KOH ET AL.
bait, another screen identified the ␨ isoform of 14-3-3, a
protein that regulates presynaptic functions of the
NMJ (Broadie et al., 1997), as a Slob binding protein
(Zhou et al., 1999). Binding of the two proteins is me-
diated by a pair of serine-containing motifs in Slob, and
abolished by mutation of the second serine in each of
these motifs. Further, CaMKII-mediated phosphoryla-
tion of Slob, which dynamically regulates the interac-
tion of Slob and 14-3-3, is also prevented by mutation of
the two Slob serines (Zhou et al., 1999).
Immunocytochemistry shows that 14-3-3, dSlo, and
Slob colocalize at presynaptic terminals. dSlo channel
activity is changed when wild type Slob and 14-3-3 are
co-expressed; however, this effect is absent when 14-
3-3 is co-expressed with the Slob serine mutant. These
results indicate that presynaptic dSlo/Slob/14-3-3 exist
in a regulatory protein complex, mediated by CaMKII-
dependent phosphorylation of Slob (Zhou et al., 1999).
I HEARD IT THROUGH THE AXON: SIGNALS
FROM THE SYNAPSE TO NUCLEUS AND
VICE VERSA
Local Protein Synthesis at the
Postsynaptic Sites
In contrast to short-term synaptic plasticity, long-
term synaptic plasticity requires structural and func-
tional changes dependent upon new protein synthesis
(Abel at al., 1998). Several hypotheses, which are not
mutually exclusive, have been suggested to explain
how long-term synaptic plasticity might be accom-
plished.
The synaptic tagging hypothesis is based on the ob-
servation that inhibition of protein synthesis at den-
dritic terminals abolishes long-term potentiation (Frey
and Morris, 1997) and facilitation (Martin et al.,
1997a). In this model, newly synthesized proteins from
the neuronal soma are transported down the axon and
then captured into specific synaptic terminals marked
by synaptic tags (reviewed in Frey and Morris, 1998).
The nature of these tags has not yet been defined;
candidate mechanisms include a change in the geome-
try of the tagged synapse (Rusakov et al., 1995) or the
phosphorylation of an early LTP-associated kinase (Os-
ten et all, 1996). An alternative hypothesis is that
synthesis of the necessary proteins occurs locally at the
post-synaptic terminal. This hypothesis is supported
by several lines of evidence, including synaptic local-
ization of several mRNAs and tRNAs (Tiedge and Bro-
sius, 1996), the existence of polyribosome-like struc-
tures and membrane organelles containing Golgi
markers within dendritic terminals (reviewed by
Huang, 1999; Steward, 1997; Tiedge et al., 1999), and
the rapid increase of CaMKII protein immunoreactiv-
ity at the dendritic terminals within 30 minutes follow-
ing repeated tetanic stimulation (Ouyang et al., 1998).
An impressive array of mRNAs has been found to
have dendritic localization. These include the high mo-
lecular weight forms of microtubule-associated protein
2 (MAP2a/b) (Garner et al., 1988), the ␣-CaMKII sub-
unit (Burgin et al., 1990), the inositol 1,4,5-trisphos-
phate receptor type 1 (Furuichi et al., 1993), neurogra-
nin (Landry et al., 1994), the cytoskeleton-associated
protein Arc (Link et al., 1995; Lyford et al., 1995), the
noncoding short RNA of polymerase III transcript BC1
(Muslimov et al., 1997), the transcription factor CREB
(Crino et al., 1998), the NMDAR1 subunit (Gazzaley et
al., 1997), and the neurotrophic factors TrkB and
BDNF (Tongiorgi et al., 1997). The localization of
mRNA at dendritic terminals raises two questions:
How are specific mRNAs translocated to the dendritic
terminals; and what are the mechanisms of local pro-
tein translation at dendritic terminals?
Insight into the first question can be found in studies
of RNA binding proteins, which have been intensely
investigated for their roles in embryonic development
(reviewed by Bashirullah et al., 1998). Specialized lo-
calization of mRNAs is required for the proper tempo-
rospatial development of embryos. Restricted spatial
localization of mRNA is achieved by cis-acting RNA
elements present in the transcript itself, usually lo-
cated in the 3’-untranslated region (UTR) of the tran-
script but sometimes within the 5’-UTR or in the coding
regions, and a variety of trans-acting factors. Many of
these trans-acting factors are RNA-binding proteins
that interact with the transcript (reviewed by Ba-
shirullah et al., 1998). In some cases, the RNA binding
proteins may regulate mRNA translation and localiza-
tion at the dendritic terminal by forming mRNA-pro-
tein complexes. For example, BC1 RNA has a 62 nu-
cleotide dendritic targeting element within its 5’-UTR
(Muslimov et al., 1997), which has a Testis-Brain RNA
binding protein (TB-RNB)-binding Y-element (Wu et
al., 1997).
Other transcripts, such as Arc mRNA, exhibit activ-
ity-dependent localization to activated dendritic termi-
nals in the presence of inhibitors of protein synthesis,
suggesting that the signal for dendritic localization
exists in the transcript itself. Therefore, newly synthe-
sized RNA binding proteins are unnecessary for appro-
priate mRNA targeting (Steward et al., 1998). Thus,
there appear to exist multiple mRNA translocation
mechanisms.
Once the mRNA has reached the synapse, it needs to
be translated into proteins required to mediate long-
term changes in synaptic plasticity. This brings us to
the second question: what are the mechanisms of local
protein translation at dendritic terminals? The regula-
tory mechanisms involved in synaptic mRNA transla-
tion are as yet poorly understood, although regulation
of the translation of ␣-CaMKII mRNA in dendritic
terminals of the mammalian brain has begun to be
characterized (Wu et al., 1998). One of the mechanisms
known to be involved is polyadenylation binding pro-
tein (CPEB)-dependent polyadenylation of the 3’-UTR
of ␣-CaMKII mRNA at dendritic terminals. CPEB-de-
pendent regulation of mRNA translation requires the
presence of a pair of CPE binding sequences upstream
of polyadenylation sequences within 3’-UTR (Wu et al.,
1998).
