The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter.

The largest central synapse in adult Drosophila is a mixed electro-chemical synapse whose gap junctions require the product of the shaking-B (shak-B) gene. Shak-B(2) mutant flies lack gap junctions at this synapse, which is between the giant fibre (GF) and the tergotrochanteral motor neuron (TTMn), but it still exhibits a long latency response upon GF stimulation. We have targeted the expression of the light chain of tetanus toxin to the GF, to block chemical transmission, in shak-B(2) flies. The long latency response in the tergotrochanteral muscle (TTM) was abolished indicating that the chemical component of the synapse mediates this response. Attenuation of GAL4-mediated labelling by a cha-GAL80 transgene, reveals the GF to be cholinergic. We have used a temperature-sensitive allele of the choline acetyltransferase gene (cha(ts2)) to block cholinergic synapses in adult flies and this also abolished the long latency response in shak-B(2) flies. Taken together the data provide evidence that both components of this mixed synapse are functional and that the chemical neurotransmitter between the GF and the TTMn is acetylcholine. Our findings show that the two components of this synapse can be separated to allow further studies into the mechanisms by which mixed synapses are built and function.

Targeted expression of shibire ts and semaphorin 1a reveals critical periods for synapse formation in the giant fiber of Drosophila.

In order to determine the timing of events during the assembly of a neural circuit in Drosophila we targeted expression of the temperature-sensitive shibire gene to the giant fiber system and then disrupted endocytosis at various times during development. The giant fiber retracted its axon or incipient synapses when endocytosis was blocked at critical times, and we perceived four phases to giant fiber development: an early pathfinding phase, an intermediate phase of synaptogenesis, a late stabilization process and, finally, a mature synapse. By co-expressing shibire(ts) and semaphorin 1a we provided evidence that Semaphorin 1a was one of the proteins being regulated by endocytosis and its removal was a necessary part of the program for synaptogenesis. Temporal control of targeted expression of the semaphorin 1a gene showed that acute excess Semaphorin 1a had a permanent disruptive effect on synapse formation.

A role for Drosophila Drac1 in neurite outgrowth and synaptogenesis in the giant fiber system.

Recent studies have shown the small GTPases, Rac1, Rho, and CDC42, to have a role in axon guidance. To assess their participation in synapse assembly and function we have expressed various forms of Drac1 in the giant fiber system of Drosophila. Overexpression of wild-type Drac1 in the giant fiber (GF) lead to a disruption in axonal morphology; axons often terminate prematurely in a large swelling in the target area but lack the normal lateral bend where the synapse with the jump motor neuron would normally be found. Electrophysiological assays revealed longer latencies and lowering following frequencies indicating defects in the synapse between the GF and the tergotrochanteral motor neuron (TTMn). Thickened abnormal GF dendrites were also observed in the brain. Overexpression of the dominant-negative form of Drac1, (N17), resulted in axons that produced extra branches in the second thoracic neuromere (T2); however, the synaptic connection to the TTMn was present and functioned normally. Conversely, expression of the constitutively active form, Drac1(V12), resulted in a complete lack of neurite outgrowth and this was also seen with overexpression of Dcdc42(V12). In the absence of a GF, these flies showed no response in the jump (TTM) or flight (DLM) muscles upon brain stimulation. Taken together these results show that the balance of actin polymerization and depolymerization determines local process outgrowth and thereby synapse structure and function.

Targeted expression of truncated glued disrupts giant fiber synapse formation in Drosophila.

Glued(1) (Gl(1)) mutants produce a truncated protein that acts as a poison subunit and disables the cytoplasmic retrograde motor dynein. Heterozygous mutants have axonal defects in the adult eye and the nervous system. Here we show that selective expression of the poison subunit in neurons of the giant fiber (GF) system disrupts synaptogenesis between the GF and one of its targets, the tergotrochanteral motorneuron (TTMn). Growth and pathfinding by the GF axon and the TTMn dendrite are normal, but the terminal of the GF axon fails to develop normally and becomes swollen with large vesicles. This is a presynaptic defect because expression of truncated Glued restricted to the GF results in the same defect. When tested electrophysiologically, the flies with abnormal axons show a weakened or absent GF-TTMn connection. In Glued(1) heterozygotes, GF-TTMn synapse formation appears morphologically normal, but adult flies show abnormal responses to repetitive stimuli. This physiological effect is also observed when tetanus toxin is expressed in the GFs. Because the GF-TTMn is thought to be a mixed electrochemical synapse, the results show that Glued has a role in assembling both the chemical and electrical components. We speculate that disrupting transport of a retrograde signal disrupts synapse formation and maturation.

Dynein-dynactin function and sensory axon growth during Drosophila metamorphosis: A role for retrograde motors.

