Glutamate receptor IIA and Glutamate receptor IIB
Long-term synaptic plasticity may be associated with structural rearrangements
within the neuronal circuitry. Although the molecular mechanisms
governing such activity-controlled morphological alterations are mostly elusive,
polysomal accumulations at the base of developing dendritic spines
and the activity-induced synthesis of synaptic components suggest that localized
translation is involved during synaptic plasticity. This study
shows that large aggregates of translational components as well as messenger
RNA of the postsynaptic glutamate receptor subunit DGluR-IIA
are localized within subsynaptic compartments of larval neuromuscular junctions
of Drosophila. Genetic models of junctional plasticity and genetic manipulations using the translation initiation factors
eIF4E and poly(A)-binding protein showed an
increased occurrence of subsynaptic translation aggregates. This was associated
with a significant increase in the postsynaptic DGluR-IIA protein levels and
a reduction in the junctional expression of the cell-adhesion molecule Fasciclin
II. In addition, the efficacy of junctional neurotransmission and the size
of larval neuromuscular junctions were significantly increased. These results
therefore provide evidence for a postsynaptic translational control of long-term
junctional plasticity (Sigrist, 2000).
Translational control is primarily exerted by regulation of the initiation
step of translation, which appears to be controlled by the
rate-limiting initiation factor eIF4E. In addition, the interaction
of the 5' cap bound eIF4E with the 3' end of mRNAs through a complex
of other initiation factors and the poly(A)-binding protein (PABP)
has been shown to synergistically facilitate translation initiation.
To assess the potential role of regulated translation during the development
of the larval neuromuscular junctions (NMJs) in Drosophila,
the subcellular expression pattern of eIF4E and PABP were analyzed in filet preparations of third instar larvae. Both antigens showed a weak and ubiquitous expression
in the cytoplasm of all larval cells, and they colocalized in strongly immunopositive aggregates up to 2microm
in length close to NMJs. The specific localization of eIF4E/PABP
aggregates close to and partially overlapping with junctional profiles revealed
that eIF4E/PABP aggregates are positioned subsynaptically within or adjacent
to the subsynaptic reticulum (SSR). No evidence was found for presynaptic
or axonal localization of such aggregates. Therefore, the almost exclusive
subsynaptic distribution of the eIF4E/PABP aggregates within larval muscles
indicates that there may be a functional relationship between NMJs and the
appearance of nearby eIF4E/PABP aggregates (Sigrist, 2000).
Ultrastructural examinations of larval NMJs revealed polysomal accumulations
within and close to the SSR. According
to their variable size, subsynaptic location and frequency of detection, the
larger of these polysomal clusters are likely to represent the eIF4E/PABP
aggregates detected by light microscopy. In addition, smaller polysomal aggregates were widely distributed
in discrete membranous compartments throughout the SSR, whereas presynaptic and axonal profiles were free
of polysomes. It is therefore concluded that mRNAs are translated within subsynaptic
compartments of larval NMJs and that local
centres of concentrated, subsynaptic translation are identified by large junctional
eIF4E/PABP aggregates (Sigrist, 2000).
To assess whether junctional translation is subject to regulation, the number was quantified of synaptic specializations (boutons) per NMJ that were labelled
by one or more translation aggregates.
Animals that overexpressed PABP in larval muscles and larvae that were mutant
in pabp showed a significantly increased occurrence of subsynaptic
eIF4E/PABP aggregates and an unaltered level of muscular PABP staining. In addition, the total PABP
levels in crude larval protein extracts were unaltered in all analysed genotypes,
even when PABP mRNA levels were significantly increased or reduced under genetic
gain-of-function or loss-of-function conditions, respectively. Such a homeostasis of total PABP levels is a well described phenomenon for PABP, and in crude protein extracts it might have masked the significant local increase in the number of PABP aggregates observable within subsynaptic compartments of NMJs. Although the exact reason for this increase in the occurrence of eIF4E/PABP aggregates is unknown, a local perturbation of PABP levels owing to a previously described overshooting compensation of the PABP-homeostasis mechanism might facilitate formation of subsynaptic translation aggregates (Sigrist, 2000).
A similar increase in the frequency of postsynaptic translation aggregates
was also observed in two mutants representing well established genetic models
of long-term synaptic plasticity in Drosophila, the hyperactive K+-channel
mutant eag, Sh and the cAMP-phosphodiesterase mutant dunce. Thus, increased neuronal activity levels (in eag, Sh)
as well as elevated cellular cAMP levels (in dunce) are capable of
inducing subsynaptic translation aggregate formation. These findings are consistent
with the hypothesis that synaptic activity can control synaptic translation (Sigrist, 2000).
To identify potential substrates and targets of subsynaptic translation
at larval NMJs, quantitative immunostainings were performed of several junctionally
expressed proteins, including the synaptic vesicle protein synaptotagmin,
the junctional anti-horseradish peroxidase (HRP) epitope, the cell-adhesion
molecule Fasciclin II (FasII), the postsynaptic glutamate receptor subunit
DGluR-IIA and the conventional myosin as a nonsynaptic protein. No obvious differences were detected in the expression levels of myosin, synaptotagmin
and the junctional anti-HRP immunoreactivity in all analysed genotypes; however, animals that showed elevated numbers of subsynaptic translation
aggregates consistently displayed increased
junctional levels of DGluR-IIA and an altered junctional distribution of FasII, which was
associated with a significant reduction of synaptic FasII levels as compared
with control animals. A similar
FasII phenotype has been described in the plasticity models eag, Sh
and dunce, and it has been shown that presynaptic FasII downregulation
is essential for increased junctional outgrowth.
Intriguingly, in Aplysia the FasII homologue apCAM is also presynaptically
downregulated after treatments that increase synaptic efficacy and growth
of new synaptic connections. This synaptic apCAM regulation
is thought to be achieved by a protein-synthesis-dependent activation of an
endocytic apCAM internalization. Given that FasII has been
detected in membranes of a subset of presynaptic vesicles,
it seems possible that subsynaptic protein synthesis affects junctional FasII
levels through similar mechanisms to those in Aplysia (Sigrist, 2000).
The postsynaptic DGluR-IIA immunoreactivities were significantly stronger
in translationally sensitized animals.
This strong increase of synaptic DGluR-IIA expression was not due to transcriptional
upregulation of dglur-IIA; the total amounts of DGluR-IIA mRNAs
were unaltered or even reduced in the analysed genotypes as compared with
controls. In situ hybridization
experiments revealed that DGluR-IIA mRNA surrounds individual type-I boutons,
with prominent staining of terminal and branch-point boutons and weak or absent staining within the SSR of interbouton connectives. Thus,
the subsynaptically localized DGluR-IIA mRNA represents a direct substrate
for the junctional translation machinery. These results can not exclude an
extrajunctional contribution to the observed synaptic DGluR-IIA increase,
but they suggest that this phenotype is due to an increased subsynaptic synthesis
of DGluR-IIA in genotypes with a higher occurrence of junctional eIF4E/PABP
aggregates (Sigrist, 2000).
To analyse the functional consequences of genetically modified subsynaptic
translation, the strength of neurotransmission at NMJs was assessed on muscle
6 of third instar larvae. The average amplitudes
of miniature excitatory junctional currents (mEJCs) and thus the quantal sizes were indistinguishable in all analysed genotypes. This finding
indicates either that the additional receptor subunits that are synaptically
localized may be functionally
silent (for example, through physiological silencing or intracellular
localization or that the amount of glutamate released from
an individual quantum is not sufficient to saturate the postsynaptic receptors. In contrast, postsynaptic responses evoked by stimulation of motor
nerve axons were substantially larger in all mutants exhibiting increased levels of subsynaptic translation. Thus, the derived
quantal content was significantly increased above control values, suggesting
that the observed larger amplitudes of evoked junctional responses arise from
an increased number of released presynaptic vesicles per action potential (Sigrist, 2000).
To investigate whether the increase in junctional efficacy was due to a
change in the number of synaptic specializations, the number
of junctional boutons per NMJ was quantified. Genotypes that
displayed an increased occurrence of subsynaptic translation aggregates had
significantly larger NMJs and reduced junctional
FasII levels. In addition, the junctional
sizes of the analysed animals correlated in a highly significant manner with
their estimated quantal contents, suggesting
that junctional efficacy and the morphological elaboration of NMJs are tightly
coupled. On the basis of light microscopic examinations of DGluR-IIA labelled
NMJs, the density of synapses within NMJs of all mutant animals appeared similar
to that of controls or even higher, indicating that the total number of synapses
increased proportionally with the junctional size. This finding indicates
that the increased quantal content in animals with facilitated subsynaptic
translation may be because of an increase in the number of vesicle release
sites per given stimulus (Sigrist, 2000).
In summary, this study has shown that translational machinery and mRNAs are associated with the subsynaptic reticulum of NMJs and that genetic manipulations that affect the occurrence of subsynaptic translation aggregates are accompanied
by changes in the levels of synaptic proteins, such as DGluR-IIA and FasII.