Local regulation of mRNA translation at the Droso-
phil a larval NMJ is a new area of inquiry, and has not
yet been intensively investigated. However, many syn-
aptic proteins show polarized localization at postsyn-
aptic elements, suggesting that such regulation may
well exist. Among these are DLG, which is highly en-
riched at type I synaptic boutons (Lahey et al., 1994);
Shaker-type K⫹ channels, partially colocalized with
DLG at postsynaptic sites (Tejedor et al., 1997); FasII,
concentrated on synaptic borders (Thomas et al., 1997);
 REGULATION OF SYNAPTIC COMPONENTS
Fig. 3. Polyribosome-like structures are observed within the SSR.
Cross-section of a type Ib bouton showing the presynaptic terminal (b)
with an active zone (AZ), and the postsynaptic SSR. Inset: High
magnification view of the boxed region. Arrowheads indicate polyri-
bosome-like structures resembling a short string of beads (Koh, un-
published data).
and Dorsal and Cactus, enriched postsynaptically
(Cantera et al., 1999).
However, it is distinctly possible that only a small
number of synaptic proteins, if any, are translated
within the SSR. Studies into the nature of localization
signals for synaptic components such as PSD-95 (Cra-
ven et al., 1999) and metabotropic glutamate receptors
(Stowell and Craig, 1999) in primary neuronal cultures
indicate that certain protein domains or amino acid
motifs are necessary for the targeting of a protein to
postsynaptic sites.
The only evidence in favor of localized postsynaptic
protein translation at the fly NMJ is the existence of
polyribosome-like structures within the SSR, as re-
vealed by EM examination of the larval NMJ (Fig. 3;
Koh and Budnik, unpublished data). Although localiza-
tion of mRNA of specific synaptic proteins at postsyn-
aptic sites has not yet been investigated, these data
indicate that some synaptic components may be trans-
lated within the SSR.
Regulation of Gene Expression
From Synaptic Terminals
The establishment of long-term memory is predi-
cated upon the expression of specific genes, and thus
requires the transmission of signals from the synapse
to the nucleus. Although it is not yet clear which sig-
nals are involved in this process, several candidates
have recently emerged, including PKA (reviewed in
Abel et al., 1998), MAPK (reviewed in Impey et al.,
1999), CREB (Crino et al., 1998), and Nuclear Factor
kappa-B (NF␬B) (reviewed by O’Neil and Kaltschmidt,
1997).
Stimulation of NMDA receptors is known to result in
activation of PKA and MAPK in the mammalian hip-
pocampus (reviewed in Abel at al., 1998), and the acti-
vated forms of these proteins have been shown to
21
translocate into the nucleus (Martin et al., 1997b). The
expression of immediate early genes, needed for new
protein synthesis after a neuron has been repeatedly
stimulated, has been shown to be regulated by CREB
transcription factors (Bailey et al., 1996; reviewed by
Abel et al., 1998). The injection of antibodies to MAPK
or inhibitors of MAPK kinase into the soma of cultured
Aplysia sensory cells prevents long-term facilitation,
without changing either basal transmission or short-
term facilitation (Martin et al., 1997b). A regulatory
link between MAPK and CREB could explain this re-
sult; CREB2 has a MAPK phosphorylation consensus
site, making it a potential substrate for activated
MAPK that has been translocated into the nucleus
(Martin et al., 1997b). CREB2 dimerizes with CREB1,
blocking CREB1-mediated transcription. MAPK phos-
phorylation of CREB2 may induce the dissociation of
CREB2 from CREB1, permitting CREB1 to form ho-
modimers, which could then be phosphorylated by the
activated PKA catalytic subunit and finally bind to the
cAMP response element (CRE) sites of immediate early
genes. One of the immediate early gene products, the
C/EBP transcription factor, may be phosphorylated by
a Rsk protein kinase and then begin late gene transla-
tion (Martin et al., 1997b; reviewed in Impey et al.,
1999).
Long-term changes in synaptic plasticity can be
directly observed in Aplysia dissociated cell culture.
Activated MAPK is found in the dendritic terminals
of Aplysia neurons (Bailey et al., 1997). One of its
known substrates is the Aplysia cell adhesion mole-
cule (ApCAM), a structural homologue of mamma-
lian NCAM and Drosophila FasII (Mayford et al.,
1992), which has a MAPK consensus site within the
C-terminal PEST sequence. ApCAM at the neuronal
cell surface is internalized when this site is phos-
phorylated (Bailey et al., 1997). Binding between the
pre- and postsynptic elements is weakened by the
downregulation of ApCAM from sensory neuronal
surfaces, allowing the expansion of synapses or ar-
borizations after long-term facilitation. Consistent
observations have been seen in the Drosophila larval
NMJ. Mutations in dunce, the gene for phosphodies-
terase II, and/or the potassium channel genes ether á
gógo or Shaker, induce hyperexcitability (Budnik et
al., 1990; Ganetzky and Wu, 1983), accompanied by
decreased levels of FasII (Schuster et al., 1996b) and
synapse overgrowth (Budnik et al., 1990; Schuster et
al., 1996b; Zhong et al., 1992). However, it has not
yet been established that MAPK plays a role in syn-
aptic plasticity at the Drosophila larval NMJ.
In primary cultures of mammalian hippocampal neu-
rons, CREB is able to carry signals directly from the
synapse to the nucleus. CREB mRNA exists within
dendritic terminals, where it is translated into protein,
and can be phosphorylated (Crino et al., 1998). Labeled
CREB microinjected into dendritic terminals is rapidly
transported into the nucleus (Crino et al., 1998). Thus,
CREB protein synthesized in dendrites could poten-
tially directly affect gene expression, serving as a mes-
senger from dendrite to nucleus.
In flies, Dorsal, a member of the Rel family of tran-
scription factors, has been detected at the larval NMJ
(Cantera et al., 1999). Rel proteins are involved in
many different cellular, molecular and physiological
22 Y.H. KOH ET AL.
processes such as immune response (reviewed by Hult-
mark, 1994), inflammation, oncogenesis, apoptosis (re-
viewed by Baeuerle and Henkel, 1994), and embryonic
development (reviewed by Belvin and Anderson, 1996).
Dorsal, along with Relish and Dif, is one of three
known Drosophila Rel proteins. All three are homo-
logues of the mammalian transcription factor nuclear
factor kappa B (NF␬B) (Dushay et al., 1996; Ip et al.,
1993; Steward, 1987). Dorsal function is inhibited by
Cactus, the Drosophila homologue of Inhibitor kappa B
(I␬B) (Geisler et al., 1992; Kidd, 1992). It has been
proposed that NF␬B and I␬B play important roles in
the mammalian nervous system, and may be involved
in NMDA receptor-mediated synaptic plasticity (Guer-
rini et al., 1995; reviewed by O’Neil and Kaltschmidt,
1997).