Mutations in the genes for components of the dynein-dynactin complex disrupt axon path finding and synaptogenesis during metamorphosis in the Drosophila central nervous system. In order to better understand the functions of this retrograde motor in nervous system assembly, we analyzed the path finding and arborization of sensory axons during metamorphosis in wild-type and mutant backgrounds. In wild-type specimens the sensory axons first reach the CNS 6-12 h after puparium formation and elaborate their terminal arborizations over the next 48 h. In Glued1 and Cytoplasmic dynein light chain mutants, proprioceptive and tactile axons arrive at the CNS on time but exhibit defects in terminal arborizations that increase in severity up to 48 h after puparium formation. The results show that axon growth occurs on schedule in these mutants but the final process of terminal branching, synaptogenesis, and stabilization of these sensory axons requires the dynein-dynactin complex. Since this complex functions as a retrograde motor, we suggest that a retrograde signal needs to be transported to the nucleus for the proper termination of some sensory neurons.

Target neuron specification of short-term synaptic facilitation and depression in the cricket CNS.

We investigated the role of retrograde signals in the regulation of short-term synaptic depression and facilitation by characterizing the form of plasticity expressed at novel synapses on four giant interneurons in the cricket cercal sensory system. We induced the formation of novel synapses by transplanting a mesothoracic leg and its associated sensory neurons to the cricket terminal abdominal segment. Axons of ectopic leg sensory neurons regenerated and innervated the host terminal abdominal ganglion forming monosynaptic connections with the medial giant interneuron (MGI), lateral giant interneuron (LGI), and interneurons 7-1a and 9-2a. The plasticity expressed by these synapses was characterized by stimulating a sensory neuron with pairs of stimuli at various frequencies or with trains of 10 stimuli delivered at 100 Hz and measuring the change in excitatory postsynaptic potential amplitude recorded in the postsynaptic neuron. Novel synapses of a leg tactile hair on 7-1a depressed, as did control synapses of cercal sensory neurons on this interneuron. Novel synapses of leg campaniform sensilla (CS) sensory neurons on MGI, like MGI's control synapses, always facilitated. The form of plasticity expressed by novel synapses is thus consistent with that observed at control synapses. Leg CS synapses with 9-2a also facilitated; however, the plasticity expressed by these sensory neurons is dependent on the identity of the postsynaptic cell since the synapses these same sensory neurons formed with LGI always depressed. We conclude that the form of plasticity expressed at these synaptic connections is determined retrogradely by the postsynaptic cell.

Presynaptic calcium/calmodulin-dependent protein kinase II regulates habituation of a simple reflex in adult Drosophila.

On repetitive stimulation, the strength of a reflex controlling leg position in Drosophila decreased, and this response decrement conformed to the parametric features of habituation. To study the presynaptic function of CaMKII in this nonassociative form of learning, we used a P[Gal4] insertion line to target the expression of mutant forms of CaMKII to the sensory neurons controlling the reflex. Targeted expression of a calcium-independent CaMKII construct (T287D) in the sensory neurons eliminated habituation. Targeted expression of a mutant CaMKII incapable of achieving calcium independence (T287A) reduced the initial reflex response, but a strong facilitation then occurred, and this eliminated most of the habituation. Finally, when a CaMKII inhibitory peptide (ala) was expressed in sensory neurons, the initial response was reduced, followed by facilitation. These results suggest that basal CaMKII levels in the presynaptic neurons set the response level and dynamics of the entire neural circuit.

Mutant molecular motors disrupt neural circuits in Drosophila.

A dominant negative mutation, Glued1, that codes for a component of the dynactin complex, disrupted the axonal anatomy of leg sensory neurons in Drosophila. To examine neuron structure in mutant animals, a P[Gal4] enhancer trap targeted expression of lacZ to the sensory neurons and thereby labeled neurons in the femoral chordotonal organ and their axons within the central nervous system. When these sensory axons were examined in the Glued1 mutant specimens, they were observed to arborize abnormally. This anatomical disruption of the sensory axons was associated with a corresponding disruption in a reflex. Normally, the tibial extensor motor neurons were excited when the femoral-tibial joint was flexed, but this resistance reflex was nearly absent in mutant animals. We used the P[Gal4] insertion strains to target expression of tetanus toxin light chain to these sensory neurons in wild-type animals and showed that this blocked the resistance reflex and produced a phenocopy of the Glued result. We conclude that disruption of the dynein-dynactin complex disrupts sensory axon path finding during metamorphosis, and this in turn disrupts synaptic connectivity.

The shaking-B2 mutation disrupts electrical synapses in a flight circuit in adult Drosophila.

