Alteration in information flow through a pair of feeding command neurons underlies a form of Pavlovian conditioning in the Drosophila brain.

Pavlovian conditioning1 is a broadly used learning paradigm where defined stimuli are associated to induce behavioral switching. To define a causal relationship between activity change in a single neuron and behavioral switching, we took advantage of a "command neuron" that connects cellular function to behavior.2 To examine the cellular and molecular basis of Pavlovian conditioning, we previously identified a pair of feeding command neurons termed "feeding neurons" in the adult Drosophila brain3 using genetic screening4 and opto- and thermo-genetic techniques.5-7 The feeding neuron is activated by sweet signals like sucrose and induces the full complement of feeding behaviors, such as proboscis extension and food pumping. Ablation or inactivation of the pair of feeding neurons abolishes feeding behavior, suggesting that this single pair of neurons is indispensable for natural feeding behaviors.2,3 Here, we describe a novel conditioning protocol to associate a signal-mediating rod removal from legs (conditioned stimulus [CS]) to feeding behavior induced by sucrose stimulation (unconditioned stimulus [US]). Calcium imaging of the feeding neuron demonstrated it acquires responsiveness to CS during conditioning, with inactivation of the feeding neuron during conditioning suppressing plasticity. These results suggest conditioning alters signals flowing from the CS into the feeding circuit, with the feeding neuron functioning as a key integrative hub for Hebbian plasticity.

Synaptotagmin 7 switches short-term synaptic plasticity from depression to facilitation by suppressing synaptic transmission.

Short-term synaptic plasticity is a fast and robust modification in neuronal presynaptic output that can enhance release strength to drive facilitation or diminish it to promote depression. The mechanisms that determine whether neurons display short-term facilitation or depression are still unclear. Here we show that the Ca2+-binding protein Synaptotagmin 7 (Syt7) determines the sign of short-term synaptic plasticity by controlling the initial probability of synaptic vesicle (SV) fusion. Electrophysiological analysis of Syt7 null mutants at Drosophila embryonic neuromuscular junctions demonstrate loss of the protein converts the normally observed synaptic facilitation response during repetitive stimulation into synaptic depression. In contrast, overexpression of Syt7 dramatically enhanced the magnitude of short-term facilitation. These changes in short-term plasticity were mirrored by corresponding alterations in the initial evoked response, with SV release probability enhanced in Syt7 mutants and suppressed following Syt7 overexpression. Indeed, Syt7 mutants were able to display facilitation in lower [Ca2+] where release was reduced. These data suggest Syt7 does not act by directly sensing residual Ca2+ and argues for the existence of a distinct Ca2+ sensor beyond Syt7 that mediates facilitation. Instead, Syt7 normally suppresses synaptic transmission to maintain an output range where facilitation is available to the neuron.

'Necessary and sufficient' in biology is not necessarily necessary - confusions and erroneous conclusions resulting from misapplied logic in the field of biology, especially neuroscience.

In this article, we describe an incorrect use of logic which involves the careless application of the 'necessary and sufficient' condition originally used in formal logic. This logical fallacy is causing frequent confusion in current biology, especially in neuroscience. In order to clarify this problem, we first dissect the structure of this incorrect logic (which we refer to as 'misapplied-N&S') to show how necessity and sufficiency in misapplied-N&S are not matching each other. Potential pitfalls of utilizing misapplied-N&S are exemplified by cases such as the discrediting of command neurons and other potentially key neurons, the distorting of truth in optogenetic studies, and the wrongful justification of studies with little meaning. In particular, the use of the word 'sufficient' in optogenetics tends to generate misunderstandings by opening up multiple interpretations. To avoid the confusion caused by the misleading logic, we now recommend using 'indispensable and inducing' instead of using 'necessary and sufficient.' However, we ultimately recommend fully articulating the limits of what our experiments suggest, not relying on such simple phrases. Only after this problem is fully understood and more rigorous language is demanded, can we finally interpret experimental results in an accurate way.

A large-scale behavioral screen to identify neurons controlling motor programs in the Drosophila brain.

Drosophila is increasingly used for understanding the neural basis of behavior through genetically targeted manipulation of specific neurons. The primary approach in this regard has relied on the suppression of neuronal activity. Here, we report the results of a novel approach to find and characterize neural circuits by expressing neuronal activators to stimulate subsets of neurons to induce behavior. Classical electrophysiological studies demonstrated that stimulation of command neurons could activate neural circuits to trigger fixed action patterns. Our method was designed to find such command neurons for diverse behaviors by screening flies in which random subsets of brain cells were activated. We took advantage of the large collection of Gal4 lines from the NP project and crossed 835 Gal4 strains with relatively limited Gal4 expression in the brain to flies carrying a UAS transgene encoding TRPM8, a cold-sensitive ion channel. Low temperatures opened the TRPM8 channel in Gal4-expressing cells, leading to their excitation, and in many cases induced overt behavioral changes in adult flies. Paralysis was reproducibly observed in the progeny of crosses with 84 lines, whereas more specific behaviors were induced with 24 other lines. Stimulation performed using the heat-activated channel, TrpA1, resulted in clearer and more robust behaviors, including flight, feeding, and egg-laying. Through follow-up studies starting from this screen, we expect to find key components of the neural circuits underlying specific behaviors, thus providing a new avenue for their functional analysis.