Intriguingly, both Dorsal and Cactus proteins are
strongly enriched in the SSR of type I boutons, with a
much lower level of distribution in the cytoplasm and
nuclei of muscle fibers (Cantera et al., 1999). The ab-
sence of Dorsal protein leads to abnormal synaptic
morphology and nuclear localization of Cactus, as well
as malformations of the body wall muscles and nuclear
hypotrophy (Cantera et al., 1999). These results sug-
gest that Rel family proteins and their I␬B inhibitors
may act as a synapse-to-nucleus signal mediating syn-
aptic plasticity.
SOMEWHERE OVER THE SYNAPSE:
CONCLUDING REMARKS AND FUTURE
DIRECTIONS
Studies using the Drosophila neuromuscular junc-
tion have shown it to be a fruitful model system for
dissecting the workings of synapses. The existence of
evolutionarily conserved synaptic components and sig-
nal transduction pathways in an easily genetically ma-
nipulated organism make this system an invaluable
tool for investigating the cellular and molecular mech-
anisms underlying synaptic plasticity and learning and
memory in higher organisms. Further, this system can
be used to discern the role of mammalian synaptic
proteins important in pathological conditions, but
whose normal function is as yet obscure. For example,
amyloid precursor protein (APP), which is cleaved to
yield Alzheimer’s disease-associated ␤-amyloid, has a
Drosophila homologue, amyloid precursor protein-like
protein (APPL) (Luo et al., 1990). Immunocytochemical
analysis of NMJs of APPL deficient larvae, together
with immunocytochemical and ultrastructural analysis
of NMJs of larvae in which full-length or truncated
Appl constructs were neurally expressed, indicate that
APPL may influence synapse formation (Torroja et al.,
1999).
Here we have reviewed the analysis of an important
synaptic organizer, DLG, a molecule first identified at
vertebrate synapses but whose functional significance
in the intact organism was uncovered by the genetic
approaches possible in the fly. Genetic analysis has
also allowed us understand the role played by CaMKII
in one of the mechanisms by which the localization of
DLG is regulated. Whether this mechanism is also
employed for vertebrate MAGUKs is a question await-
ing future studies.
A particularly intriguing and long-standing question
has been the role of the SSR, and what, if any, function
it has in synaptic plasticity. The finding of polyribo-
some-like structures within the SSR indicates that one
function of the SSR may be post-transcriptional regu-
lation of synaptic proteins. Which mRNAs are localized
within the SSR, as well as what complement of post-
transcriptional machineries can be found there to
translate and process those messages, is an area of
inquiry that has scarcely begun.
The interplay of molecular and genetic approaches
available in the fruit fly may provide the key to resolv-
ing this question, and other aspects of regulation of the
biochemical and molecular components involved in
synaptic plasticity.
ACKNOWLEDGMENTS
We thank Ulrich Thomas and Michael Gorczyca for
permission to cite their unpublished results. We thank
Michael Gorczyca, Ulrich Thomas, Laurie Quysner,
Mary Packard, and David Epstein for their invaluable
comments on this manuscript. L. Sian Gramates is
supported by an NIH predoctoral fellowship (F31
NS10861-01).
REFERENCES
Abel T, Martin KC, Bartsch D, Kandel ER. 1998. Memory suppressor
genes: inhibitory constraints on the storage of long term memory.
Science 279:338 –341.
Adelman JP, Shen KZ, Kavanaugh MP, Warren RA, Wu YN Lagrutta
A Bond, CT, North RA. 1992. Calcium-activated potassium chan-
nels expressed from cloned complementary DNA. Neuron 9:209 –
216.
Anderson MS, Halpern ME, Keshishian H. 1988. Identification of the
neuropeptide transmitter proctolin in Drosophila larval character-
ization of fiber-specific neuromuscular endings. J Neurosci 8:242–
255.
Angaut-Petit D, Toth P, Rogero O, Faille L, Tejedor FJ, Ferŕus A.
1998. Enhanced neurotransmitter release is associated with reduc-
tion of neuronal branching in a Drosophila mutant overexpresssing
frequenin. Eur J Neurosci 10:423– 434.
Atkinson NS, Robertson GA, Ganetzky B. 1991. A component of cal-
cium-activated potassium channels encoded by the Drosophila slo
locus. Science 253:551–555.
Atwood HL, Govind CK. and Wu C-F. 1993. Differential ultrastruc-
ture of synaptic terminals on ventral longitudinal muscles in larval
Drosophila. J Neurobiol 24:1009 –1044.
Atwood HL, Karunanithi S, Georgiou J, Charlton MP. 1997. Strength
of synaptic transmission at neuromuscular junctions of crustaceans
and insects in relation to calcium entry. Invert Neurosci 3:81– 87.
Bading H, Greenberg ME. 1991. Stimulation of protein tyrosine phos-
phorylation by NMDA receptor activation. Science 253:912–914.
Baeuerle PA, Henkel T 1994. Function and activation of NF-B in the
immune system. Annu Rev Immunol 12:141–179.
Bailey CH, Kandel ER. 1993. Structural changes accompanying mem-
ory storage. Ann Rev Physiol 55:397– 426.
Bailey CH, Bartsch D, Kandel ER. 1996. Toward a molecular defini-
tion of long-term memory storage. Proc Natl Acad Sci 93:13445–
13452.
Bailey CH, Kaang BK, Chen M, Martin KC, Lim CS, Casadio A,
Kandel ER. 1997. Mutation in the phosphorylation sites of MAP
Kinase blocks learning related internalization of apCAM in Aplysia
sensory neurons. Neuron 18:913–924.
Bashirullah A, Cooperstock RL, Lipshitz HD. 1998. RNA localization
in development. Annu Rev Biochem 67:335–394.
Belvin MP, Anderson KV. 1996. A conserved signaling pathway: the
Drosophila Toll-Dorsal pathway. Annu Rev Cell Dev Biol 12:393–
416.
Bredt DS, Snyder SH. 1992. Nitric oxide, a novel neuronal messenger.
Neuron, 8:3–11.