These same manipulations also affected the function and morphology of NMJs,
suggesting that subsynaptic translation can instruct junctional growth and
synaptic reorganization and thereby long-term functional changes. These results
further suggest that subsynaptic translation can be regulated by altered levels
of neuronal activity, indicating that the regulation of postsynaptic translation
participates in activity-dependent junctional plasticity. Thus, the inducible
recruitment of postsynaptic protein synthesis appears to render individual
synapses competent to instruct long-term changes in their functions and morphological
organization. Given that localized protein synthesis has been shown to act
in a synapse specific stabilization of long-term facilitation in central neurons
of Aplysia, it emerges that synaptic translation
might represent a common principle of long-term alterations of neuronal function
and connectivity (Sigrist, 2000).
It was hypothesized that cystine/glutamate transporters (xCTs) might be critical regulators of ambient extracellular glutamate levels in the nervous system and that misregulation of this glutamate pool might have important neurophysiological and/or behavioral consequences. To test this idea, a novel Drosophila xCT gene was identified and functionally characterized, that has been named 'genderblind' (gb). Genderblind is expressed in a previously overlooked subset of peripheral and central glia. Genetic elimination of gb causes a 50% reduction in extracellular glutamate concentration, demonstrating that xCT transporters are important regulators of extracellular glutamate. Consistent with previous studies showing that extracellular glutamate regulates postsynaptic glutamate receptor clustering, gb mutants show a large (200%-300%) increase in the number of postsynaptic glutamate receptors. This increase in postsynaptic receptor abundance is not accompanied by other obvious synaptic changes and is completely rescued when synapses are cultured in wild-type levels of glutamate. Additional in situ pharmacology suggests that glutamate-mediated suppression of glutamate receptor clustering depends on receptor desensitization. Together, these results suggest that (1) xCT transporters are critical for regulation of ambient extracellular glutamate in vivo; (2) ambient extracellular glutamate maintains some receptors constitutively desensitized in vivo; and (3) constitutive desensitization of ionotropic glutamate receptors suppresses their ability to cluster at synapses (Augustin, 2007).
The primary physiological role of xCT transporters remains controversial. Although xCT transporters mediate 1:1 exchange between extracellular cystine and intracellular glutamate, glutamate excretion is generally ignored, and xCT transporters are often assumed to function primarily as a cystine-uptake mechanism for glutathione synthesis and protection from oxidative stress. However, this bias ignores several important facts: (1) xCT transporters also export glutamate. (2) Mammalian brain xCT appears most abundant in 'border areas between the brain proper and periphery', specifically 'several regions facing the CSF,' including ventricle walls and meninges, consistent with the idea that xCT transporters are important for regulation of free glutamate content of CSF but not for cystine uptake in all brain cells. (3) Mammalian xCT transporters appear to be dispensable for cystine uptake and glutathione synthesis. Instead, glutathione synthesis in neurons and glia may be regulated by excitatory amino acid transport (EAAT) family proteins. EAATs are best known as sodium-dependent transporters for glutamate uptake, but EAATs also efficiently import cysteine, the reduced form of cystine used in glutathione synthesis. In agreement, overexpression of Drosophila gb (Tub-Gal4;UAS-gb) causes shortened lifespan and neurodegeneration, consistent with increased glutamate secretion but the exact opposite phenotype that one would expect if the role of GB were cystine uptake for neuroprotection. (4) Microdialysis of rat brains with inhibitors of xCT function leads to a decrease in nonvesicular glutamate secretion (Augustin, 2007).
Accordingly, it is argued that glutamate export by xCT transporters is at least as important as cystine import, particularly in the nervous system. Full acceptance of this idea, however, requires one to accept the idea that xCT transporters maintain ambient extracellular glutamate in the nervous system for good reasons and that extracellular glutamate in the brain is not merely a potentially pathological byproduct of glutamatergic transmission. The data suggest that ambient extracellular glutamate regulates constitutive receptor desensitization for control of synaptic glutamate receptor abundance (Augustin, 2007).
A link between glutamate receptor desensitization and clustering has not previously been demonstrated. It is well known that desensitization functionally eliminates glutamate receptors on a short time scale (tens to hundreds of milliseconds). The data suggest that constitutive desensitization is, on a longer time scale (hours), also associated with removal of receptors from the synapse. The EC50 for activation of Drosophila larval muscle glutamate receptors is ~2 mM, and significant numbers of receptors can be desensitized at considerably lower concentrations. Because 2 mM is near the concentration of glutamate bathing NMJ receptors in vivo, it must be concluded that one-half or more of Drosophila larval muscle glutamate receptors are constitutively desensitized, and therefore delocalized, in vivo. This conclusion is consistent with the 200%-300% increase in postsynaptic glutamate receptor abundance that we observe after switching NMJs to culture media containing 0 mM glutamate (Augustin, 2007).
At first, the idea that many glutamate receptors should be desensitized (and subsequently delocalized) in vivo seems surprising. However, constitutive desensitization (and subsequent delocalization) of ligand-gated ion channels by ambient ligand is analogous to constitutive inactivation of voltage-gated ion channels by resting membrane potential. Constitutive inactivation of voltage-gated channels is a common and important regulator of membrane excitability. For example, at a typical rat skeletal muscle resting potential of -90 mV, approximately two-thirds of rat skm-1 skeletal muscle sodium channels are inactivated. As a result, only one-third of channels in the membrane are normally available for generation of action potentials. However, if resting membrane potential is modified or the voltage dependence of sodium channel inactivation is slightly shifted by (for example) channel phosphorylation, then the number of functionally available sodium channels in the membrane can change quickly and dramatically, with consequent large effects on cell excitability. In the case of glutamate receptors, the number of functionally available receptors at a synapse, and therefore synaptic strength, could similarly be quickly and effectively altered by relatively minor changes in ambient glutamate levels (perhaps because of regulation of xCT-mediated transport) or changes in the concentration dependence of receptor desensitization as a result of (for example) receptor phosphorylation. These possibilities have not been explored (Augustin, 2007).
A physiological role for ambient extracellular glutamate also has medical implications. Abnormal levels of CSF glutamate have been linked to a variety of human neurodevelopmental and neurodegenerative disorders, including anxiety/stress-related disorders, Rett syndrome, autism, and all forms (both familial and sporadic) of amyotrophic lateral sclerosis. Furthermore, xCT and 4F2hc have specifically been implicated in development, behavior, and disease. For example, lysinuric protein intolerance, a recessive disorder characterized by severe mental retardation, is caused by mutations in the human xCT gene SLC7A7 [solute carrier family 7 (cationic amino acid transporter, y+ system), member 7]. Similarly, 4F2hc is required for tumor transformation in human cancers. Finally, human xCT protein was recently identified as the fusion-entry receptor for Kaposi's sarcoma-associated herpes virus. Not surprisingly, therefore, extracellular glutamate and xCT transporters are beginning to be targeted for pharmacological inhibition. Our results suggest that pharmacological inhibition of xCT transport could considerably ameliorate neuropathologies exacerbated by extracellular glutamate but raise the caveat that tampering with extracellular glutamate could have unexpected developmental and/or psychotropic effects (Augustin, 2007 and references therein).
Two distinct mechanisms regulate synaptic efficacy at the Drosophila neuromuscular junction: a
PKA-dependent modulation of quantal size and a retrograde regulation of presynaptic release. Postsynaptic expression of a constitutively active PKA catalytic subunit decreases quantal size, whereas overexpression of a mutant PKA regulatory subunit (inhibiting PKA activity) increases
quantal size. Increased PKA activity also decreases the response to direct iontophoresis of glutamate
onto postsynaptic receptors. The PKA-dependent modulation of quantal size (or response of the muscle to the
spontaneous release of a single synaptic vesicle) requires the presence of
the muscle-specific glutamate receptor DGluRIIA, since PKA-dependent modulation of quantal size is
lost in viable homozygous DGluRIIA- mutants. The DGluRIIA sequence contains an optimal PKA consensus phosphorylation site on the C-terminal tail (RRXS), believed to be located in the cytoplasmic portion of the protein. Elevated postsynaptic PKA reduces the quantal amplitude and the time constant of miniature excitatory junctional potential (mEJP) decay to values that are nearly identical to those observed in DGluRIIA mutants. PKA modulation of quantal size is sensitive to the copy number of DGluRIIA. Larvae heterozygous for a deletion of DGluRIIA show significantly less modulation by PKA than wild-type controls. This suggests that PKA-dependent modulation of receptor function may be influenced by the subunit composition of postsynaptic receptors. PKA activity appears to constitutively regulate synaptic function at the wild-type synapse. The demonstration that inhibition of PKA leads to a large increase in quantal size suggests that there is a high basal phosphorylation of DGluRIIA at the wild-type synapse. The PKA-dependent reduction in quantal size is accompanied developmentally by an increase in presynaptic quantal content, indicating the presence of a retrograde signal that regulates presynaptic release. A retrograde regulation of presynaptic transmitter release may serve to maintain postsynaptic excitation during the developmental growth of this synapse (Davis, 1998).