A single pair of interneurons commands the Drosophila feeding motor program.

Many feeding behaviours are the result of stereotyped, organized sequences of motor patterns. These patterns have been the subject of neuroethological studies, such as electrophysiological characterization of neurons governing prey capture in toads. However, technical limitations have prevented detailed study of the functional role of these neurons, a common problem for vertebrate organisms. Complexities involved in studies of whole-animal behaviour can be resolved in Drosophila, in which remote activation of brain cells by genetic means enables us to examine the nervous system in freely moving animals to identify neurons that govern a specific behaviour, and then to repeatedly target and manipulate these neurons to characterize their function. Here we show neurons that generate the feeding motor program in Drosophila. We carried out an unbiased screen using remote neuronal activation and identified a critical pair of brain cells that induces the entire feeding sequence when activated. These 'feeding neurons' (here abbreviated to Fdg neurons for brevity) are also essential for normal feeding as their suppression or ablation eliminates sugar-induced feeding behaviour. Activation of a single Fdg neuron induces asymmetric feeding behaviour and ablation of a single Fdg neuron distorts the sugar-induced feeding behaviour to become asymmetric, indicating the direct role of these neurons in shaping motor-program execution. Furthermore, recording neuronal activity and calcium imaging simultaneously during feeding behaviour reveals that the Fdg neurons respond to food presentation, but only in starved flies. Our results demonstrate that Fdg neurons operate firmly within the sensorimotor watershed, downstream of sensory and metabolic cues and at the top of the feeding motor hierarchy, to execute the decision to feed.

Regulation of postsynaptic retrograde signaling by presynaptic exosome release.

Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. Here we show that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling.

Simultaneous recording of calcium signals from identified neurons and feeding behavior of Drosophila melanogaster.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP(1), to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons(2-5). Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding(6) (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER(7) with a calcium imaging technique(8) using GCaMP3.0(1, 9), I have established an experimental system, where we can monitor activity of neurons in the feeding center - the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca(2+) imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Differential regulation of synchronous versus asynchronous neurotransmitter release by the C2 domains of synaptotagmin 1.

Synaptic vesicle fusion at many synapses has been kinetically separated into two distinct Ca(2+)-dependent temporal components consisting of a rapid synchronous phase followed by a slower asynchronous component. Mutations in the synaptic vesicle Ca(2+) sensor Synaptotagmin 1 (Syt 1) reduce synchronous neurotransmission while enhancing the slower asynchronous phase of release. Syt 1 regulation of vesicle fusion requires interactions mediated by its tandem cytoplasmic C2 domains (C2A and C2B). Although Ca(2+) binding by Syt 1 is predicted to drive synchronous release, it is unknown if Ca(2+) interactions with either C2 domain is required for suppression of asynchronous release. To determine if Ca(2+) binding by Syt 1 regulates these two phases of release independently, we performed electrophysiological analysis of transgenically expressed Syt 1 mutated at Ca(2+) binding sites in C2A or C2B in the background of Drosophila Syt 1-null mutants. Transgenic animals expressing mutations that disrupt Ca(2+) binding to C2A fully restored the synchronous phase of neurotransmitter release, whereas the asynchronous component was not suppressed. In contrast, rescue with Ca(2+)-binding mutants in C2B displayed little rescue of the synchronous release component, but reduced asynchronous release. These results suggest that the tandem C2 domains of Syt 1 play independent roles in neurotransmission, as Ca(2+) binding to C2A suppresses asynchronous release, whereas Ca(2+) binding to C2B mediates synchronous fusion.

Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth.

The molecular pathways involved in retrograde signal transduction at synapses and the function of retrograde communication are poorly understood. Here, we demonstrate that postsynaptic calcium 2+ ion (Ca2+) influx through glutamate receptors and subsequent postsynaptic vesicle fusion trigger a robust induction of presynaptic miniature release after high-frequency stimulation at Drosophila neuromuscular junctions. An isoform of the synaptotagmin family, synaptotagmin 4 (Syt 4), serves as a postsynaptic Ca2+ sensor to release retrograde signals that stimulate enhanced presynaptic function through activation of the cyclic adenosine monophosphate (cAMP)-cAMP-dependent protein kinase pathway. Postsynaptic Ca2+ influx also stimulates local synaptic differentiation and growth through Syt 4-mediated retrograde signals in a synapse-specific manner.