Brenman JE, Bredt DS. 1996. Synaptic signaling by nitric oxide. Curr
Opin Neurobiol 7:374 –378.
Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano
DR, Wu Z, Huang F, Xia H, Peters MF, Froehner SC, Bredt DS.
1996. Interaction of nitric oxide synthase with the postsynaptic
density protein PSD–95 and 1-syntrophin mediated by PDZ do-
mains. Cell 84:757–767.
 REGULATION OF SYNAPTIC COMPONENTS
Broadie K, Rushton E, Skoulakis EM.C, Davis RL. 1997. Leonardo, a
Drosophila 14 –3–3 protein involved in learning, regulates presyn-
aptic function. Neuron 19:391– 402.
Budnik V. 1996. Synapse maturation and structural plasticity at
Drosophila neuromuscular junctions. Curr Opin Neurobiol 6:858 –
867.
Budnik V, Zhong Y, Wu C -F. 1990. Morphological plasticity of motor
axons in Drosophila mutants with altered excitability. J Neurosci
10:3754 –3768.
Budnik V, Koh YH, Guan B, Hartmann B, Hough C, Woods D, Gorc-
zyca M. 1996. Regulation of synapse structure and function by the
Drosophila tumor suppressor gene dlg. Neuron 17:627– 640.
Burgin KE, Waxham MN, Rickling S, Westgate SA, Mobley WC, Kelly
PT. 1990. In situ hybridization histochemistry of Ca2⫹/calmodulin-
dependent protein kinase in developing rat brain. J Neurosci 10:
1788 –1798.
Cantera R, Nassel DR. 1992. Segmental peptidergic innervation of
abdominal targets in larval and adult dipteran insects revealed
with an antiserum against leucokinin I. Cell Tissue Res 269:459 –
471.
Cantera R, Kozolva T, Barillas-Mury C, Kafatos FC. 1999. Muscle
structure and innervation are affected by loss of dorsal in the fruit
fly Drosophila melanogaster. Mol Cell Neurosci 13:131–141.
Chen HJ, Rojas-Soto M, Oguni A, Kennedy MB. 1998. A synaptic
Ras-GTPase activating protein (p135 SynGAP) inhibited by CaM
kinase II. Neuron 20:895–904.
Cho K-O Wall JB, Pugh PC, Ito M, Muller SA, Kennedy MB. 1991. The
␣-subunit of type II Ca2⫹/calmodulin dependent protein kinase is
highly conserved in Drosophila. Neuron 7:439 – 450.
Cho K-O, Hunt CA, Kennedy MB. 1992. The rat brain postsynaptic
density fraction contains a homolog of the Drosophila discs-large
tumor suppressor protein. Neuron 9:929 –942.
Cobb MH, Goldsmith EJ. 1995. How MAPKinases are regulated.
J Biol Chem 270:14843–14846.
Coffino P, Bourne HR, Friedrich U, Hochman J, Insel PA, Lemaire I,
Melmon, K.L, Tomkins GM. 1976. Molecular mechanisms of cyclic
AMP action: a genetic approach. Recent Prog Horm Res 32:669 –
684.
Craven SE, El-Husseini AE, Bredt DS. 1999. Synaptic targeting of the
postsynaptic density protein PSD–95 mediated by lipid and protein
motifs. Neuron 22:497–509.
Crino P, Khodakhah K, Becker K, Ginsberg S, Hemby S, Eberwine J.
1998. Presence and phosphorylation of transcription factors in de-
veloping dendrites. Proc Natl Acad Sci 95:2313–2318.
Davis GW, Goodman CS. 1998. Synapse-specific control of synaptic
efficacy at the terminals of a single neuron. Nature 392:82– 86.
Davis GW, DiAntonio A, Petersen SA, Goodman CS. 1998. Postsyn-
aptic PKA controls quantal size and reveals a retrograde signal that
regulates presynaptic transmitter release in Drosophila. Neuron
20:305–315.
Davis RL, Cherry J, Dauwalder B, Han PL, Skoulakis E 1995. The
cyclic AMP system and Drosophila learning. Mol Cell Biochem
149/150:271–278.
Dimitratos SD, Woods DF, Bryant PJ. 1997. Camguk Lin–2, and
CASK: novel membrane-associated guanylate kinase homologs that
also contain CaM kinase domains. Mech Dev 63:127–30.
Downward J, Riehl R, Wu L, Weinberg RA. 1990. Identification of a
nucleotide exchange-promoting activity for p21ras. Proc Natl Acad
Sci 87:5998 – 6002.
Doyle DA, Lee A, Lewis J, Kim E, Sheng M, MacKinnon R. 1996.
Crystal structures of a complexed and peptide-free membrane pro-
tein-binding domain: molecular basis of peptide recognition by PDZ.
Cell 85:1067–1076.
Dubnau J, Tully T. 1998. Gene discovery in Drosophila: new insight
for learning and memory. Ann Rev Neurosci 21:407– 444.
Dushay MS, Asling B, Hultmark D. 1996. Origins of immunity: Rel-
ish, a compound rel-like gene in the antibacterial defense of Dro-
sophila. Proc Natl Acad Sci 93:10343–10347.
English JD, Sweatt JD. 1996. Activation of p42 mitogen-activated
protein kinase in hippocampal long term potentiation. J Biol Chem
271:24329 –24332.
English JD, Sweatt JD. 1997. A requirement for the mitogen-acti-
vated protein kinase cascade in hippocampal long term potentia-
tion. J Biol Chem 272:19103–19106.
Fields RD, Itoh K. 1996. Neural cell adhesion molecules in activity-
dependent development and synaptic plasticity. Trends Neurosci
19:473– 480
Frey U, Morris RGM. 1997. Synaptic tagging and long-term potenti-
ation. Nature 385:533–536.
23
Frey U, Morris RGM. 1998. Synaptic tagging: implications for late
maintenance of hippocampal long term potentiation. Trends Neu-
rosci 21:181–188.
Furuichi T, Samon-Chazottes D, Fujino I, Yamada N, Hasegawa M,
Miyaki A, Yoshikawa S, Guenet J-L, Mikoshiba K. 1993. Wide
spread expression of inositol 1,4,5-triphosphate receptor type 1 gene
(Insp3r1. in the mouse central nervous system. Receptors Chan-
nels, 1:11–24.