During the development of the Drosophila NMJ, a large increase in muscle volume is tightly coupled to increases in
both presynaptic structure and presynaptic function. This coupling assures that the presynaptic motoneuron is able to
appropriately excite postsynaptic muscle. It has been proposed that this correlation between presynaptic and postsynaptic
growth is maintained by a signal from muscle to motoneuron (Schuster, 1996a). This
hypothesis was tested by manipulating postsynaptic excitation by increasing PKA activity and then assaying whether there is a
presynaptic compensation for changes in postsynaptic excitation (Davis, 1998).
The hypothesis that an activity-dependent retrograde signal regulates presynaptic release at this synapse is supported by the data.
There is a significant increase in quantal content (>50% increase compared to wild-type and genetic controls) that
accompanies the PKA-dependent decrease in quantal size.
Quantal content is a measure of the number of vesicles released by the nerve.
This increase in quantal content is observed in each
of the three experimental genotypes in which the PKA activator decreases quantal size. Furthermore, this increase in
quantal content is observed in both voltage-clamp and current-clamp experiments. Since
presynaptic release is increased in response to a decrease in the postsynaptic sensitivity to transmitter, it is proposed that
there exists a retrograde signal capable of regulating presynaptic transmitter release at this synapse. These results agree
with those of Petersen (1997), who observed an increased presynaptic release in glutamate receptor mutants
that decrease quantal size (Davis, 1998).
The PKA-dependent increase in quantal size due to PKA inhibition is not compensated for by a change in presynaptic
release, however. There is no change in presynaptic quantal content despite a substantial (>40%) increase in quantal size
when PKA is inhibited in muscle. As a result, there is a significant increase in the average compound extrajunctional potential (EJC), when
compared to wild type. PKA activity was inhibited in muscle by expression of a mutant PKA regulatory subunit under UAS control.
These regulatory subunits carry a mutated cAMP binding site and as a result have reduced sensitivity to cAMP resulting
in decreased PKA catalytic activity.
Again, these results are in agreement with those of Petersen (1997), who showed that an
overexpression of DGluRIIA increases quantal size without a compensatory change in presynaptic release. Thus, under
these conditions, there does not appear to be a presynaptic compensation for increased postsynaptic excitation (Davis, 1998).
A retrograde signal from muscle to motoneuron could influence presynaptic release by increasing presynaptic structure.
Bouton number was quantified for the junction at muscles 6 and 7 in abdominal segment A3. There was no
change in bouton number comparing wild type and with larvae expressing increased
PKA in muscle. Similarly, there is no change in presynaptic bouton number in larvae in which
PKA is inhibited in muscle. Thus, a retrograde signal most likely regulates either the number of
presynaptic active zones present in each presynaptic bouton or regulates some aspect of the presynaptic release
mechanism (Davis, 1998).
The rate of spontaneous release events is altered by postsynaptic PKA.
In both current clamp and voltage clamp experiments, a reduction in quantal size (due to increased PKA in the muscle) correlates with
a significant reduction in the frequency of spontaneous mEJP events. This reduction in mEJP frequency may originate postsynaptically,
since presynaptic release is actually increased, not decreased, in these mutants. It is possible that a PKA-dependent
decrease in quantal size reduces the amplitude of many release events below the noise level. Alternately, these results
may indicate that postsynaptic receptors are functionally silenced by increased postsynaptic PKA activity. There was no
significant change in miniature EJC frequency when PKA activity was inhibited in muscle, indicating that silent synapses, or
very small events, are not revealed by increasing quantal size (Davis, 1998).
In Drosophila, mutations in specific ion channel genes can increase or decrease the level of neural/synaptic activity. These genetic tools have been used, in combination with classical pharmacological agents, to modulate neural activity during embryogenesis and examine effects on the differentiation of an identified neuromuscular junction. Electrical
activity is found to be required for the neural induction of transmitter receptor expression during synaptogenesis. Likewise, neural electrical activity is required to localize transmitter receptors to the synaptic site. In muscles with activity-blocked synapses, a low level of receptors is expressed homogeneously in the muscle membrane as in muscles developing without innervation. Thus, presynaptic electrical activity is required to mediate the neural induction of the transmitter receptor field in the postsynaptic membrane (Broadie, 1993).
Pronounced alterations in synaptic activity have a relatively small impact on functional glutamate receptor expression during neurogenesis. Null mutations in paralytic (para) (which codes for a sodium channel), significantly reduce synaptic activity but does not significantly alter GluR expression by the end of embryogenesis. Likewise, single mutations in Shaker or ether a-go-go, a structural gene encoding a fast voltage-gated potassium channel and another potassium channel respectively, do not significantly alter synaptic GluR expression by the time of hatching. However, more dramatic changes in synaptic activity have a small but significant effect on GluR expression during synaptogenesis. For example, a deficiency in the para locus reduces GluR expression at the neuromuscular junction. Suppression of presynaptic electrical activity, whether by using mutations or injection tetrodotoxin, which binds neuronal sodium channels and so blocks the propagation of action potentials, reduces the expression of GluRs during synaptogenesis. Different alleles of para block synaptic transmission at different temperatures. Embryos raised at these restrictive temperatures show a dramatic reduction in GluR expression at the NMJ at the end of embryogenesis. Evidence is also provided that presynaptic electrical activity is required to localize GluRs to the synaptic site during synaptogenesis. Synaptic morphogenesis is independent of synaptic activity during embryogenesis, and it is concluded that postsynaptic activity, mediated through GluRs, similarly plays no role in synaptic morphogenesis in the embryo. Unlike synaptic structure, the size of the synaptic zone is significantly altered in line with alterations in presynaptic electrical activity. Hyperactive synapses expand to occupy a large area of the muscle surface relative to wild type. Likewise, inactive synapses occupy an even smaller synaptic domain relative to hyperactive synapses, although inactive synapses are not significantly smaller than wild type. Thus, activity modulates the size of the synaptic domain during synaptogenesis. The decreased synaptic density of hyperactive synapses explains the decrease in GluR expression in the postsynaptic membrane observed in eag Sh double mutants; GluR expression is not suppressed by increased activity but, rather, GluRs are localized over a larger surface area and so decrease in density at any given synaptic site (Broadie, 1993)
Glutamate receptor channels in Drosophila embryos and larvae were examined with the patch-clamp technique in various
configurations. In the cell-attached mode, only one type of channel is observed in the extrajunctional region at any stage.
The burst duration histogram was fit with three exponentials. The burst duration of long component lengthens with increasing
glutamate concentration. In excised outside-out patches the unitary channel current is 7.1 pA at -60 mV and the direction of
current reversed at zero membrane potential. In contrast, junctional receptor channels have different properties. In the whole-cell
configuration, spontaneous synaptic currents with steps on the falling phase are observed. The step amplitudes have two
discrete values of 9.4 and 18.5 pA at -60 mV, due to the opening of junctional glutamate receptor channels. Synaptic currents
change amplitudes linearly with the membrane potential in the negative potential range but nonlinearly above zero. With 1 mM
glutamate in the bath, synaptic currents are no longer observed. Instead, there are single channel events with the current
amplitude varying between 8 and 12 pA at -60 mV. Their long burst duration depends on glutamate concentration, indicating
that they are glutamate receptor channel events. The extrapolated reversal potential of these channel currents is around +12
mV. These junctional receptor channels are strictly localized at the junction. These findings suggest that the channel conversion
mechanism in Drosophila is different from that observed in vertebrates. Further close examination of other intermediate steps
during neuromuscular junction formation is needed (Nishikawa, 1995).
Outside-out patches of membrane were excised from muscle fibers 6 and 7 of third-instar wild-type Drosophila larvae.
Channels were observed to open in response to short pulses of L-glutamate. At a holding potential of -60 mV, the channels
open to one main conductance level of about 120 pS. The current-voltage plot for the channels is linear and reverses
around 0 mV holding potential. The channels are also activated by quisqualate but not by aspartate, N-methyl-D-aspartate
(NMDA), kainate, glycine, gamma-aminobutyric acid (GABA) and acetylcholine. At high glutamate concentrations (3 or 10
mM), channel activation reached a peak within 0.3 ms. The channels open in 'bursts', flickering between open and closed
states. The channels opened only for a few milliseconds after switching on the glutamate and channel activity declines to zero after
the initial surge, with time constants between 5 and 20 ms. During applications of low glutamate concentrations
(0.2-0.5 mM) to the same patch the channels open much less frequently and during most applications no openings are
observed. The openings observed are short and 'bursts' of openings are rare. Two exponential components were
identified in the open-time distribution obtained with pulsed applications of glutamate (0.5-10 mM) with time constants of
about 0.1 and 2.0 ms. The kinetics of the channels seem to be similar to the kinetics of certain glutamate gated channels
found on the muscles of crayfish and locust (Heckmann, 1995).
