Postsynaptic translation affects the efficacy and morphology of neuromuscular junctions.

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. Here we show 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 melanogaster. 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. Our results therefore provide evidence for a postsynaptic translational control of long-term junctional plasticity.

Genetic analysis of the mechanisms controlling target selection: target-derived Fasciclin II regulates the pattern of synapse formation.

In Drosophila, motoneuron growth cones initially probe many potential muscle targets but later withdraw most of these contacts to form stereotypic synapses with only one or a few muscles. Prior to synapse formation, Fasciclin II (Fas II) is expressed at low levels on muscle. During synapse formation, Fas II concentrates at the synapse and disappears from the rest of the muscle. We previously showed that Fas II is required both pre- and postsynaptically for synaptic stabilization. Here, we show that the differential expression of target-derived Fas II has a profound influence on the patterning of synapse formation. A transient increase in muscle Fas II stabilizes growth cone contacts and leads to novel synapses that are functional and stable; targets that normally receive two inputs can now receive up to six inputs. Changing the relative levels of Fas II on neighboring muscles leads to dramatic shifts in target selection.

Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth.

The glutamatergic neuromuscular synapse in Drosophila forms and differentiates into distinct boutons in the embryo and grows by sprouting new boutons throughout larval life. We demonstrate that two axons form approximately 18 boutons on muscles 7 and 6 by hatching and grow to approximately 180 boutons by third instar. We further show that, after synapse formation, the homophilic cell adhesion molecule Fasciclin II (Fas II) is localized both pre- and postsynaptically where it controls synapse stabilization. In FasII null mutants, synapse formation is normal, but boutons then retract during larval development. Synapse elimination and resulting lethality are rescued by transgenes that drive Fas II expression both pre- and postsynaptically; driving Fas II expression on either side alone is insufficient. Fas II can also control synaptic growth; various FasII alleles lead to either an increase or decrease in sprouting, depending upon the level of Fas II.

Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity.

Increased neuronal activity (eag Shaker mutants) and cAMP concentration (dunce mutants) lead to increased synaptic structure and function at the Drosophila neuromuscular junction. Here, we show that the increase in synaptic growth is accompanied by an approximately 50% decrease in synaptic levels of the cell adhesion molecule Fasciclin II (Fas II). This decrease in Fas II is both necessary and sufficient for presynaptic sprouting; FasII mutants that decrease Fas II levels by approximately 50% lead to sprouting similar to eag Shaker and dunce, while transgenes that maintain synaptic Fas II levels suppress sprouting in eag Shaker and dunce. However, FasII mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons.

Genetic dissection of structural and functional components of synaptic plasticity. III. CREB is necessary for presynaptic functional plasticity.

Increased cAMP (in dunce mutants) leads to an increase in the structure and function of the Drosophila neuromuscular junction. Synaptic Fasciclin II (Fas II) controls this structural plasticity, but does not alter synaptic function. Here, we show that CREB, the cAMP response element-binding protein, acts in parallel with Fas II to cause an increase in synaptic strength. Expression of the CREB repressor (dCREB2-b) in the dunce mutant blocks functional but not structural plasticity. Expression of the CREB activator (dCREB2-a) increases synaptic strength only in FasII mutants that increase bouton number. This CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release. Expression of dCREB2-a in a FasII mutant background genetically reconstitutes this cAMP-dependent plasticity. Thus, cAMP initiates parallel changes in CREB and Fas II to achieve long-term synaptic enhancement.

Homeostasis of synaptic transmission in Drosophila with genetically altered nerve terminal morphology.

We present a new test of the hypothesis that synaptic strength is directly related to nerve terminal morphology through analysis of synaptic transmission at Drosophila neuromuscular junctions with a genetically reduced number of nerve terminal varicosities. Synaptic transmission would decrease in target cells with fewer varicosities if there is a relationship between the number of varicosities and the strength of synaptic transmission. Animals that have an extreme hypomorphic allele of the gene for the cell adhesion molecule Fasciclin II possess fewer synapse-bearing nerve terminal varicosities; nevertheless, synaptic strength is maintained at a normal level for the muscle cell as a whole. Fewer failures of neurotransmitter release and larger excitatory junction potentials from individual varicosities, as well as more frequent spontaneous release and larger quantal units, provide evidence for enhancement of transmitter release from varicosities in the mutant. Ultrastructural analysis reveals that mutant nerve terminals have bigger synapses with more active zones per synapse, indicating that synaptic enlargement and an accompanying increase in synaptic complexity provide for more transmitter release at mutant varicosities. These results show that morphological parameters of transmitting nerve terminals can be adjusted to functionally compensate for genetic perturbations, thereby maintaining optimal synaptic transmission.

Glutamate receptors of Drosophila melanogaster. Primary structure of a putative NMDA receptor protein expressed in the head of the adult fly.

The NMDA subtype of ionotropic glutamate receptors has been implicated in the activity-dependent modification of synaptic efficacy in the mammalian brain. Here we describe a cDNA isolated from Drosophila melanogaster which encodes a putative invertebrate NMDA receptor protein (DNMDAR-I). The deduced amino acid sequence of DNMDAR-I displays 46% amino acid identity to the rat NMDAR1 polypeptide and shows significant homology (16-23%) to other vertebrate and invertebrate glutamate receptor proteins. The DNMDAR-I gene maps to position 83AB of chromosome 3R and is highly expressed in the head of adult flies. Our data indicate that the NMDA subtype of glutamate receptors evolved early during phylogeny and suggest the existence of activity-dependent synaptic plasticity in the insect brain.

Molecular analysis of Drosophila glutamate receptors.

Insects and other invertebrates use L-glutamate as a neurotransmitter in the central nervous system and at the neuromuscular junction. In contrast to the well-studied effects of L-glutamate on invertebrate muscle cells, relatively little is known about the physiological role of glutamate receptors (GluRs) in the invertebrate central nervous system. We have applied a molecular cloning approach to elucidate the molecular structure of neuronal and muscle-specific Drosophila glutamate receptor subunits (DGluRs). Several domains conserved between rat GluR subunits and DGluRs indicate regions of high functional significance. Drosophila genetics may now be used as a valuable experimental tool to gain further insight into the role of DGluRs in development, synaptic plasticity and control of gene expression.

Glutamate receptors of Drosophila melanogaster: cloning of a kainate-selective subunit expressed in the central nervous system.

We report the isolation and functional characterization of cDNAs encoding a Drosophila kainate-selective glutamate receptor. The deduced mature 964-residue protein (DGluR-I) is 108,482 Da and exhibits significant homology to mammalian glutamate receptor subunits. Injection of DGluR-I cRNA into Xenopus oocytes generated kainate-operated ion channels which were blocked by the selective non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione and philanthotoxin. DGluR-I transcripts are differentially expressed during Drosophila development and, in late embryogenesis, accumulate in the central nervous system.

Molecular cloning of an invertebrate glutamate receptor subunit expressed in Drosophila muscle.

Insects and other invertebrates use glutamate as a neurotransmitter in the central nervous system and at the neuromuscular junction. A complementary DNA from Drosophila melanogaster, designated DGluR-II, has been isolated that encodes a distant homolog of the cloned mammalian ionotropic glutamate receptor family and is expressed in somatic muscle tissue of Drosophila embryos. Electrophysiological recordings made in Xenopus oocytes that express DGluR-II revealed depolarizing responses to L-glutamate and L-aspartate but low sensitivity to quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate. The DGluR-II protein may represent a distinct glutamate receptor subtype, which shares its structural design with other members of the ionotropic glutamate receptor family.