The synaptotagmins: calcium sensors for vesicular trafficking.

The synaptotagmin family of vesicle proteins is believed to mediate calcium-dependent regulation of membrane trafficking. Detailed biochemical and in vivo studies of the most characterized isoform, synaptotagmin 1 (syt 1), have provided compelling evidence that it functions as a calcium sensor for fast neurotransmitter release at synapses. However, the function of the remaining isoforms is unclear, and multiple roles have been hypothesized for several of these. Recent evidence in Drosophila has given insight into the function of some of the remaining synaptotagmin family members. Of the five evolutionarily conserved isoforms in Drosophila, only two, syt 1 and syt 4, localize to most, if not all, synapses. The former is localized to presynaptic terminals, whereas the latter is predominantly postsynaptic. This suggests an intriguing possibility that syt 4 may mediate a postsynaptic vesicle trafficking pathway, providing a molecular basis for an evolutionarily conserved bidirectional vesicular trafficking communication system at synapses.

Synaptotagmins are trafficked to distinct subcellular domains including the postsynaptic compartment.

The synaptotagmin family has been implicated in calcium-dependent neurotransmitter release, although Synaptotagmin 1 is the only isoform demonstrated to control synaptic vesicle fusion. Here, we report the characterization of the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologues of mammalian Synaptotagmins 4, 7, 12, and 14. Like Synaptotagmin 1, Synaptotagmin 4 is ubiquitously present at synapses, but localizes to the postsynaptic compartment. The remaining isoforms were not found at synapses (Synaptotagmin 7), expressed at very low levels (Synaptotagmins 12 and 14), or in subsets of putative neurosecretory cells (Synaptotagmins alpha and beta). Consistent with their distinct localizations, overexpression of Synaptotagmin 4 or 7 cannot functionally substitute for the loss of Synaptotagmin 1 in synaptic transmission. Our results indicate that synaptotagmins are differentially distributed to unique subcellular compartments. In addition, the identification of a postsynaptic synaptotagmin suggests calcium-dependent membrane-trafficking functions on both sides of the synapse.

Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington's disease.

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein that results in intracellular aggregate formation and neurodegeneration. Pathways leading from polyglutamine tract expansion to disease pathogenesis remain obscure. To elucidate how polyglutamine expansion causes neuronal dysfunction, we generated Drosophila transgenic strains expressing human huntingtin cDNAs encoding pathogenic (Htt-Q128) or nonpathogenic proteins (Htt-Q0). Whereas expression of Htt-Q0 has no discernible effect on behavior, lifespan, or neuronal morphology, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan, and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and lead to disruption of axonal transport and accumulation of aggregates at synapses. In contrast, Drosophila expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking. Our findings indicate that nonnuclear events induced by cytoplasmic huntingtin aggregation play a central role in the progressive neurodegeneration observed in Huntington's disease.

Presynaptic N-type calcium channels regulate synaptic growth.

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.

Is synaptotagmin the calcium sensor?

After much debate, recent progress indicates that the synaptic vesicle protein synaptotagmin I probably functions as the calcium sensor for synchronous neurotransmitter release. Following calcium influx into presynaptic terminals, synaptotagmin I rapidly triggers the fusion of synaptic vesicles with the plasma membrane and underlies the fourth-order calcium cooperativity of release. Biochemical and genetic studies suggest that lipid and SNARE interactions underlie synaptotagmin's ability to mediate the incredible speed of vesicle fusion that is the hallmark of fast synaptic transmission.

Synaptotagmin I functions as a calcium sensor to synchronize neurotransmitter release.

To characterize Ca(2+)-mediated synaptic vesicle fusion, we analyzed Drosophila synaptotagmin I mutants deficient in specific interactions mediated by its two Ca(2+) binding C2 domains. In the absence of synaptotagmin I, synchronous release is abolished and a kinetically distinct delayed asynchronous release pathway is uncovered. Synapses containing only the C2A domain of synaptotagmin partially recover synchronous fusion, but have an abolished Ca(2+) cooperativity. Mutants that disrupt Ca(2+) sensing by the C2B domain have synchronous release with normal Ca(2+) cooperativity, but with reduced release probability. Our data suggest the Ca(2+) cooperativity of neurotransmitter release is likely mediated through synaptotagmin-SNARE interactions, while phospholipid binding and oligomerization trigger rapid fusion with increased release probability. These results indicate that synaptotagmin is the major Ca(2+) sensor for evoked release and functions to trigger synchronous fusion in response to Ca(2+), while suppressing asynchronous release.