Superresolution imaging of Drosophila tissues using expansion microscopy.

The limited resolving power of conventional diffraction-limited microscopy hinders analysis of small, densely packed structural elements in cells. Expansion microscopy (ExM) provides an elegant solution to this problem, allowing for increased resolution with standard microscopes via physical expansion of the specimen in a swellable polymer hydrogel. Here, we apply, validate, and optimize ExM protocols that enable the study of Drosophila embryos, larval brains, and larval and adult body walls. We achieve a lateral resolution of ∼70 nm in Drosophila tissues using a standard confocal microscope, and we use ExM to analyze fine intracellular structures and intercellular interactions. First, we find that ExM reveals features of presynaptic active zone (AZ) structure that are observable with other superresolution imaging techniques but not with standard confocal microscopy. We further show that synapses known to exhibit age-dependent changes in activity also exhibit age-dependent changes in AZ structure. Finally, we use the significantly improved axial resolution of ExM to show that dendrites of somatosensory neurons are inserted into epithelial cells at a higher frequency than previously reported in confocal microscopy studies. Altogether, our study provides a foundation for the application of ExM to Drosophila tissues and underscores the importance of tissue-specific optimization of ExM procedures.

Transcriptomics of aged Drosophila motor neurons reveals a matrix metalloproteinase that impairs motor function.

The neuromuscular junction (NMJ) is responsible for transforming nervous system signals into motor behavior and locomotion. In the fruit fly Drosophila melanogaster, an age-dependent decline in motor function occurs, analogous to the decline experienced in mice, humans, and other mammals. The molecular and cellular underpinnings of this decline are still poorly understood. By specifically profiling the transcriptome of Drosophila motor neurons across age using custom microarrays, we found that the expression of the matrix metalloproteinase 1 (dMMP1) gene reproducibly increased in motor neurons in an age-dependent manner. Modulation of physiological aging also altered the rate of dMMP1 expression, validating dMMP1 expression as a bona fide aging biomarker for motor neurons. Temporally controlled overexpression of dMMP1 specifically in motor neurons was sufficient to induce deficits in climbing behavior and cause a decrease in neurotransmitter release at neuromuscular synapses. These deficits were reversible if the dMMP1 expression was shut off again immediately after the onset of motor dysfunction. Additionally, repression of dMMP1 enzymatic activity via overexpression of a tissue inhibitor of metalloproteinases delayed the onset of age-dependent motor dysfunction. MMPs are required for proper tissue architecture during development. Our results support the idea that matrix metalloproteinase 1 is acting as a downstream effector of antagonistic pleiotropy in motor neurons and is necessary for proper development, but deleterious when reactivated at an advanced age.

Rejuvenation of the aged neuromuscular junction by exercise.

Age-dependent declines in muscle function are observed across species. The loss of mobility resulting from the decline in muscle function represents an important health issue and a key determinant of quality of life for the elderly. It is believed that changes in the structure and function of the neuromuscular junction are important contributors to the observed declines in motor function with increased age. Numerous studies indicate that the aging muscle is an important contributor to the deterioration of the neuromuscular junction but the cellular and molecular mechanisms driving the degeneration of the synapse remain incompletely described. Importantly, growing data from both animal models and humans indicate that exercise can rejuvenate the neuromuscular junction and improve motor function. In this review we will focus on the role of muscle-derived neurotrophin signaling in the rejuvenation of the aged neuromuscular junction in response to exercise.

TrpA1 activation in peripheral sensory neurons underlies the ionic basis of pain hypersensitivity in response to vinca alkaloids.