Outside-out patches from wild-type Drosophila larval muscle were exposed briefly to L-Glutamate (Glu) using a piezo-driven
application system. Glu in concentrations of 0.1 to 30 mM was applied and the responses to repeated applications of a given
concentration were averaged. The peak current, i, and the current rise time, tr, from 0.1 i to 0.9 i were determined from the
averages. Half-maximum activation of the channels is reached with approximately 2 mM Glu. i increases proportional to the
power n = 3.5 to n = 5.8 (average of four experiments, n = 4.4) for Glu concentrations between 0.3 and 0.5 mM. tr increases
from approximately 0.2 ms at 10 mM Glu to a value of approximately 3.5 ms at 0.2 mM Glu. A linear reaction scheme with
five binding steps preceding the channel-opening conformational change is proposed as the kinetic mechanism of channel
activation: this scheme was investigated in computer simulations. A set of rate constants assuming the same affinity for each binding site is
found to describe the data better than one assuming positive cooperativity. The results are very similar to those for Glu-gated
channels of crayfish and locust muscle, which is evidence of a common kinetic mechanism for these channels (Heckmann, 1996).
Glutamate receptor channels are ubiquitous agonist-gated channels. Pharmacologically they are classified into several subtypes
but may have common fundamental channel properties. To build a foundation for future molecular biological and genetic
studies, kinetics of the glutamate receptor channel were studied in embryonic Drosophila myotubes in culture using the patch
clamp technique. There are many channel events of brief duration, together with prolonged ones. Brief events are frequently
observed in low concentrations whereas the frequency of prolonged events increases with agonist concentrations. Long
openings (> 5 ms) were often interrupted by brief closures, most of which lasted less than 100 mu s, thus showing a bursting
behavior. At all agonist concentrations, the burst duration was fitted with three exponential components (brief, intermediate
and long). The mean duration of the long component increases linearly with the glutamate concentration. The mean closed time
and number of brief closures per ms within long bursts are independent of agonist concentration. The mean burst durations
of the brief (30-250 mu s) and intermediate component (300-1050 mu s) do not change significantly with agonist
concentration. The closing episodes within bursts are rare in the brief and intermediate burst components. The ratio of the
fractional areas of the brief or intermediate and long burst components increases linearly with agonist concentration in the
log-log plot with a slope of one. These findings suggest that the brief and intermediate components are due to singly-liganded
openings and the long component is the result of doubly-liganded openings (Chang, 1996).
Focal extracellular excitatory postsynaptic currents were recorded to investigate short-term depression at glutamatergic
Drosophila neuromuscular synapses. The amplitudes of quantal excitatory postsynaptic currents (qEPSCs) elicited before and
after depolarizations eliciting large release were compared. Depression reduces the amplitude of the qEPSCs to 0.65 +/- 0.14
of control. Recovery from depression and of the receptor channels from desensitization follow a similar time course. Thus
receptor desensitization seems to be involved in short-term depression at Drosophila neuromuscular junctions (Adelsberger, 1997).
Outside-out patches from wild-type Drosophila larval muscle were exposed to L-glutamate (glu) using a piezo-driven
application system. Glu receptor channels open and desensitize in response to rapid applications of 10 mM glu.
Desensitization was fitted with an exponential function with a mean time constant of desensitization (tau d) of 15 ms in
response to 10 mM glu. The tau d is concentration dependent and decreases to 6 ms (on average) with 0.7 mM glu and
increases again to 12 ms (on average) in response to 0.5 mM glu. Desensitization in response to longer applications of glu is
almost complete, but surprisingly, even a 1-ms pulse of 3 mM glu produces about 30% desensitization. In the presence of low
glu concentrations, the response to a pulse is reduced and is about halved by preequilibration with 30 microM glu.
Recovery from desensitization is not concentration dependent and was fitted with an exponential function with a mean time
constant of 150 ms. During recovery the channels rarely open. Kinetic schemes were fitted to these results, and a circular
reaction scheme was found to fit the data best. An important feature of the scheme is desensitization from a lower ligated
closed state. This allows substantial desensitization of synaptic receptor channels in response to quantal release of transmitter,
in part without opening of the channels. Desensitization reduces the probability of the channels opening in response to a
subsequent release for a period of time determined by the rate of recovery from desensitization and might serve as a form of
molecular short-term memory (Heckmann, 1997).
Evoked excitatory postsynaptic currents (EPSC) were recorded with an extracellular macropatch electrode from glutamatergic
neuromuscular junctions of Drosophila larvae. At 20 degrees C quantal current amplitude is about -400 pA and the 10%-90%
rise time is slightly below 0.2 ms for the fastest rising events and on average 0.3+/-0.1 ms in the best recordings. The quantal
currents often have 'shoulders' but decay approximately monoexponentially from half amplitude. The time constant of the
exponential fit varies with mean values ranging from 2.5 ms to 7.7 ms in 13 experiments and an average value of 4.4+/-1.6 ms.
Comparison of these results with data obtained earlier with outside-out patches of larval muscle membrane leads to the conclusion that glutamate has to reach a saturating peak concentration of
at least 10 mM in the synaptic cleft to allow the observed short quantal current rise times. To account for the time course of the
quantal current decay one has to assume that the glutamate concentration in the synaptic cleft remains in the millimolar range for
more than a millisecond and that the time course of the decay of the quantal currents is in part due to desensitization of the
postsynaptic receptor channels (Heckmann, 1998).
Mutations in rho-type guanine exchange factor (rt/GEF), also called dpix, were recovered from a large-scale screen in Drosophila for genes that control synaptic structure. dPix/rtGEF is homologous to mammalian Pix. dPix plays a major role in regulating postsynaptic structure and protein localization at the Drosophila glutamatergic neuromuscular junction. dpix mutations lead to decreased synaptic levels of the PDZ protein Discs large, the cell adhesion molecule Fas II, and the glutamate receptor subunit GluRIIA, and to a complete reduction of the serine/threonine kinase Pak and the subsynaptic reticulum. The electrophysiology of these mutant synapses is nearly normal. Many, but not all, dpix defects are mediated through dPak, a member of the family of Cdc42/Rac1-activated kinases. Direct interaction of mammalian Pix with Pak has been detected. Thus, a Rho-type GEF (Pix) and Rho-type effector kinase (Pak) regulate postsynaptic structure (Parnas, 2001).
In mammals, the Pix family contains two members: alphaPix (Cool-2) and ßPix (Cool-1). Pix has an SH3 domain, a DBL-homology GEF domain, and a pleckstrin homology domain. The Cool (for cloned-out of library)/Pix (for PAK-interactive exchange factor)
proteins directly bind to members of the PAK family of serine/threonine kinases
and regulate their activity. In Drosophila, dPix is localized to the PSD: dpix mutations lead to the loss of synaptic Pak kinase. Paks are a family of Cdc42/Rac1-activated serine/threonine kinases important in regulating actin-containing structures. In the fly NMJ, Pak kinase is localized to the PSD. In mammals, Pak is recruited to focal complexes in a Cdc42-, Rac1-, and Pix- dependent manner (Parnas, 2001).
In addition to the absence of Pak kinase at the synapse, dpix mutations lead to the decrease in synaptic levels of the PDZ protein Discs-large (Dlg), the cell adhesion molecule Fasciclin II (Fas II), the glutamate receptor (GluR) subunit GluRIIA, and to the elimination of the subsynaptic reticulum (SSR). In Drosophila, the PSD-95 homolog Dlg has been shown to be directly responsible for the clustering of the Shaker potassium channel and to partially control the clustering of the cell adhesion molecule Fas II to the NMJ. Many, but not all, dpix defects are mediated through Pak kinase. Thus, the data suggest a pathway for synaptic clustering from dPix to Pak kinase to Dlg to Shaker and to Fas II (Parnas, 2001).
The dpix phenotype is consistent with at least two functions at the postsynaptic compartment: targeting and stabilization of postsynaptic components. In dpix mutants, Pak kinase is completely missing from the synapse. Since Pix is known to directly interact with Pak in mammals and target it to focal complexes, the data best fit with the model in which dPix targets Pak kinase to the synapse via a direct interaction. Furthermore, overexpressing either Pak kinase or a membrane-tethered gain-of-function form of Pak kinase does not result in any accumulation of Pak kinase at the synapse. Still, it is possible that Pak kinase is targeted to the synapse via a different mechanism and fails to stabilize in dpix mutants (Parnas, 2001).
In contrast to Pak kinase, Dlg and GluRIIA are not completely eliminated from the synapse in dpix mutants, but rather, their levels are reduced. In the case of Dlg, its localization pattern is also disrupted, indicating that dPix controls the postsynaptic targeting of Dlg at least to some extent, as well as its stabilization at the synapse. The localization pattern of GluRIIA (in subsynaptic domains opposite active zones) is intact. Thus, dPix is not necessary for the synaptic targeting of GluRIIA per se, but rather, it is important for maintenance of its levels and/or stabilization (Parnas, 2001).