Furuyashiki T, Fujisawa K, Fujita A, Madaule P, Uchino S, Mishina
M, Bito H, Narumiya S 1999. Citron, a Rho-target, interacts with
PSD–95/SAP–90 at glutamatergic synapses in the thalamus. J Neu-
rosci 19:109 –118.
Ganetzky B, Wu C-F. 1983. Neurogenetic analysis of potassium cur-
rents in Drosophila: synergistic effects on neuromuscular transmis-
sion in double mutants. J Neurogenet 1:17–28.
Garcia EP, Mehta S, Blair LA.C, Wells DG, Shang J, Fukushima, T,
Fallon JR, Garner C, Marshall J. 1998. SAP90 binds and clusters
kainate receptors causing incomplete desensitization. Neuron 21:
727–739.
Garner C, Kindler S. 1996. Synaptic proteins and the assembly of the
postsynaptic apparatus. Trends Cell Biol 6:429 – 433.
Garner CC, Tucker RP, Matus A. 1988. Selective localization of mes-
senger RNA for the cytoskeletal protein MAP2 in dendrites. Nature
336:674 – 677.
Gazzaley, A.H, Benson DL, Huntley GW, Morrison JH. 1997. Differ-
ential subcellular regulation of NMDAR1 protein and mRNA in
dendrites of dentate gyrus granule cells after perforant path tran-
section. J Neurosci 17:2006 –2017.
Geisler R, Bergmann A, Hiromi Y, Nusslein-Volhard C. 1992. cactus,
a gene involved in dorsoventral pattern formation of Drosophila, is
related to the I kappa B gene family of vertebrates. Cell 71:613–
621.
Gorczyca M, Augart C, Budnik V. 1993. Insulin-like receptor and
insulin-like peptide are localized at neuromuscular junctions in
Drosophila. J. Neurosci, 3692–3704.
Gramates LS, Budnik V. 1999a. Assembly and maturation of the
Drosophila larval neuromuscular junction. In: Budnik V, Gramates
LS, editors. Neuromuscular junctions in Drosophila. San Diego:
Academic Press, Int Rev Neurobiol 43:93–117.
Gramates LS, Budnik V. 1999b. Genetics Society of America Annual
Drosophila Research Conference 40:a190 (Abstract).
Greengard P, Valtora F, Czernik AJ, Benfenati F. 1993. Synaptic
vesicle phosphoproteins and regulation of synaptic function. Sci-
ence 259:780 –785.
Griffith LC, Greenspan RJ. 1993. The diversity of calcium/calmodulin-
dependent protein kinase II isoforms in Drosophila is generated by
alternative splicing of a single gene. J Neurochem 61:1534 –1537.
Griffith LC, Verselis LM, Aitken KM, Kyriacou CP, Danho Wand
Greenspan RJ. 1994. Inhibition of calcium/calmodulin-dependent
protein kinase in Drosophila disrupts behavioral plasticity. Neuron
10:501–509.
Guan B, Hartmann B, Koh YH, Gorczyca M, Budnik V. 1996. The
Drosophila tumor suppressor gene, dlg, is involved in structural
plasticity at a glutamatergic synapse. Curr Biol 6:695–706.
Guerrini L, Blasi F, Denis-Donini S. 1995. Synaptic activation of
NF-kappa B by glutamate in cerebellar granule neurons in vitro.
Proc Natl Acad Sci 92:9077–9081.
Hannan F, Zhong Y. 1999. Second messenger systems underlying
plasticity at the neuromuscular junction. In: Budnik V, Gramates
LS, editors. Neuromuscular junction in Drosophila. San Diego: Ac-
ademic Press, Int Rev Neurobiol 43:119 –138.
Harish OE, Poo MM. 1992. Retrograde modulation at developing
neuromuscular synapses: involvement of G protein and arachidonic
acid cascade. Neuron 9:1201–1209.
Hata Y, Butz S, Sudhof TC. 1996. CASK: a novel dlg/PSD95 homolog
with an N-terminal calmodulin-dependent protein kinase domain
identified by interaction with neurexins. J Neurosci 16:2488 –2494.
Hawkins RD, Zhuo M, Arancio O. 1994. Nitric oxide and carbon
monoxide as possible retrograde messengers in hippocampal long-
term potentiation. Neurobiology 25:652– 65.
Hawkins RD, Son H, Arancio O. 1998. Nitric oxide as a retrograde
messenger during long-term potentiation in hippocampus. Prog
Brain Res 118:155–172.
Hoskins R, Hajnal AF, Harp SA, Kim SK. 1996. The C. elegans vulval
induction gene lin–2 encodes a member of the MAGUK family of cell
junction proteins. Development, 122:97–111.
Huang EP. 1999. Synaptic plasticity: regulated translation in den-
drites. Curr Biol 9: R168-R170.
Hultmark D. 1994. Ancient relationship. Nature 367:116 –117.
24 Y.H. KOH ET AL.
Impey S, Obrietan K, Storm DR. 1999. Making new connections: Role
of ERK/MAP Kinase signaling in neuronal plasticity. Neuron 23:
11–14.
Ip YT, Reach M, Engstrom Y, Kadalayil L, Cai H, Gonzales-Crespo S,
Tatei K, Levine M. 1993. Dif, a dorsal related gene that mediates an
immune response in Drosophila. Cell 75:753–763.
Irie M, Hata Y, Takeuchi M, Ichtchenko K, Toyoda A, Hirao K, Takai
Y, Rosahl TW, Sudhof TC. 1997. Binding of neuroligins to PSD–95.
Science 277:1511–1515.
Jaffrey SR, Snowman AM, Eliasson MJ, Cohen NA, Snyder SH. 1998.
CAPON: a protein associated with neuronal nitric oxide synthase
that regulates its interactions with PSD95. Neuron 20:115–124.
Jia X, Gorczyca M, Budnik V. 1993. Ultrastructure of neuromuscular
junctions in Drosophila: comparison of wild-type and mutants with
increased excitability. J Neurobiol 24:1025–1044.
Kennedy M. 1997. The postsynaptic density at glutamatergic syn-
apses. Trends Neurosci 20:264 –268.
Keshishian H, Chiba A, Chang TN, Halfon MS, Harkins EW, Jarecki
J, Wang L, erson M, Cash S, Halpern ME, Johansen J. 1993.
Cellular mechanisms governing synaptic development in Drosoph-
ila melanogaster. J Neurobiol 24:757–787.