Chemotherapy induced peripheral neuropathy (CIPN), a side effect of many anti-cancer drugs including the vinca alkaloids, is characterized by a severe pain syndrome that compromises treatment in many patients. Currently there are no effective treatments for this pain syndrome except for the reduction of anti-cancer drug dose. Existing data supports the model that the pain associated with CIPN is the result of anti-cancer drugs augmenting the function of the peripheral sensory nociceptors but the cellular mechanisms underlying the effects of anti-cancer drugs on sensory neuron function are not well described. Studies from animal models have suggested a number of disease etiologies including mitotoxicity, axonal degeneration, immune signaling, and reduced sensory innervations but these outcomes are the result of prolonged treatment paradigms and do not necessarily represent the early formative events associated with CIPN. Here we show that acute exposure to vinca alkaloids results in an immediate pain syndrome in both flies and mice. Furthermore, we demonstrate that exposure of isolated sensory neurons to vinca alkaloids results in the generation of an inward sodium current capable of depolarizing these neurons to threshold resulting in neuronal firing. These neuronal effects of vinca alkaloids require the transient receptor potential ankyrin-1 (TrpA1) channel, and the hypersensitization to painful stimuli in response to the acute exposure to vinca alkaloids is reduced in TrpA1 mutant flies and mice. These findings demonstrate the direct excitation of sensory neurons by CIPN-causing chemotherapy drugs, and identify TrpA1 as an important target during the pathogenesis of CIPN.

The Drosophila LC8 homolog cut up specifies the axonal transport of proteasomes.

Because of their functional polarity and elongated morphologies, microtubule-based transport of proteins and organelles is critical for normal neuronal function. The proteasome is required throughout the neuron for the highly regulated degradation of a broad set of protein targets whose functions underlie key physiological responses, including synaptic plasticity and axonal degeneration. Molecularly, the relationship between proteasome transport and the transport of the targets of proteasomes is unclear. The dynein motor complex is required for the microtubule-based motility of numerous proteins and organelles in neurons. Here, we demonstrate that microtubule-based transport of proteasomes within the neuron in Drosophila utilizes a different dynein light chain to that used by synaptic proteins. Live imaging of proteasomes and synaptic vesicle proteins in axons and synapses finds that these cargoes traffic independently, and that proteasomes exhibit significantly reduced retrograde transport velocities compared to those of synaptic vesicle proteins. Genetic and biochemical analyses reveals that the Drosophila homolog of the LC8 dynein light chains (mammalian DYNLL1 and DYNLL2), called Cut up, binds proteasomes and functions specifically during their transport. These data support the model that Cut up functions to specify the dynein-mediated transport of neuronal proteasomes.

Ciberial Muscle 9 (CM9) Electrophysiological Recordings in Adult Drosophila melanogaster.

The complexity surrounding presynaptic recordings in mammals is a significant barrier to the study of presynaptic mechanisms during neurotransmission in the mammalian central nervous system (CNS). Here we describe an adult fly neuromuscular junction (NMJ), the ciberial muscle 9 (CM9) NMJ, which allows for the recording of both evoked (EPSPs) and spontaneous postsynaptic excitatory potentials (mEPSPs) at a mature glutamatergic synapse. Combined with CM9-specific genetic technologies, the CM9 NMJ provides a powerful experimental system to better understand the regulation of neurotransmitter release at a mature synapse.

Extension of Health Span and Life Span in Drosophila by S107 Requires the calstabin Homologue FK506-BP2.

The accumulation of oxidative damage is strongly linked to age-dependent declines in cell function, but the contribution of oxidative damage to morbidity is still debated. Many organisms seem to tolerate oxidative damage, and the extension of health span and life span by augmenting antioxidant activity has been inconsistent. Here we use the Drosophila model system to investigate the relationship among oxidative stress, health span, and life span. The oxidation-dependent dissociation of the Calstabin protein from the ryanodine receptor has been shown to result in reduced muscle function in mammals. The S107 molecule is able to reestablish this binding resulting in improved muscle function. We find that S107 is able to restore motor function in aging Drosophila to young levels, and this effect of S107 is absent in calstabin (FK506-BP2) mutants. Interestingly, FK506-BP2 mutant flies have reduced sensitivity to the effects of age and oxidative stress on motor function between 7 and 35 days of age. Muscle expression of FK506-BP2 in FK506-BP2 mutants completely restores the sensitivity of motor function to both age and oxidative stress, supporting the idea that the age-dependent decline in motor function in Drosophila requires FK506-BP2 function within the muscle. Although FK506-BP2 mutant flies are found to have less sensitivity to oxidative stress, FK506-BP2 mutants do not live longer than wild type. These results demonstrate that the deleterious effects of oxidation on motor function early in life are the result of a singular event that does not compromise survival.

Insulin signaling controls neurotransmission via the 4eBP-dependent modification of the exocytotic machinery.