Constitutive activation of Galphas in the Drosophila
brain abolishes associative learning, a behavioral disruption far worse than that observed in any single cAMP metabolic mutant, suggesting that Galphas is essential for synaptic plasticity. The intent of this study was to examine the role of Galphas in regulating synaptic function by targeting constitutively active Galphas to either pre- or postsynaptic cells and by examining loss-of-function Galphas mutants (dgs) at the glutamatergic neuromuscular junction (NMJ) model synapse. Surprisingly, both loss of Galphas and activation of Galphas in either pre- or postsynaptic compartment similarly increases basal neurotransmission, decreases short-term plasticity (facilitation and augmentation), and abolished posttetanic potentiation. Elevated synaptic function is specific to an evoked neurotransmission pathway because both spontaneous synaptic vesicle fusion frequency and amplitude are unaltered in all mutants. In the postsynaptic cell, the glutamate receptor field is regulated by Galphas activity; based on immunocytochemical studies, GluRIIA receptor subunits are dramatically downregulated (>75% decrease) in both loss and constitutive active Galphas genotypes. In the presynaptic cell, the synaptic vesicle cycle is regulated by Galphas activity; based on FM1-43 dye imaging studies, evoked vesicle fusion rate is increased in both loss and constitutively active Galphas genotypes. An important conclusion of this study is that both increased and decreased Galphas activity very similarly alters pre- and post-synaptic mechanisms. A second important conclusion is that Galphas activity induces transynaptic signaling; targeted Galphas activation in the presynapse downregulates postsynaptic GluRIIA receptors, whereas targeted Galphas activation in the postsynapse enhances presynaptic vesicle cycling (Renden, 2003).
This study provides no evidence that postsynaptic function is regulated by the level of Galphas activity or that alterations in the postsynaptic glutamate receptor field play any role in short-term plasticity at the Drosophila NMJ. In both gain- and loss-of-function Galphas mutants, there is no substantial change in glutamate receptor conductance, density, or distribution based on mEJC amplitude analyses and direct assay of glutamate-gated currents in the muscle. This finding is extremely surprising in light of the dramatic alteration of the molecular character of the postsynaptic glutamate receptor field in both loss and gain of function Galphas mutants. Two different antibodies were used to assay the GluR fields: a polyclonal antibody against all GluRII subunits showed a significant reduction of signal in all Galphas mutants and a monoclonal antibody specific to GluRIIA showed a nearly complete loss of signal in all Galphas mutants. Immunoreactivity against DGluRIIA in the embryo appeared normal in dgsR60 homozygous mutants, indicating a postembryonic modification of DGluRIIA expression under the control of dgs. At a minimum, these analyses reveal a striking molecular alteration of the GluR field downstream of Galphas, possibly to the extent of nearly eliminating GluRIIA subunits (Renden, 2003).
Complete loss of GluRIIA has been shown to cause significantly decreased mEJC amplitudes, whereas a nearly complete loss of GluRIIA immunoreactivity, using two antibodies, is reported here without a similar change in mEJC amplitudes. One way to rationalize this apparent contradiction is to postulate that the reduced presence of GluRIIA after Galphas manipulation is not sufficient to alter significantly mEJC kinetics or amplitudes. The present report shows a 75% reduction of receptor abundance, whereas GluRIIA genetic nulls were examined previously. More recently, the effect of graded expression levels of GluRIIA was examined, revealing that low levels of GluRIIA, in the absence of GluRIIB, results in an overcompensation of presynaptic transmitter release, doubling the amplitude of glutamatergic transmission at the NMJ. At higher levels of GluRIIA expression, this phenotype was eliminated. If the levels of DGluRIIB were also downregulated (or eliminated) by altered Galphas signaling, these findings would be in agreement with those of the previous study. A second possibility is that the loss of GluRIIA immunoreactivity caused by Galphas manipulation may represent epitope masking rather than loss of GluRIIA subunits. Extracellular binding of an auxiliary protein to glutamate receptors has recently been reported in C. elegans, and an essential auxiliary subunit of mammalian AMPA receptors (stargazin) has recently been found. Stargazin is essential for proper insertion and localization of receptors with the postsynaptic density and is modulated by PKA phosphorylation, thereby controlling receptor number. Interaction with such proteins, or other changes in the accessibility/confirmation of GluRIIA in the postsynaptic compartment, might alter its recognition by antibodies. A final possibility is that there may be compensatory increases in the levels of the other GluRII subunits present at the NMJ. Such a compensatory mechanism might permit loss of GluRIIA subunits without an appreciable change in mEJC amplitudes. The loss of GluRIIA immunoreactivity demonstrates conclusively that the postsynaptic GluR field is strikingly controlled by the level of Galphas activity, but the functional significance of this regulation remains elusive and awaits further investigation (Renden, 2003).
Numerous lines of evidence have demonstrated the existence of both anterograde and retrograde transynaptic signals at the Drosophila NMJ. Such signals are involved in induction of postsynaptic receptor fields, pruning of postsynaptic receptor fields, and compensatory regulation of presynaptic quantal content. The present study shows that increasing Galphas function either pre- or postsynaptically results in nearly identical phenotypes, and independent assays of presynaptic and postsynaptic function indicate similar mechanisms. Specifically, presynaptic Galphas activation modifies the postsynaptic GluRIIA receptor field, and postsynaptic Galphas activation heightens presynaptic vesicle cycling. Moreover, global loss-of-function Galphas mutants also modify the postsynaptic GluRIIA field. Are these paired pre- and post-synaptic alterations a form of compensation or are they independent, Galphas-dependent mechanisms? What signals are used to communicate the level of Galphas activity in both directions across the synaptic cleft (Renden, 2003)?
The identity of the messenger(s) is still unclear, but there are a few likely suspects. Glutamate itself has been shown to act as an anterograde regulatory message at the Drosophila NMJ; presynaptic glutamatergic tone inversely controls the levels of DGluRIIA postsynaptically. Thus it is possible that the elevated glutamatergic transmission in Galphas mutants directly causes the downregulation of GluRIIA expression. At the Drosophila NMJ and in mammalian systems, integrin function has been shown to be required for functional synaptic plasticity. Integrins are known to signal between cells within a short period of time through activation of associated intracellular cascades. At the Drosophila NMJ, the hypertonicity response is mediated in part by integrins dependent on intracellular cAMP levels, and in Xenopus cultured neurons, PKA-dependent transmission is inhibited by disintegrin. These studies suggest that integrins may function as anterograde and/or retrograde messengers mediating physical transynaptic signaling. Another possible retrograde messenger is nitric oxide, produced by phosphorylation of nitric oxide synthase (NOS). There is evidence that NOS is present in Drosophila and is localized to epithelial and neuronal tissues . Application of nitric oxide to the NMJ induces presynaptic vesicle fusion, making it a formal candidate as a retrograde messenger (Renden, 2003).
In conclusion, tissue-specific expression of constitutively active Galphas on either side of the Drosophila NMJ synaptic cleft greatly enhances basal neurotransmission to disrupt expression of short-term synaptic plasticity, specifically in low [Ca2+]bath conditions. This Galphas-dependent alteration does not affect the probability of spontaneous vesicle fusion or the basal function of the postsynaptic receptor field and so is specific to evoked release of neurotransmitter. Increases in Galphas activity on either side of the synapse greatly increases evoked amplitude in low Ca2+, primarily due to a cAMP-dependent increased synaptic vesicle mobility, but also dramatically reduce GluRIIA receptor levels. When Galphas activity is decreased, neurotransmission is similarly enhanced, GluRIIA receptor levels are similarly downregulated, but synaptic vesicle mobility is not detectably altered. It is clear that there is a bidirectional transynaptic communication network at the Drosophila NMJ that responds to altered Galphas activity to modify both pre- and post-synaptic compartments. However, the functional significance of some of these changes remains unclear, and the messengers mediating transynaptic signaling remain to be identified (Renden, 2003).
The subunit composition of postsynaptic neurotransmitter receptors is a key determinant of synaptic physiology. Two glutamate receptor subunits, Drosophila glutamate receptor IIA (DGluRIIA) and DGluRIIB, are expressed at the Drosophila neuromuscular junction and are redundant for viability, yet differ in their physiological properties. A third glutamate receptor subunit at the Drosophila neuromuscular junction, DGluRIII, has been identified that is essential for viability. DGluRIII is required for the synaptic localization of DGluRIIA and DGluRIIB and for synaptic transmission. Either DGluRIIA or DGluRIIB, but not both, is required for the synaptic localization of DGluRIII. DGluRIIA and DGluRIIB compete with each other for access to DGluRIII and subsequent localization to the synapse. These results are consistent with a model of a multimeric receptor in which DGluRIII is an essential component. At single postsynaptic cells that receive innervation from multiple motoneurons, DGluRIII is abundant at all synapses. However, DGluRIIA and DGluRIIB are differentially localized at the postsynaptic density opposite distinct motoneurons. Hence, innervating motoneurons may regulate the subunit composition of their receptor fields within a shared postsynaptic cell. The capacity of presynaptic inputs to shape the subunit composition of postsynaptic receptors could be an important mechanism for synapse-specific regulation of synaptic function and plasticity (Marrus, 2004a).
