Impact of insulin signaling and proteasomal activity on physiological output of a neuronal circuit in aging Drosophila melanogaster.

The insulin family of growth factors plays an important role in development and function of the nervous system. Reduced insulin and insulin-growth-factor signaling (IIS), however, can improve symptoms of neurodegenerative diseases in laboratory model organisms and protect against age-associated decline in neuronal function. Recently, we showed that chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber escape response circuit. Here, we expand our initial findings by demonstrating that reduced functional output in the giant fiber system of aging flies can be prevented by increasing proteasomal activity within the circuit. Manipulations of IIS in neurons can also affect longevity, underscoring the relevance of the nervous system for aging.

Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies.

Lowered insulin/insulin-like growth factor (IGF) signaling (IIS) can extend healthy lifespan in worms, flies, and mice, but it can also have adverse effects (the "insulin paradox"). Chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber system (GFS), a simple escape response neuronal circuit, by increasing targeting of the gap junctional protein innexin shaking-B to gap junctions (GJs). Endosomal recycling of GJs was also stimulated in cultured human cells when IIS was reduced. Furthermore, increasing the activity of the recycling small guanosine triphosphatases (GTPases) Rab4 or Rab11 was sufficient to maintain GJs upon elevated IIS in cultured human cells and in flies, and to rescue age-related loss of GJs and of GFS function. Lowered IIS thus elevates endosomal recycling of GJs in neurons and other cell types, pointing to a cellular mechanism for therapeutic intervention into aging-related neuronal disorders.

Electrophysiological recordings from the giant fiber pathway of D. melanogaster.

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Inhibition of GSK-3 ameliorates Abeta pathology in an adult-onset Drosophila model of Alzheimer's disease.

Abeta peptide accumulation is thought to be the primary event in the pathogenesis of Alzheimer's disease (AD), with downstream neurotoxic effects including the hyperphosphorylation of tau protein. Glycogen synthase kinase-3 (GSK-3) is increasingly implicated as playing a pivotal role in this amyloid cascade. We have developed an adult-onset Drosophila model of AD, using an inducible gene expression system to express Arctic mutant Abeta42 specifically in adult neurons, to avoid developmental effects. Abeta42 accumulated with age in these flies and they displayed increased mortality together with progressive neuronal dysfunction, but in the apparent absence of neuronal loss. This fly model can thus be used to examine the role of events during adulthood and early AD aetiology. Expression of Abeta42 in adult neurons increased GSK-3 activity, and inhibition of GSK-3 (either genetically or pharmacologically by lithium treatment) rescued Abeta42 toxicity. Abeta42 pathogenesis was also reduced by removal of endogenous fly tau; but, within the limits of detection of available methods, tau phosphorylation did not appear to be altered in flies expressing Abeta42. The GSK-3-mediated effects on Abeta42 toxicity appear to be at least in part mediated by tau-independent mechanisms, because the protective effect of lithium alone was greater than that of the removal of tau alone. Finally, Abeta42 levels were reduced upon GSK-3 inhibition, pointing to a direct role of GSK-3 in the regulation of Abeta42 peptide level, in the absence of APP processing. Our study points to the need both to identify the mechanisms by which GSK-3 modulates Abeta42 levels in the fly and to determine if similar mechanisms are present in mammals, and it supports the potential therapeutic use of GSK-3 inhibitors in AD.

Electrophysiological recordings from the Drosophila giant fiber system (GFS).

INTRODUCTION: The giant fiber system (GFS) of Drosophila is a well-characterized neuronal circuit that mediates the escape response in the fly. It is one of the few adult neural circuits from which electrophysiological recordings can be made routinely. This protocol describes a simple procedure for stimulating the giant fiber neurons directly in the brain of the adult fly and obtaining recordings from the output muscles of the GFS: the tergotrochanteral "jump" muscle (TTM) and the large indirect flight muscles (dorsal longitudinal muscles, or DLMs). It is a relatively noninvasive method that allows the investigator to stimulate the giant fibers in the brain and assay the function of several central synapses within this neural circuit by recording from the thoracic musculature.

A modifier screen in the Drosophila eye reveals that aPKC interacts with Glued during central synapse formation.

BACKGROUND: The Glued gene of Drosophila melanogaster encodes the homologue of the vertebrate p150Glued subunit of dynactin. The Glued1 mutation compromises the dynein-dynactin retrograde motor complex and causes disruptions to the adult eye and the CNS, including sensory neurons and the formation of the giant fiber system neural circuit. RESULTS: We performed a 2-stage genetic screen to identify mutations that modified phenotypes caused by over-expression of a dominant-negative Glued protein. We screened over 34,000 flies and isolated 41 mutations that enhanced or suppressed an eye phenotype. Of these, 12 were assayed for interactions in the giant fiber system by which they altered a giant fiber morphological phenotype and/or altered synaptic function between the giant fiber and the tergotrochanteral muscle motorneuron. Six showed interactions including a new allele of atypical protein kinase C (aPKC). We show that this cell polarity regulator interacts with Glued during central synapse formation. We have mapped the five other interacting mutations to discrete chromosomal regions. CONCLUSION: Our results show that an efficient way to screen for genes involved in central synapse formation is to use a two-step strategy in which a screen for altered eye morphology precedes the analysis of central synaptogenesis. This has highlighted a role for aPKC in the formation of an identified central synapse.

Molecular mechanism of rectification at identified electrical synapses in the Drosophila giant fiber system.

Electrical synapses are neuronal gap junctions that mediate fast transmission in many neural circuits. The structural proteins of gap junctions are the products of two multigene families. Connexins are unique to chordates; innexins/pannexins encode gap-junction proteins in prechordates and chordates. A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons. Some electrical synapses are bidirectional; others are rectifying junctions that preferentially transmit depolarizing current anterogradely. The phenomenon of rectification was first described five decades ago, but the molecular mechanism has not been elucidated. Here, we demonstrate that putative rectifying electrical synapses in the Drosophila Giant Fiber System are assembled from two products of the innexin gene shaking-B. Shaking-B(Neural+16) is required presynaptically in the Giant Fiber to couple this cell to its postsynaptic targets that express Shaking-B(Lethal). When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification. These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.

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.

What makes a fly enter diapause?

Diapause is a dormant state that insects may undergo as a response to changing environmental conditions. In flies, like many insects inhabiting temperate zones, diapause occurs generally during the winter months when ambient temperatures are cool and food sources scarce. Whilst the environmental factors involved in determining diapause have been known for a long time, the genes and molecular events controling its initiation are poorly understood. Here I outline the factors that initiate diapause and highlight recent studies that implicate insulin signaling in its control.

Making an escape: development and function of the Drosophila giant fibre system.

Flies escape danger by jumping into the air and flying away. The giant fibre system (GFS) is the neural circuit that mediates this simple behavioural response to visual stimuli. The sensory signal is received by the giant fibre and relayed to the leg and wing muscle motorneurons. Many of the neurons in the Drosophila GFS are uniquely identifiable and amenable to cell biological, electrophysiological and genetic studies. Here we review the anatomy and development of this system and highlight its utility for studying many aspects of nervous system biology ranging from neural development and synaptic plasticity to the aetiology of neural disorder.

Differential localization of glutamate receptor subunits at the Drosophila neuromuscular junction.

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. We now identify a third glutamate receptor subunit at the Drosophila neuromuscular junction, DGluRIII, which 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.