Altered insulin signaling has been linked to widespread nervous system dysfunction including cognitive dysfunction, neuropathy and susceptibility to neurodegenerative disease. However, knowledge of the cellular mechanisms underlying the effects of insulin on neuronal function is incomplete. Here, we show that cell autonomous insulin signaling within the Drosophila CM9 motor neuron regulates the release of neurotransmitter via alteration of the synaptic vesicle fusion machinery. This effect of insulin utilizes the FOXO-dependent regulation of the thor gene, which encodes the Drosophila homologue of the eif-4e binding protein (4eBP). A critical target of this regulatory mechanism is Complexin, a synaptic protein known to regulate synaptic vesicle exocytosis. We find that the amounts of Complexin protein observed at the synapse is regulated by insulin and genetic manipulations of Complexin levels support the model that increased synaptic Complexin reduces neurotransmission in response to insulin signaling.

Neuronal epigenetics and the aging synapse.

Two of the most salient phenotypes of aging are cognitive decline and loss of motor function, both of which are controlled by the nervous system. Cognition and muscle contraction require that neuronal synapses develop and maintain proper structure and function. We review the literature on how normal physiological aging disrupts central and peripheral synapse function including the degradation of structure and/or control of neurotransmission. Here we also attempt to connect the work done on the epigenetics of aging to the growing literature of how epigenetic mechanisms control synapse structure and function. Lastly, we address possible roles of epigenetic mechanisms to explain why the basal rates of age-related dysfunction vary so widely across individuals.

Coordination and fine motor control depend on Drosophila TRPγ.

Motor coordination is broadly divided into gross and fine motor control, both of which depend on proprioceptive organs. However, the channels that function specifically in fine motor control are unknown. Here we show that mutations in trpγ disrupt fine motor control while leaving gross motor proficiency intact. The mutants are unable to coordinate precise leg movements during walking, and are ineffective in traversing large gaps due to an inability in making subtle postural adaptations that are requisite for this task. TRPγ is expressed in proprioceptive organs, and is required in both neurons and glia for gap crossing. We expressed TRPγ in vitro, and found that its activity is promoted by membrane stretch. A mutation eliminating the Na(+)/Ca(2+) exchanger suppresses the gap-crossing phenotype of trpγ flies. Our findings indicate that TRPγ contributes to fine motor control through mechanical activation in proprioceptive organs, thereby promoting Ca(2+) influx, which is required for function.

The guanine exchange factor Gartenzwerg and the small GTPase Arl1 function in the same pathway with Arfaptin during synapse growth.

The generation of neuronal morphology requires transport vesicles originating from the Golgi apparatus (GA) to deliver specialized components to the axon and dendrites. Drosophila Arfaptin is a membrane-binding protein localized to the GA that is required for the growth of the presynaptic nerve terminal. Here we provide biochemical, cellular and genetic evidence that the small GTPase Arl1 and the guanine-nucleotide exchange factor (GEF) Gartenzwerg are required for Arfaptin function at the Golgi during synapse growth. Our data define a new signaling pathway composed of Arfaptin, Arl1, and Garz, required for the generation of normal synapse morphology.

An age-dependent change in the set point of synaptic homeostasis.

Homeostatic plasticity functions within the nervous system to maintain normal neural functions, such as neurotransmission, within predefined optimal ranges. The defined output of these neuronal processes is referred to as the set point, which is the value that the homeostatic system defends against fluctuations. Currently, it is unknown how stable homeostatic set points are within the nervous system. In the present study we used the CM9 neuromuscular junctions (NMJs) in the adult Drosophila to investigate the stability of the set point of synaptic homeostasis across the lifespan of the fly. At the fly NMJ, it is believed that the depolarization of the muscle by neurotransmitter during an action potential, represented by the EPSP, is a homeostatic set point that is precisely maintained via changes in synaptic vesicle release. We find that the amplitude of the EPSP abruptly increases during middle age and that this enhanced EPSP is maintained into late life, consistent with an age-dependent change to the homeostatic set point of the synapse during middle age. In support of this, comparison of the homeostatic response at the young versus the old synapse shows that the magnitude of the homeostatic response at the older synapse is significantly larger than the response at the young NMJ, appropriate for a synapse at which the set point has been increased. Our data demonstrate that the amplitude of the EPSP at the Drosophila NMJ increases during aging and that the homeostatic signaling system adjusts its response to accommodate the new set point.