The localization of glutamate receptors is essential for the formation and plasticity of excitatory synapses. These receptors cluster opposite neurotransmitter release sites of glutamatergic neurons, but these release sites have heterogeneous structural and functional properties. At the Drosophila neuromuscular junction, receptors expressed in a single postsynaptic cell are confronted with an array of hundreds of apposed active zones. Hence, this is an ideal preparation for the investigation of whether receptor clustering is sensitive to the morphological and physiological properties of the apposed active zones. To investigate the relationship between the localization of glutamate receptors and the properties of the apposed active zones, receptor localization was investigated in mutants in which receptors are limited. It was found that receptors are not uniformly distributed opposite the full array of active zones but that some active zones have a disproportionately large share of receptors as assayed by receptor levels and response to transmitter. The active zones at which receptors preferentially cluster are larger and have a higher neurotransmitter release probability than the average active zone. A similar relationship is found between glutamate receptor clusters and active-zone size at wild-type synapses.
It is concluded that when confronted with an array of active zones, glutamate receptors preferentially cluster opposite the largest and most physiologically active sites. These results suggest an activity-dependent matching of pre- and post-synaptic function at the level of a single active zone (Marrus, 2004b).
A novel ionotropic glutamate receptor, DGluRIII, has been identified and characterized at the Drosophila NMJ (Marrus, 2005a). DGluRIII is an essential subunit that is required for the synaptic localization of the two previously described receptors, DGluRIIA and DGluRIIB. A strong hypomorphic mutant was generated that expresses low levels of wild-type DGluRIII protein. In this mutant, where receptor levels are limited, glutamate receptors are found to localize opposite the largest and most physiologically active release sites. These results demonstrate a matching of pre- and post-synaptic function at individual active zones. Such a mechanism could ensure the alignment of receptors opposite functional active zones during development and maximize synaptic strength at this high-fidelity synapse (Marrus, 2004b).
A strong hypomorphic mutation of DGluRIII was generated (Marrus, 2004a) by rescuing a genetic null for DGluRIII with low-level expression of wild type DGluRIII protein (referred to as the DGluRIII mutant). Analysis of glutamate receptor (GluR) expression in this DGluRIII mutant revealed faint puncta of staining within the synaptic region (Marrus, 2004a). To investigate whether these puncta could represent functional receptors, it was first asked whether they localize opposite active zones. NMJs were double stained for an active-zone marker (Wucherpfennig, 2003) and DGluRIII in wild-type and DGluRIII mutant larvae. DGluRIII staining is dramatically reduced in the mutant, but those puncta that are visible colocalize with active zones. Because DGluRIII is thought to function as a component of a heteromultimeric glutamate receptor (Marrus, 2004a), double staining was performed for DGluRIII and a second subunit, DGluRIIA. Each punctum of DGluRIII colocalizes with DGluRIIA. Because the DGluRIII receptor puncta in the DGluRIII mutant localize opposite presynaptic release sites and appear to coassemble with other glutamate receptor subunits, they likely represent functional glutamate receptor complexes (Marrus, 2004b).
To quantify the reduction in DGluRIII levels in the DGluRIII mutant, the intensity of DGluRIII immunoreactivity opposite each active zone was measured. There is an 18-fold reduction in DGluRIII levels in the mutant. Although the intensity of all receptor puncta is down, it appears that some active zones have relatively high levels of apposed receptors, whereas many others have no detectable receptor. It was enquired whether residual receptors are uniformly localized opposite active zones or, alternatively, whether receptors preferentially cluster opposite certain active zones. To investigate this question, the distribution of glutamate receptor intensities was compared opposite each active zone from wild-type and mutant synapses. If receptors are allocated opposite active zones in the same manner in the mutant as in the wild-type, then scaling the wild-type distribution by 18 (the difference in mean intensities) should mimic the mutant distribution. However, these distributions are significantly different. The mutant has many more active zones with relatively brighter apposed GluR puncta, and many more with no detectable GluR puncta, than would be expected from a uniform 18-fold scaling. It is concluded that receptors preferentially localize opposite certain active zones in the mutant (Marrus, 2004b).
To investigate the function of these receptors, electrophysiology was performed at the NMJ. To assess the function of the entire postsynaptic receptor field, ionophoretic glutamate was applied to the synapses. The ionophoretic response in the DGluRIII mutant is approximately 13-fold less than that of wild-type larvae. Although the precise amplitude of ionophoretic responses is somewhat variable, there is good agreement between the reduction in receptor function (13-fold) and receptor staining (18-fold). This observation suggests that there are no unknown receptors that are capable of mediating a substantial glutamate response in the DGluRIII mutant (Marrus, 2004b).
It is concluded that when receptors are limited, receptor patches are most likely to cluster opposite the largest and most active release sites. Even when receptors are in excess, there is a correlation between the density of postsynaptic receptors and the size of the apposed active zone. These findings suggest a model of activity-dependent matching of the functional properties of the pre- and postsynaptic specializations at individual release sites (Marrus, 2004b).
A number of findings support this model. (1) In the DGluRIII mutant the remaining receptors are not evenly distributed opposite each active zone. Instead, certain receptor puncta contain a disproportionate number of receptors as assayed by immunocytochemistry and electrophysiology. (2) Sites with more receptors are located opposite larger active zones. The range of sizes in active zones in the mutant is very similar to that in the wild-type, but receptors are preferentially located opposite the larger active zones. For hippocampal neurons, the largest active zones have been found to have the highest probability of release. (3) In the DGluRIII mutant, the EJC saturates at a lower calcium level than does the wild-type EJC, even though presynaptic release is not saturated. This change in the calcium dependence of the EJC cannot be explained by postsynaptic saturation of a limited pool of receptors that are evenly distributed across the array of active zones. Release of a single vesicle may saturate the receptors in the apposed puncta (explaining the decrease in mEJP amplitude). However, the postsynaptic response to each release site is independent, so this type of saturation would affect the EJC in low and high calcium equally. Instead, the disparity in release rates and EJC amplitude at different calcium concentrations strongly suggests that there is preferential localization of receptors opposite sites of relatively higher release rates. If most receptors are utilized at lower calcium levels, then the progressive recruitment of additional release sites at higher calcium levels will have a reduced impact on EJC amplitude as a result of the relative paucity of receptors at these sites. (4) At the wild-type synapse there is a correlation between the brightest receptor puncta and the largest active zones. It has been demonstrated that the intensity of glutamate receptor staining correlates with both the amount and function of glutamate receptors (Marrus, 2004a). Based on the results from the DGluRIII mutant, it is suggested that these larger active zones at the wild-type synapse are also more active. The physiological dissociation of postsynaptic response and presynaptic release is not seen at the wild-type synapse because there are a sufficient number of receptors to localize opposite each active zone. Nonetheless, the correlation between receptor levels and active-zone size suggests that this matching mechanism functions during normal synaptic development and is not exclusively a homeostatic compensation used by the DGluRIII mutant (Marrus, 2004b).
Three possible mechanisms are considered for the matching of presynaptic release with postsynaptic receptor levels. (1) Presynaptic activity, potentially mediated by neurotransmitter release, could promote the local clustering or stabilization of apposed receptors. More active release sites would be more likely to activate postsynaptic receptors that would, in turn, promote the retention or growth of that receptor patch. During initial synapse formation, there is evidence that presynaptic activity does regulate the global localization of glutamate receptors to the synaptic region. The role of neurotransmitter release in regulating this global localization of receptors in the embryo is controversial, but its role during synaptic growth has not been investigated. (2) The second model postulates a retrograde specification of presynaptic release properties. In this view, larger receptor patches may initially localize opposite active zones in a random manner but then induce an increase in presynaptic function during development. There is good evidence for retrograde regulation of presynaptic properties at this synapse, but there is no evidence that such mechanisms act locally at a single active zone. (3) It is possible that the matching of pre- and postsynaptic functional properties is not, in fact, activity dependent. A transynaptic signal could coordinately regulate pre- and post-synaptic development and thus simultaneously control the function of the pre- and post-synaptic specializations. Although this third model is a formal possibility, the more parsimonious explanation, that the matching of activity levels is an activity-dependent process, is preferred (Marrus, 2004b).
What might be the purpose of an activity-dependent matching of pre- and post-synaptic function at individual release sites? During development, it would be an effective fail-safe mechanism for ensuring that receptors only localize opposite properly developed, i.e., functional, release sites. In addition, for synapses such as the NMJ, which demands high-fidelity synaptic transmission, it would increase synaptic strength by placing the most receptors at the sites of highest release (Marrus, 2004b).
A Drosophila forward genetic screen for mutants with defective synaptic
development has been identified and termed bad reception (brec). Homozygous brec mutants are
embryonic lethal, paralyzed, and show no detectable synaptic transmission at the
glutamatergic neuromuscular junction (NMJ). Genetic mapping, complementation
tests, and genomic sequencing show that brec mutations disrupt a previously
uncharacterized ionotropic glutamate receptor subunit, named here 'GluRIID.'