The shaking-B2 mutation was used to analyze synapses between haltere afferents and a flight motoneuron in adult Drosophila. We show that the electrical synapses among many neurons in the flight circuit are disrupted in shaking-B2 flies, suggesting that shaking-B expression is required for electrical synapses throughout the nervous system. In wild-type flies haltere afferents are dye-coupled to the first basalar motoneuron, and stimulation of these afferents evokes electromyograms from the first basalar muscle with short latencies. In shaking-B2 flies dye coupling between haltere afferents and the motoneuron is abolished, and afferent stimulation evokes electromyograms at abnormally long latencies. Intracellular recordings from the motoneuron confirm that the site of the defect in shaking-B2 flies is at the synapses between haltere afferents and the flight motoneuron. The nicotinic cholinergic antagonist mecamylamine blocks the haltere-to-flight motoneuron synapses in shaking-B2 flies but does not block those synapses in wild-type flies. Together, these results show that the haltere-to-flight motoneuron synapses comprise an electrical component that requires shaking-B and a chemical component that is likely to be cholinergic.

Mutations in the 8 kDa dynein light chain gene disrupt sensory axon projections in the Drosophila imaginal CNS.

Mutations in an 8 kDa (8x10(3) Mr) cytoplasmic dynein light chain disrupt sensory axon trajectories in the imaginal nervous system of Drosophila. Weak alleles are behaviorally mutant, female-sterile and exhibit bristle thinning and bristle loss. Null alleles are lethal in late pupal stages and alter neuronal anatomy within the imaginal CNS. We utilized P[Gal4] inserts to examine the axon projections of stretch receptor neurons and an engrailed-lacZ construct to characterize the anatomy of tactile neurons. In mutant animals both types of sensory neurons exhibited altered axon trajectories within the CNS, suggesting a defect in axon pathfinding. However, the alterations in axon trajectory did not prevent these axons from reaching their normal termination regions. In the alleles producing these neuronal phenotypes, expression of the cytoplasmic dynein 8 kDa light chain gene is completely absent. These results demonstrate a new function for the cytoplasmic dynein light chain in the regulation of axonogenesis and may provide a point of entry for studies of the role of cellular motors in growth cone guidance.

The effect of neuronal growth on synaptic integration.

The way in which the dimensions of neurons change during postembryonic development has important effects on their electrotonic structures. Theoretically, only one mode of growth can conserve the electrotonic structures of growing neurons without employing changes in membrane electrical properties. If the dendritic diameters of a neuron increase as the square of the increase in dendritic lengths, then the neuron's electrotonic structure is conserved. We call this special mode of allometric growth "isoelectrotonic growth." In this study we compared the developmental changes in morphology of two identified invertebrate neurons with theoretical growth curves. We found that a cricket neuron, MGI, grows isoelectrotonically and thereby preserves its electrotonic properties. In contrast, the crayfish neuron, LG, grows in nearly isometric manner resulting in an increase in its electrotonic length.

Retrograde signaling and the development of transmitter release properties in the invertebrate nervous system.

The dynamics of presynaptic transmitter release are often matched to the functional properties of the postsynaptic cell. In organisms ranging from cats to crickets, evidence suggests that retrograde signaling is essential for matching these presynaptic release properties to individual postsynaptic partners. Retrograde interactions appear to control the development of presynaptic, short-term facilitation and depression.

Long-term regulation of short-term transmitter release properties: retrograde signaling and synaptic development.

The dynamics of presynaptic transmitter release are often matched to the physiological properties and functions of the postsynaptic cell. In organisms ranging from cats to crickets, evidence suggests that retrograde signaling is essential for matching these presynaptic release properties to individual postsynaptic partners. Retrograde interactions appear to control the development of presynaptic, short-term facilitation and homosynaptic depression through local, retrograde signaling at the synapse.

Transplantation of neurons reveals processing areas and rules for synaptic connectivity in the cricket nervous system.

In order to assess the nature of spatial cues in determining the characteristic projection sites of sensory neurons in the CNS, we have transplanted sensory neurons of the cricket Acheta domesticus to ectopic locations. Thoracic campaniform sensilla (CS) function as proprioceptors and project to an intermediate layer of neuropil in thoracic ganglia while cercal CS transduce tactile information and project into a ventral layer in the terminal abdominal ganglion (TAG). When transplanted to ectopic locations, these afferents retain their modality-specific projection in the host ganglion and terminate in the layer of neuropil homologous to that of their ganglion of origin. Thus, thoracic CS neurons project to intermediate neuropil when transplanted to the abdomen and cercal CS neurons project to a ventral layer of neuropil when transplanted to the thorax. We conclude that CS can be separated into two classes based on their characteristic axonal projections within each segmental ganglion. We also found that the sensory neurons innervating tactile hairs project to ventral neuropil in any ganglion they encounter after transplantation. Ectopic sensory neurons can form functional synaptic connections with identified interneurons located within the host ganglia. The new contacts formed by these ectopic sensory neurons can be with normal targets, which arborize within the same layer of neuropil in each segmental ganglion, or with novel targets, which lack dendrites in the normal ganglion and are thus normally unavailable for synaptogenesis. These observations suggest that a limited set of molecular markers are utilized for cell-cell recognition in each segmentally homologous ganglion. Regenerating sensory neurons can recognize novel postsynaptic neurons if they have dendrites in the appropriate layer of neuropil. We suggest that spatial constraints produced by the segmentation and the modality-specific layering of the nervous system have a pivotal role in determining synaptic specificity.