Kidd S. 1992. Characterization of the Drosophila cactus locus and
analysis of interactions between cactus and dorsal proteins. Cell
71:623– 635.
Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M. 1995. Clus-
tering of Shaker-type K⫹ channels by interaction with a family of
membrane-associated guanylate kinases. Nature 378:85– 88.
Kim E, Cho KO, Rothschild A, Sheng M. 1996. Heteromultimerization
and NMDA receptor-clustering activity of Chapsyn–110, a member
of the PSD–95 family of proteins. Neuron 17:103–113.
Kim E, Naisbitt S, Hsueh YP, Rao A, Rothschild A, Craig AM, Sheng
M. 1997. GKAP, a novel synaptic protein that interacts with the
guanylate kinase-like domain of the PSD–95/SAP90 family of chan-
nel clustering molecules. J Cell Biol 136:669 – 678.
Kim JH, Liao D, Lau L-F, Huganir R 1998. SynGAP: a synaptic
RasGAP that associates with the PSD–95/SAP90 protein family.
Neuron 20:683– 691.
Kistner U, Wenzel BM, Veh RW, Cases-Langhoff C, Garner AM,
Appeltauer U, Voss B, Gundelfinger ED, Garner CC. 1993. SAP90,
a rat presynaptic protein related to the product of the Drosophila
tumor suppressor gene dlg-A. J Biol Chem 268:4580 – 4583.
Koh YH, Popova G, Thomas U, Griffith LC, Budnik V. 1999. Regula-
tion of DLG localization at synapses by CaMKII-dependent phos-
phorylation. Cell 98:353–363.
Kornau HC, Shenker LT, Kennedy MB, Seeburg PH. 1995. Domain
interaction between NMDA receptor subunits and the postsynaptic
density protein PSD-95. Science 269:1737–1740.
Kryl D, Yacoubian T, Haapasalo A, Castren E, Lo D, Barker PA. 1999.
Subcellular localization of full-length and truncated Trk receptor
isoforms in polarized neurons and epithelial cells. J Neurosci 19:
5823–5833.
Kurdyak P, Atwood H L, Stewart BA, Wu C-F. 1994. Differential
physiology and morphology of motor axons to ventral longitudinal
muscles in larval Drosophila. J Comp Neurol 350:463– 472.
Lahey T, Gorczyca M, Jia X, Budnik V. 1994. The Drosophila tumor
suppressor gene dlg is required for normal synaptic bouton struc-
ture. Neuron 13:823– 835.
Landry CF, Watson JB, Kashima T, Campagnoni AT. 1994. Cellular
influences on RNA sorting in neurons and glia: an in situ hybrid-
ization histochemical study. Mol Brain Res 27:1–11.
Link W, Konietzko U, Kauselmann G, Krug M, Schwanke B, Frey U,
Kuhl D. 1995. Somatodendritic expression of an immediate early
gene is regulated by synaptic activity. Proc Natl Acad Sci 92:5734 –
5738
Lisman J. 1994. The CaM kinase II hypothesis for the storage of
synaptic memory. Trends Neurosci 17:406 – 412.
Lue RA, Brandin E, Chan EP, Branton D. 1996. Two independent
domains of hDlg are sufficient for subcellular targeting: the PDZ1–2
conformational unit and an alternatively spliced domain. J Cell Biol
135:1125–1137.
Luo L, Martin-Morri LE, White K. 1990. Identification, secretion, and
neural expression of APPL, a Drosophila protein similar to human
amyloid protein precursor. J Neurosci 10:3849 –3861.
Lyford GL, Yamagata K, Kaufmann WE, Barnes CA, Sanders LK,
Copeland NG, Gilbert DJ, Jenkins NA, Lanahan AA, Worley PF.
1995. Arc, a growth factor and activity-regulated gene, encodes a
novel cytoskeleton-associated protein that is enriched in neuronal
dendrites. Neuron 15:433– 445.
Madaule P, Furuyashiki T, Reid T, Ishizaki T, Watanabe G, Morii N,
Narumiya S. 1995. A novel partner for the GTP-bound forms of rho
and rac. FEBS Lett 377:243–248.
Madaule P, Eda M, Watanabe N, Fujisawa K, Matsuoka T, Bito H,
Ishizaki T, Narumiya S. 1998. Role of citron kinase as a target of the
small GTPase Rho in cytokinesis. Nature 394:491– 494.
Malenka RC, Kauer JA, Perkel DJ, Mauk MD, Kelly PT, Nicoll RA,
Waxham MN. 1989. An essential role for postsynaptic calmodulin
and protein kinase activity in long-term potentiation. Nature 340:
554 –557.
Martin KC, Casadio A, Zhu H, Yaping E, Rose JC, Kandel ER. 1997a.
Synapse specific, long term facilitation of Aplysia sensory to motor
synapses: A function for local protein synthesis in memory storage.
Cell 91:927–938.
Martin KC, Michael D, Rose JC, Barad M, Casadio A, Zhu H, Kandel
ER. 1997b. MAP Kinase translocates into the nucleus of the pre-
synaptic cell and is required for long term facilitation in Aplysia.
Neuron 18:899 –912.
Masuko N, Makino K, Kuwahara H, Fukunaga K, Sudo T, Araki N,
Yamamoto N, Yamada Y, Miyamoto E, Saya H. 1999. Tissue-spe-
cific membrane-associated guanylate kinase protein, with calmod-
ulin and PSD–95/SAP90 a possible regulatory role in molecular
clustering at synaptic sites. J Biol Chem 274:5872–5790.
Mayford M, Barzilai A, Keller F, Schacher S, Kandel ER. 1992. Mod-
ulation of an NCAM-related adhesion molecule with long-term syn-
aptic plasticity in Aplysia. Science 272:1020 –1023.
Mayford M, Bach ME, Kandel ER.1996. CaMKII function in the
nervous system explored from a genetic perspective. Cold Spring
Harbor Symp Quant Biol LXI:219 –224.
Meyer T, Hanson PI, Stryer L, Schulman H. 1992. Calmodulin trap-
ping by calcium-calmodulin-dependent protein kinase. Science 256:
1199 –1202.
Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM,
Makhinson M, He Y, Ramsay MF, Morris RG.M, Morrison JH,
O’Dell TJ, Grant SGN. 1999. Enhanced long-term potentiation and
impaired learning in mice with mutant postsynaptic density–95
protein. Nature 396:433– 439.