Stimulus discrimination by the polymodal sensory neuron.

Polymodal sensory neurons inform organisms about the nature of the physical world around them. The activity of these cells guide behaviors including the withdrawal from nocifensive stimuli such as intense heat or harsh force to feeling the comforting weight of a warm blanket. Molecular and genetic analysis of the channel proteins required for these divers behavioral responses have revealed an elaborate and disparate collection of channel proteins within the polymodal sensory neuron. Recent data supports that the biophysical traits of the channel proteins combined with the collection of channels activated during stimulation is sufficient to describe the nature of the stimulus. It is currently unclear what the functional arrangement of channel proteins are during perception. Specifically, are channel proteins arranged in parallel and function independently during perception, or are these channel proteins arranged in functional sensory networks. We propose a hierarchal functional arrangement of channels within polymodal sensory neurons that incorporates aspects of both parallel and serial arrangements of channel proteins.

Normal dynactin complex function during synapse growth in Drosophila requires membrane binding by Arfaptin.

Mutations in DCTN1, a component of the dynactin complex, are linked to neurodegenerative diseases characterized by a broad collection of neuropathologies. Because of the pleiotropic nature of dynactin complex function within the neuron, defining the causes of neuropathology in DCTN1 mutants has been difficult. We combined a genetic screen with cellular assays of dynactin complex function to identify genes that are critical for dynactin complex function in the nervous system. This approach identified the Drosophila homologue of Arfaptin, a multifunctional protein that has been implicated in membrane trafficking. We find that Arfaptin and the Drosophila DCTN1 homologue, Glued, function in the same pathway during synapse growth but not during axonal transport or synapse stabilization. Arfaptin physically associates with Glued and other dynactin complex components in the nervous system of both flies and mice and colocalizes with Glued at the Golgi in motor neurons. Mechanistically, membrane binding by Arfaptin mediates membrane association of the dynactin complex in motor neurons and is required for normal synapse growth. Arfaptin represents a novel dynactin complex-binding protein that specifies dynactin complex function during synapse growth.

Patch-clamping Drosophila sensory neurons.

Electrophysiological studies provide essential clues about the regulation and physiological function of ion channel proteins. Probing ion channel activity in vivo, though, often is challenging. This can limit the usefulness of such model organisms as Drosophila for electrophysiological studies. This is unfortunate because these genetically tractable organisms represent powerful research tools that facilitate elaboration of complex questions of physiology. Here, we describe a recently developed method for recording ion channel activity in Drosophila sensory neurons. This approach is based on patch-clamping primary neuron cultures from Drosophila embryos. Such cultures allow the study of ion channels in different genetic backgrounds. In addition to describing how to prepare a primary neuronal cell culture from Drosophila embryos, we discuss, as an example of utility, analysis of Na(+) currents in cultured class IV multidendritic (md) sensory neurons with the patch clamp technique. Excitability of md sensory neurons, manifested as action potential firing, is revealed with whole-cell current-clamping. Voltage-clamping class IV md neurons revealed the activity of the voltage-gated Na(+) channel, paralytic. Moreover, challenging class IV md neurons with acidic pH activates acid-sensing inward Na(+) currents. Genetic manipulation of Drosophila combined with this electrophysiological readout of activity identifies pickpocket1 (Ppk1), a member of the Deg/ENaC channel family, as responsible for conducting an acid-sensing Na(+) current in class IV md sensory neurons.

Inhibition of neuronal degenerin/epithelial Na+ channels by the multiple sclerosis drug 4-aminopyridine.

The voltage-gated K(+) (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na(+) and Ca(2+) currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na(+) (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.

Modulation of methuselah expression targeted to Drosophila insulin-producing cells extends life and enhances oxidative stress resistance.