GluRIID is expressed in the postsynaptic domain of the NMJ, as well as widely
throughout the synaptic neuropil of the CNS. In the NMJ of null brec mutants,
all known glutamate receptor subunits are undetectable by immunocytochemistry,
and all functional glutamate receptors are eliminated. Thus, it is concluded that
GluRIID is essential for the assembly and/or stabilization of glutamate
receptors in the NMJ. In null brec mutant embryos, the frequency of periodic
excitatory currents in motor neurons is significantly reduced, demonstrating
that CNS motor pattern activity is regulated by GluRIID. Although synaptic
development and molecular differentiation appear otherwise unperturbed in null
mutants, viable hypomorphic brec mutants display dramatically undergrown NMJs by
the end of larval development, suggesting that GluRIID-dependent central pattern
activity regulates peripheral synaptic growth. These studies reveal GluRIID as a
newly identified glutamate receptor subunit that is essential for glutamate
receptor assembly/stabilization in the peripheral NMJ and required for properly
patterned motor output in the CNS (Featherstone, 2005 ).
The Drosophila genome encodes 30 putative ionotropic glutamate receptor subunits
(Littleton, 2000), but only 21 genes contain amino acid sequences
thought to be required for pore formation (Sprengel, 2001). Three genes,
called 'GluRIIA,' 'GluRIIB,' and 'GluRIII' (also known as 'GluRIIC') (Sprengel,
2001), have been shown to encode functional ionotropic glutamate
receptor subunits localized to the NMJ. GluRIIC null mutants are embryonic lethal, and
strong hypomorphs have many fewer GluRs at the larval NMJ (Marrus,
2004a; Marrus, 2004b). GluRIIA null mutants are viable but display reduced
receptor channel open time, smaller miniature excitatory junction potentials,
and reduced sensitivity to the antagonist argiotoxin 636.
GluRIIB null mutants are also viable but show no
significant change in receptor function (DiAntonio, 1999), suggesting
that GluRIIB is less important for channel function or that most native
receptors lack GluRIIB. Simultaneous deletion of both GluRIIA and GluRIIB causes
embryonic lethality (Petersen, 1997; DiAntonio, 1999) and a
presumed complete loss of functional glutamate receptors. Antibody staining
suggests that GluRIIA and GluRIIB occupy adjacent partially overlapping domains
(Marrus, 2004b; Chen, 2005), indicating that at least
some receptors contain either GluRIIA or GluRIIB but not both. Thus, it has been
proposed that glutamate receptors at the Drosophila NMJ are composed of GluRIIC
plus either GluRIIA or GluRIIB (Featherstone, 2005).
Another study (G. Qin, 2005) supports the findings reported here
and also introduces a fifth NMJ subunit, GluRIIE. Thus, the Drosophila NMJ
contains, at least, five different ionotropic glutamate receptor subunits, each
encoded by a different gene: GluRIIA, GluRIIB, GluRIIC, GluRIID, and GluRIIE.
Null mutations in GluRIIC, GluRIID, and GluRIIE each cause embryonic lethality,
loss of functional NMJ glutamate receptors, and decreased immunoreactivity for
other subunits. This
suggests that GluRIIC, GluRIID, and GluRIIE are essential subunits contained by
each glutamate receptor at the NMJ. In contrast, GluRIIA and GluRIIB are each
individually dispensable, although at least one of these subunits is required
for a functional receptor because deletion of both GluRIIA and GluRIIB is lethal.
The subunit stoichiometry of
mammalian non-NMDA glutamate receptors has never been definitively solved, but
recent evidence from partial crystal structures strongly suggests that each
ionotropic glutamate receptor is a 'dimer of dimers', e.g., composed of four
subunits (Sun, 2002; Gouaux, 2004; Mayer, 2004). If
Drosophila NMJ glutamate receptors are similarly tetrameric, then all existing
data suggest that they are heterotetramers composed of one GluRIIC subunit, one
GluRIID subunit, and one GluRIIE subunit, plus either one subunit of GluRIIA or
one subunit of GluRIIB (but not both GluRIIA and GluRIIB). In other words, the
Drosophila NMJ contains two subclasses of ionotropic glutamate receptor: (1)
GluRIIA-containing receptors and (2) GluRIIB-containing receptors. This model is
consistent with immunocytochemical and genetic results: (1) immunoreactivity for
GluRIIA and GluRIIB is segregated such that clusters appear to contain either
GluRIIA or GluRIIB but not both (Marrus, 2004b; Chen,
2005); (2) only some GluRIID clusters are immunoreactive for GluRIIA (Chen, 2005),
and (3) GluRIIA and GluRIIB are
differentially trafficked and stabilized (Chen, 2005). If GluRIIA and GluRIIB can
be found in the same receptor (which presumably also contains the required
subunits GluRIIC, GluRIID, and GluRIIE), then it must be concluded that Drosophila
NMJ glutamate receptors are likely pentameric (Featherstone, 2005).
Because at least four different GluR subunits are required in the Drosophila NMJ
in vivo, it suggests that there are at least four distinct subunit-dependent
requirements for receptor assembly, trafficking, and/or stabilization. It is not
clear how the different subunits play these roles. Amino acid sequence alignment
shows that GluRIIA, GluRIIB, GluRIIC, GluRIID, and GluRIIE subunits differ most
from each other near their N termini, a region that is known to be involved in
ligand binding and possibly receptor assembly (Gouaux, 2004; Mayer, 2004).
However, GluRIIA and GluRIIB show no obvious similarity in
this region to explain why they might to be able to substitute for each other.
GluRIIC has a class II C-terminal consensus PDZ (PSD-95/DLG/zona
occludens-1)-binding domain (Marrus, 2004b), suggesting that GluRIIC might
have a unique anchoring role. Neither GluRIIA nor GluRIIB contains recognizable
PDZ-binding motifs, although stabilization of GluRIIB-containing receptors
requires (apparently indirectly) the presence of the PDZ domain protein DLG
(Chen, 2005). Thus, it remains unclear how individual
Drosophila GluR subunits contribute to receptor assembly and function. For
mammalian receptors, answers to this question have typically been sought using
heterologously expressed receptor subunits. However, this study suggests that
there may be important differences in the mechanisms of receptor assembly and
function in vivo. GluRIIA forms functional homomeric receptors when expressed in
Xenopus oocytes, but in vivo overexpression of GluRIIA
in muscle is essentially unable to overcome the requirement for GluRIID or form
functional GluRs. It is conceivable that Xenopus oocytes
contain endogenous proteins similar to kainite receptor subunits, and these
proteins are sufficient for GluRIIA assembly and/or stabilization. Indeed,
Xenopus oocytes are known to contain an endogenous protein (XenU1) that can
substitute for the mammalian NMDA receptor subunit NR2.
Alternatively, homomeric GluRIIA receptors may be only very inefficiently
formed in oocytes, similar to the in vivo situation, but this inefficiency is
not apparent outside of a synaptic context. Heterologous expression of other
Drosophila NMJ GluR subunits has not been reported. Thus, these results suggest
caution when interpreting some results using expressed subunits and highlight
the importance of in vivo studies (Featherstone, 2005).
This study also suggests a functional role for glutamate
receptors in the Drosophila CNS. Uniquely among known fly GluR subunits, GluRIID
is expressed both in the NMJ and at central synapses. Excitatory transmission in
the Drosophila CNS is thought to be predominantly cholinergic, although in situ
data for several putative ionotropic glutamate receptor subunits shows that many
subunits are expressed in the CNS. GluRIID is expressed at high levels throughout the synaptic
neuropil of the ventral nerve cord, indicating that glutamatergic synapses in
Drosophila might be much more widespread and pervasive than has been speculated
previously. Consistent with this, it is shown that, in the absence of GluRIID, there
is severe disruption of endogenous central motor pattern output. Interestingly,
the only glutamategated responses that have been demonstrated in Drosophila
neurons are inhibitory; glutamate-gated currents in voltage-clamped larval CNS
neurons are prolonged (2-5 s), reverse at -55mV, and are blocked by picrotoxin.
Nevertheless, GluRIID in the CNS could be part of
an excitatory receptor that remains functionally unidentified. More intriguing
is the possibility that GluRIID is an essential component of a kainate
receptor-like glutamate-gated cation channel in Drosophila muscle but part of a
glutamate-gated anion channel in the CNS. Glutamate-gated currents in embryos
are, unfortunately, so far undetectable in embryos; thus, it has not been possible to
determine whether GluRIID is required for CNS glutamate-gated anion currents.
Although the nature of the GluRIID-containing receptor is unknown, loss of its
function clearly causes dramatic changes in endogenous patterned activity within
motor neurons. In mutants, many motor neurons lack detectable patterned motor
output, and all cells show a striking reduction in the frequency of patterned
motor output. This result minimally demonstrates that GluRIID-dependent
glutamatergic transmission plays a vital modulatory role in controlling motor
output from the CNS (Featherstone, 2005).