A role for postsynaptic neurons in determining presynaptic release properties in the cricket CNS: evidence for retrograde control of facilitation.

Intracellular recording sin the cricket cercal system show that the synaptic terminals of a single sensory neuron can facilitate at one target, the medial giant interneuron (MGI), and simultaneously depress at another target, interneuron 10-3. A quantal analysis of transmission at these synapses demonstrates that facilitation and depression are properties of the presynaptic cell. For facilitating synapses contacting MGI, the mean quantal content (m), determined from the probability of the failures, increases for the second EPSP, while the quantal size (q) remains constant. Similarly, an analysis of depression for those synapses contacting 10-3 supports a presynaptic mechanism for depression. Since facilitation and depression are presynaptic and their expression at the synapses of a single, identified sensory neuron are correlated with the target interneuron, we conclude that these properties are regulated locally, at the synapse, possibly by an interaction with the postsynaptic cell.

Isolation of mutations affecting neural circuitry required for grooming behavior in Drosophila melanogaster.

We have developed a screen for the isolation of mutations that produce neural defects in adult Drosophila melanogaster. In this screen, we identify mutants as flies unable to remove a light coating of applied dust in a 2-hr period. We have recovered and characterized six mutations and have found that they produce coordination defects and some have reduced levels of reflex responsiveness to the stimulation of single tactile sensory bristles. The grooming defects produced by all six of the mutations are recessive, and each of the mutations has been genetically mapped. We have also used our assay to test the grooming ability of stocks containing mutations that produce known neural defects.

Projections of leg proprioceptors within the CNS of the fly Phormia in relation to the generalized insect ganglion.

We previously reported a modality-specific layering of leg sensory axons in the CNS of the flies Phormia regina and Drosophila melanogaster with tactile and gustatory axons projecting into a ventral layer and the proprioceptive hair plate axons into an intermediate layer. Here the description is expanded to include the afferent projections of campaniform sensilla on the legs and wings of Phormia. The leg campaniform sensilla produce a number of patterns of projections within an intermediate layer of their ganglion, some of which project intersegmentally into the other thoracic ganglia. One of these patterns is shared by the hair plate sense organs. Selected wing campaniform sensilla were also stained and showed that there is little or no overlap between the projections of leg and wing campaniform sensilla. Similarities with the arrangement of campaniform sensilla and their central processes in Drosophila melanogaster are discussed. To apply the results of this study to a broader range of insects we provide an atlas of the fly CNS and compare it with the locust, which has been the model for much insect neuroanatomy and neurophysiology.

Connectivity of identified central synapses in the cricket is normal following regeneration and blockade of presynaptic activity.

Cercal sensory neurons in the cricket innervate interneurons in the central nervous system (CNS) and provide a model system for studying the formation of central synapses. When axons of the sensory neurons were transected during larval development, the cell bodies and the soma-bearing portion of axons, which are located within the cercus, survived but lost their excitability for 9-10 days. During this period, the sensory neurons grew new axons and reinnervated the terminal abdominal ganglion. Physiological recordings showed that sensory neurons of known identity reestablished monosynaptic contacts with their normal postsynaptic interneuron. Moreover, each synapse exhibited a characteristic strength indistinguishable from the intact synapse in an unoperated cricket. Since this selective connectivity was apparent immediately after the excitability of the axotomized sensory neurons was restored, action potentials in the sensory neurons appear to be unnecessary for normal synaptic regeneration to occur. Consistent with this, the reinnervation process was unaffected even when action potentials in the sensory neurons were blocked by tetrodotoxin (TTX) immediately following axotomy until just before testing. During the normal course of development, the characteristic strength of individual synapses changes systematically, resulting in the developmental rearrangement of these synapses (Chiba et al., 1988). This synaptic rearrangement was also unaffected when action potentials in the sensory neurons were blocked by TTX for the last 30% of larval development. Therefore, in the cricket cercal sensory system, both regeneration of the central synapses following axotomy of the presynaptic sensory neurons and the normal rearrangement of connectivity during larval development appear not to require axonal action potentials.