Milner B, Squire LR, Kandel ER. 1998. Cognitive neuroscience and
the study of memory. Neuron 20:445– 468.
Monastriani M, Gorczyca M, Rapus J, Eckert M, White K, Budnik V.
1995. Octopamine immunoreactivity in the fruit fly Drosophila
melanogaster. J Comp Neurol 356:275–287.
Muller BM, Kistner U, Veh RW, Cases-Langhoff C, Becker B, Gun-
delfinger ED, Garner CC. 1995. Molecular characterization and
spatial distribution of SAP97, a novel presynaptic protein homolo-
gous to SAP90 and the Drosophila discs-large tumor suppressor
protein. J Neurosci 15:2354 –2366.
Muller BM. Kistner U, Kindler S, Chung WJ, Kuhlendahl S, Fenster
SD, Lau LF, Veh RW Huganir RL, Gundelfinger ED, Garner CC.
1996. SAP102, a novel postsynaptic protein that interacts with
NMDA receptor complexes in vivo. Neuron 17:255–265.
Muslimov IA, Santi E, Homel P, Perini S, Higgins D, Tiedge H. 1997.
RNA transport in dendrites: a cis-acting targeting element is con-
tained within neuronal BC1 RNA. J Neurosci 17:4722– 4733.
Naisbitt S, Kim E, Weinberg RJ, Rao A, Yang FC, Craig AM, Sheng
M. 1997. Characterization of guanylate kinase-associated protein, a
postsynaptic density protein at excitatory synapses that interacts
directly with postsynaptic density–95/synapse-associated protein
90. J Neurosci 17:5687–5696.
Nguyen QT, Lichtman JW. 1996. Mechanism of synapse disassembly
at the developing neuromuscular junction. Curr Opin Neurobiol
6:104 –112.
Nguyen T, Sudhof TC. 1997. Binding properties of neuroligin 1 and
neurexin 1beta reveal function as heterophilic cell adhesion mole-
cules. J Biol Chem 272:26032–26039.
Niethammer M, Valtschanoff JG, Kapoor TM, Allison DW, Weinberg
TM, Craig AM, Sheng M. 1998. CRIPT, a novel postsynaptic protein
that binds to the third PDZ domain of PSD–95/SAP90. Neuron
20:693–707.
O’Dell TJ, Hawkins RD, Kandel ER, Arancio O. 1991. Tests of the
roles of two diffusible substances in long-term potentiation: evi-
dence for nitric oxide as a possible early retrograde messenger. Proc
Natl Acad Sci 88:11285–11289.
O’Neill LA, Kaltschmidt. C. 1997. NF-kappa B: a crucial transcription
factor for glial and neuronal cell function. Trends Neurosci 20:252–
258.
Osten P, Valsamis L, Harris A, Sacktor TC. 1996. Protein synthesis-
dependent formation of protein kinase Mzeta in long-term potenti-
ation. J Neurosci 16:2444 –2451.
Ouyang Y, Kantor D, Harris KM, Schuman EM, Kennedy MB. 1997.
Visualization of the distribution of autophosphorylated calcium/
calmodulin-dependent protein kinase II after tetanic stimulation in
the CA1 area of the hippocampus. J Neurosci 14:5416 –5427.
 REGULATION OF SYNAPTIC COMPONENTS
Park JH, Straub VA, O’Shea M. 1998. Anterograde signaling by nitric
oxide: characterization and in vitro reconstitution of an identified
nitrergic synapse. J Neurosci 18:5463–5476.
Petersen S,A, Fetter RD, Noordermeer JN, Goodman CS, DiAntonio
A. 1997. Genetic analysis of glutamate receptors in Drosophila
reveals a retrograde signal regulating presynaptic transmitter re-
lease. Neuron 19:1237–1248.
Regulski M, Tully T. 1995. Molecular and biochemical characteriza-
tion of dNOS: A Drosophila Ca2⫹/calmodulin-dependent nitric oxide
synthase. Proc Natl Acad Sci 92: 9072–9076.
Rheuben MB, Yoshihara M, Kidokoro Y. 1999. Ultrastructural corre-
lates of neuromuscular junction development. In: Budnik V, Gra-
mates LS, editors. Neuromuscular junctions in Drosophila. San
Diego: Academic Press, Int Rev Neurobiol 43:69 –92.
Rusakov DA, Stewart MG, Sojka M, Richter-Levin G, Bliss TV. 1995.
Dendritic spines form ’collars’ in hippocampal granule cells. Neu-
roreport 6:1557–1561.
Sattler R, Xiong Z, Lu W, Hafner M, MacDonald J F, Tymianski M.
1999. Specific coupling of NMDA receptor activation to nitric oxide
neurotoxicity by PSD–95 Protein. Science 284:1845–1848.
Schopperle WM, Holmqvist MH, Zhou Y, Wang J, Wang Z, Griffith
LC, Keselman I, Kusinitz F, Dagan D, Levitan IB. 1998. Slob, a
novel protein that interacts with the Slowpoke calcium-dependent
potassium channel. Neuron 20:565–573.
Schuster CM, Ultsch A, Schloss P, Cox JA, Schmitt B, Betz H. 1991.
Molecular cloning of an invertebrate glutamate receptor subunit
expressed in Drosophila muscle. Science 254:112–114.
Schuster C.M, Davis GW, Fetter RD, Goodman CS. 1996a. Genetic
dissection of structural and functional components of synaptic plas-
ticity. I. Fasciclin II controls synaptic stabilization and growth.
Neuron 17:641– 654
Schuster C.M, Davis GW, Fetter RD, Goodman CS. 1996b. Genetic
dissection of structural and functional components of synaptic plas-
ticity. II. Fasciclin II controls presynaptic structural plasticity.
Neuron 17:655– 667.
Shen K, Meyer T. 1999. Dynamic control of CaMKII translocation and
localization in hippocampal neurons by NMDA receptor stimula-
tion. Science 284:162–166.
Sheng M. 1996. PDZs and receptor/channel clustering: rounding up
the latest suspects. Neuron 17:575–578.
Steward O. 1997. mRNA localization in neurons: a multipurpose
mechanism? Neuron 18:9 –12.
Steward O, Wallace C S, Lyford G L, Worley P F. 1998. Synaptic
activation causes the mRNA for the IEG arc to localize selectively
near activated postsynaptic sites on dendrites. Neuron 21:741–751.