Ubiquitously reduced signaling via Methuselah (MTH), a G-protein-coupled receptor (GPCR) required for neurosecretion, has previously been reported to extend life and enhance stress resistance in flies. Whether these effects are due to reduced MTH signalling in specific tissues remains unknown. We determined that reduced expression of mth targeted to the insulin-producing cells (IPCs) of the fly brain was sufficient to extend life and enhance oxidative stress resistance. Paradoxically, we discovered that overexpression of mth targeted to the same cells has similar phenotypic effects to reduced expression due to MTH's interaction with ß-arrestin, which uncouples GPCRs from their G-proteins. We confirmed the functional relationship between MTH and ß-arrestin by finding that IPC-targeted overexpression of ß-arrestin alone mimics the longevity phenotype of reduced MTH signaling. As reduced MTH signaling also inhibits insulin secretion from the IPCs, the most parsimonious mechanistic explanation of its longevity and stress-resistance enhancement might be through reduced insulin/IGF signaling (IIS). However, examination of phenotypic features of long-lived IPC-mth modulated flies as well as several downstream IIS targets implicates enhanced activity of the JNK stress-resistance pathway more directly than insulin signaling in the longevity and stress-resistance phenotypes.

Adult neuronal Arf6 controls ethanol-induced behavior with Arfaptin downstream of Rac1 and RhoGAP18B.

Alcohol use disorders affect millions of individuals. However, the genes and signaling pathways involved in behavioral ethanol responses and addiction are poorly understood. Here we identify a conserved biochemical pathway that underlies the sedating effects of ethanol in Drosophila. Mutations in the Arf6 small GTPase signaling pathway cause hypersensitivity to ethanol-induced sedation. We show that Arf6 functions in the adult nervous system to control ethanol-induced behavior. We also find that the Drosophila Arfaptin protein directly binds to the activated forms of Arf6 and Rac1 GTPases, and mutants in Arfaptin also display ethanol sensitivity. Arf6 acts downstream of Rac1 and Arfaptin to regulate ethanol-induced behaviors, and we thus demonstrate that this conserved Rac1/Arfaptin/Arf6 pathway is a major mediator of ethanol-induced behavioral responses.

Pickpocket1 is an ionotropic molecular sensory transducer.

The molecular transformation of an external stimulus into changes in sensory neuron activity is incompletely described. Although a number of molecules have been identified that can respond to stimuli, evidence that these molecules can transduce stimulation into useful neural activity is lacking. Here we demonstrate that pickpocket1 (ppk1), a Drosophila homolog of mammalian Degenerin/epithelial sodium channels, encodes an acid-sensing sodium channel that conducts a transient depolarizing current in multidendritic sensory neurons of Drosophila melanogaster. Stimulation of Ppk1 is sufficient to bring these sensory neurons to threshold, eliciting a burst of action potentials. The transient nature of the neural activity produced by Ppk1 activation is the result of Ppk1 channel gating properties. This model is supported by the observation of enhanced bursting activity in neurons expressing a gain of function ppk1 mutant harboring the degenerin mutation. These findings demonstrate that Ppk1 can function as an ionotropic molecular sensory transducer capable of transforming the perception of a stimulus into phasic neuronal activity in sensory neurons.

Effects of diet on synaptic vesicle release in dynactin complex mutants: a mechanism for improved vitality during motor disease.

Synaptic dysfunction is considered the primary substrate for the functional declines observed within the nervous system during age-related neurodegenerative disease. Dietary restriction (DR), which extends lifespan in numerous species, has been shown to have beneficial effects on many neurodegenerative disease models. Existing data sets suggest that the effects of DR during disease include the amelioration of synaptic dysfunction but evidence of the beneficial effects of diet on the synapse is lacking. Dynactin mutant flies have significant increases in mortality rates and exhibit progressive loss of motor function. Using a novel fly motor disease model, we demonstrate that mutant flies raised on a low calorie diet have enhanced motor function and improved survival compared to flies on a high calorie diet. Neurodegeneration in this model is characterized by an early impairment of neurotransmission that precedes the deterioration of neuromuscular junction (NMJ) morphology. In mutant flies, low calorie diet increases neurotransmission, but has little effect on morphology, supporting the hypothesis that enhanced neurotransmission contributes to the effects of diet on motor function. Importantly, the effects of diet on the synapse are not because of the reduction of mutant pathologies, but by the increased release of synaptic vesicles during activity. The generality of this effect is demonstrated by the observation that diet can also increase synaptic vesicle release at wild-type NMJs. These studies reveal a novel presynaptic mechanism of diet that may contribute to the improved vigor observed in mutant flies raised on low calorie diet.