Although embryonic synaptogenesis appears normal in the absence of GluRIID,
partial loss of GluRIID in viable brec mutants dramatically reduces
postembryonic synaptic growth and differentiation. The loss of glutamate
receptors in either the CNS or NMJ could cause NMJ morphology defects in two
ways: (1) loss of muscle depolarization could disrupt a retrograde signal that
induces presynaptic growth, or (2) disruption of endogenous central motor
pattern activity could alter electrical activity-dependent presynaptic growth.
In Drosophila, there is not good support for the former mechanism, because
inhibition of muscle depolarization does not detectably alter NMJ arborization.
In contrast, it is well established
that neuronal electrical activity is positively correlated with the growth of
the Drosophila presynaptic motor terminal. Thus the second model is the most
parsimonious explanation. This conclusion raises the exciting prospect that the
endogenous pattern of central electrical activity plays a critical role in
sculpting postembryonic NMJ development (Featherstone, 2005).
NF-κB signaling has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. However, the cellular and molecular activity of NF-κB signaling within the nervous system remains to be clearly defined. This study shows that the NF-κB and IκB homologs Dorsal and Cactus surround postsynaptic glutamate receptor (GluR) clusters at the Drosophila NMJ. Mutations in dorsal, cactus, and IRAK/pelle kinase specifically impair GluR levels, assayed immunohistochemically and electrophysiologically, without affecting NMJ growth, the size of the postsynaptic density, or homeostatic plasticity. Additional genetic experiments support the conclusion that cactus functions in concert with, rather than in opposition to, dorsal and pelle in this process. Finally, evidence is provided that Dorsal and Cactus act posttranscriptionally, outside the nucleus, to control GluR density. Based upon these data it is speculated that Dorsal, Cactus, and Pelle function together, locally at the postsynaptic density, to specify GluR levels (Heckscher, 2007).
NF-κB signaling has been implicated in the mechanisms of neural plasticity, learning, epilepsy, neurodegeneration, and the adaptive response to neuronal injury. The data presented in this study advance the understanding of neuronal NF-κB signaling in two ways. First, multiple lines of evidence are presented that NF-κB/Dorsal signaling is required for the control of GluR density at the NMJ. These data provide a synaptic function for NF-κB signaling that may be directly relevant to the diverse activities ascribed to NF-κB in the nervous system. Second, molecular and genetic evidence is provided that Dorsal, Cactus, and Pelle may function posttranscriptionally, at the postsynaptic side of the NMJ, to specify GluR density during postembryonic development (Heckscher, 2007).
Several independent lines of experimentation suggest that Cactus, Dorsal, and Pelle function together at the PSD to specify GluR density. Evidence is provided that Cactus and Dorsal localize to a similar postsynaptic domain. In addition, overexpression of a GFP-tagged Pelle protein that is sufficient to rescue a pelle mutation, can traffic to the PSD where Cactus and Dorsal reside. Next, genetic evidence is presented that cactus, dorsal, and pelle function together, in the same genetic pathway, to control GluR density. It is particularly surprising that mutations in cactus behave similarly to dorsal and pelle. In other systems (embryonic patterning and immunity), Cactus inhibits Dorsal-mediated transcription by binding and sequestering cytoplasmic Dorsal protein. As a result, in these other systems, cactus mutations cause phenotypes that are opposite to those observed in dorsal mutations. This study used the same cactus and dorsal mutations that previously have been observed to generate the predicted opposing phenotypes during embryonic patterning, and yet it was observed that cactus phenocopies the dorsal mutations. In addition, genetic epistasis experiments indicate that these genes function together to facilitate GluR density. Thus, at the NMJ, Cactus functions in concert with, rather than in opposition to, Dorsal (Heckscher, 2007).
One explanation for this observation could be that Dorsal does not function as a nuclear transcription factor during the control of GluR levels. In support of this idea it has been demonstrated that (1) Dorsal protein is not detected in the nucleus, (2) reporters of Dorsal-dependent transcription fail to show activity in muscle nuclei, and (3) mutation of the Dorsal transactivation domain, dlU5 does not impair GluR abundance even though this same mutation has been shown to impair transcription-dependent patterning during embryogenesis. An alternative explanation for the observation that dorsal and cactus have similar phenotypes at the NMJ could be that Cactus and Dorsal act synergistically to control the transcription of GluRs at the NMJ. Indeed, there is evidence in other systems that IκB can shuttle with NF-κB to the nucleus. A previous study shows Cactus accumulation in Drosophila larval muscle nuclei in a dorsal mutant background (Cantera, 1999). However, this result could not be repeated despite examination of Cactus localization in five allelic combinations of dorsal. Furthermore, the data from vertebrate systems suggest that IκB should shuttle into the nucleus with NF-κB, not in its absence. Thus, a model is favored in which Dorsal and Cactus function together at the postsynaptic membrane to facilitate GluR abundance during development (Heckscher, 2007).
If this model is correct, then it is predicted that NF-κB does not control GluR density through transcriptional regulation. This prediction is supported by two experimental observations: (1) GluR transcript levels (assessed by QT PCR) are not statistically different from wild-type in dorsal and cactus mutations that cause an ~50% decrease in GluR abundance; (2) it was demonstrated that overexpression of a myc-tagged GluRIIA cDNA using a heterologous, muscle-specific promoter is not able to restore synaptic GluRIIA levels in either the cactus or dorsal mutant backgrounds. These data are consistent with Dorsal and Cactus acting posttranscriptionally to control GluR density at the NMJ. There are two general mechanisms by which GluR levels could be controlled posttranscriptionally: (1) altered receptor delivery to the NMJ or (2) altered receptor internalization/degradation. If receptor internalization/degradation were enhanced in the cactus, dorsal, or pelle mutant backgrounds, one might expect GluRIIA-myc overexpression to overcome this change and restore normal receptor levels. In addition, less myc-tagged protein might be seen in the mutants in comparison to wild-type. This is not what was observed. Therefore, the hypothesis is favored that Cactus, Dorsal, and Pelle function together to promote the delivery of glutamate receptors to the NMJ during development (Heckscher, 2007).
The possibility that Cactus, Dorsal, and Pelle act posttranscriptionally to control GluR density raises many questions. For example, do Dorsal and Cactus exist as a protein complex at the PSD? If so, is this complex regulated and how might such a complex influence GluR density? Since pelle kinase-dead mutants impair GluR density, it is possible that Dorsal and Cactus recruit Pelle to the PSD. If so, what are the targets of Pelle kinase that are relevant to establishing or maintaining the proper density of glutamate receptors at the PSD? Finally, the demonstration that cytoplasmic NF-κB/Dorsal can influence GluR density does not rule out the possibility that NF-κB/Dorsal may also translocate to the muscle nucleus at the Drosophila NMJ under certain stimulus conditions. Indeed, in both the vertebrate central and peripheral nervous systems NF-κB is found within neuronal and muscle nuclei, and nuclear translocation can be stimulated by neuronal activity, glutamate, injury, and disease. For nuclear entry of Dorsal, two events must occur: (1) Cactus must be degraded and (2) Dorsal must be phosphorylated. It remains possible that one or both of these criteria are not met during the normal development of the Drosophila NMJ but could be met under as-yet-to-be-identified stimulus conditions. The possibility that NF-κB acts both locally at the synapse and globally via the nucleus is not unique to this signaling pathway. A similar organization has been documented for wingless/wnt signaling where noncanonical cytoplasmic signaling can impact cytoskeletal organization while canonical signaling involves the nuclear translocation of downstream beta-catenin and TCF-dependent gene transcription (Heckscher, 2007).
It remains unknown how NF-κB signaling is activated at the Drosophila NMJ. In Drosophila embryonic patterning and innate immunity, NF-κB signaling is initiated through activation of Toll or Toll-like receptors. There are nine Toll and Toll-like receptors encoded in the Drosophila genome. However, none of these receptors appear to be present in Drosophila larval muscle. The Toll receptor is expressed in a subset of embryonic muscle fibers, but is absent from larval muscle. None of the Toll-like receptors are expressed in Drosophila embryonic muscle and none appear to be expressed in larval muscle. An alternative possibility is that TNF-α receptors activate NF-κB in Drosophila muscle as has been observed in vertebrate skeletal muscle. Indeed, a TNF-α receptor homolog (Wengen) has been identified, and it is expressed in Drosophila skeletal muscle. The possibility that TNF-α signaling is mediated via NF-κB is intriguing given the recent demonstration that TNF-α regulates GluR abundance in the vertebrate central nervous system. In both cultured neurons and hippocampal slices glial-derived TNF-α signaling is required for the increase in postsynaptic AMPA receptor abundance observed following chronic activity blockade. Thus, the current data in combination with work in the vertebrate CNS raise the possibility that a conserved TNFα/NF-κB signaling system controls GluR abundance at both neuromuscular and central synapses during development and in response to chronic activity blockade (Heckscher, 2007).
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Glutamate receptor IIA and Glutamate receptor IIB:
Biological Overview
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| Effects of Mutation
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