Steward R 1987. Dorsal, an embryonic polarity gene in Drosophila, is
homologous to the vertebrate proto-oncogene, c-rel. Science 238:
692– 694.
Stowell JN, Craig AM. 1999. Axon/dendrite targeting of metabotropic
glutamate receptors by their cytoplasmic carboxy-terminal do-
mains. Neuron 22:525–536.
Takeuchi M, Hata Y, Hirao K, Toyoda A, Irie M, Takai Y. 1997.
SAPAPs. A family of PSD–95/SAP90-associated proteins localized
at postsynaptic density. J Biol Chem 272:11943–11951.
Tejedor FJ, Bokhari A, Rogero O, Gorczyca M, Zhang J, Kim E, Sheng
M and Budnik V. 1997. Essential role for dlg in synaptic clustering
of Shaker K⫹ channels in vivo. J Neurosci 17:152–159.
Tezuka T, Umemori H, Akiyama T, Nakanishi S, Yamamoto T. 1999.
PSD–95 promotes Fyn-mediated tyrosine phosphorylation of the
N-methyl-D-aspartate receptor subunit NR2A. Proc Natl Am Sci
96:435– 440.
Thomas U, Kim E, Kuhlendahl S, Koh YH, Gundelfinger ED, Sheng
M, Garner CC, Budnik V. 1997. Synaptic clustering of the cell
adhesion molecule Fasciclin II by Discs-Large and its role in the
regulation of presynaptic structure. Neuron 19:787–799.
Tiedge H, Bloom FE, Richter D 1999. RNA, whither goest thou?
Science, 283:186 –187.
25
Tiedge H, Brosius J 1996. Translational machinery in hippocampal
neurons in culture. J Neurosci 16: 7171–7181.
Tongiorgi E, Righi M, Cattaneo A. 1997. Activity-dependent dendritic
targeting of BDNF and TrkB mRNAs in hippocampal neurons.
J Neurosci 17:9492–9505.
Torroja L, Packard M, Gorczyca M, White K, Budnik V. 1999. The
Drosophila beta-amyloid precursor protein homolog promotes syn-
apse differentiation at the neuromuscular junction. J Neurosci 19:
7793–7803.
Trahey M, Wong G, Halenbeck R, Rubinfeld B, Martin GA, Ladner M,
Long CM, Crosier WJ, Watt K, Koths K, McCormick F. 1988.
Molecular cloning of two types of GAP complementary DNA from
human placenta. Science 242:1697–1700.
Turner KM, Burgoyne RD, Morgan A. 1999. Protein phosphorylation
and the regulation of synaptic membrane traffic. TINS 22:459 – 464.
Vogel US, Dixon RA, Schaber MD, Diehl RE, Marshall MS, Scolnick
EM, Sigal IS, Gibbs JB. 1988. Cloning of bovine GAP and its
interaction with oncogenic ras p21. Nature 335:90 –93.
Wang J, Renger JJ, Griffith LC, Greenspan RJ, Wu C-F. 1994. Con-
comitant alteration of physiological and developmental plasticity in
Drosophila CaMKII inhibited synapses. Neuron 13:1373–1384.
Wang Z Palmer G, Griffith LC. 1998. Regulation of Drosophila Ca2⫹/
calmodulin-dependent protein kinase II by autophosphorylation an-
alyzed by site-directed mutagenesis. J Neurochem 71:378 –387.
Wigler MH. 1990. Oncoproteins. GAPs in understanding Ras. Nature
346:696 – 697.
Wildemann B, Bicker G. 1999a. Nitric oxide and cyclic GMP induce
vesicle release at Drosophila neuromuscular junction. J Neurobiol
39:337–346.
Wildemann B, Bicker G 1999b) Developmental expression of nitric
oxide/cyclic GMP synthesizing cells in the nervous system of Dro-
sophila melanogaster. J Neurobiol 38:1–15.
Woods DF, Bryant PJ. 1991. The discs-large tumor suppressor gene of
Drosophila encodes a guanylate kinase homolog localized at septate
junctions. Cell 66:451– 464.
Woods DF, Bryant PJ. 1993. ZO1 DlgA and PSD–95/SAP90: homolo-
gous proteins in tight, septate and synaptic cell junctions. Mech Dev
44:85– 89.
Wu X -Q, Gu W, Meng X, Hecht NB. 1997. The RNA-binding protein
TB-RBP, is the mouse homologue of translin, a recombination pro-
tein associated with chromosomal translocations. Proc Natl Acad
Sci 94:5640 –5645.
Wu L, Wells D, Tay J, Mendis D, Abbott M-A, Barnitt A, Quinlan E,
Heynen A, Fallon JR, Richter JD. 1998. CPEB-mediated cytoplas-
mic polyadenylation and the regulation of experience-dependent
translation of CaMKII mRNA at synapses. Neuron 21:1129 –1139.
Zhang W, Vazquez L, Apperson M, Kennedy MB. 1999. Citron binds
to PSD–95 at glutamatergic synapses on inhibitory neurons in the
hippocampus. J Neurosci 19:96 –108.
Zhong Y, Pena L. 1995. A novel synaptic transmission mediated by a
PACAP-like neuropeptide in Drosophila. Neuron 15:2354 –2366.
Zhong Y, Wu C-F. 1991. Altered synaptic plasticity in Drosophila
memory mutants with a defective cyclic AMP cascade. Science,
251:198 –201.
Zhong Y, Budnik V, Wu C-F. 1992. Synaptic plasticity in Drosophila
memory and hyperexcitable mutants: role of cAMP cascade. J Neu-
rosci 12:644 – 651.
Zhou Y, Schopperle WM, Murrey H, Jaramillo A, Dagan D, Griffith
LC, Levitan IB. 1999. A dynamically regulated 14 –3–3 Slob, and
Slowpoke potassium channel complex in Drosophila presynaptic
nerve terminals. Neuron 22:809 – 818.
Zipkin ID, Kindt RM, Kenyon CJ. 1997. Role of new Rho family
member in cell migration and axon guidance in C. elegans. Cell
90:883– 894.
Zito K, Fetter RD, Goodman CS, Isacoff EY. 1997. Synaptic clustering
of Fasciclin II and Shaker: Essential targeting sequences and role of
Dlg. Neuron 19:1007–1016.