Search

The neuropil, the plexus of axons and dendrites, plays a critical role in operating the circuit processing of the nervous system. Revealing the spatiotemporal activity pattern within the neuropil would clarify how the information flows throughout the nervous system. However, calcium imaging to examine the circuit dynamics has mainly focused on the population of somas due to their discrete distribution. Development of a methodology to analyze the calcium imaging data of the densely packed neuropil would provide us with new insights into the circuit dynamics. Here, we propose a new method to decompose calcium imaging data of the neuropil into populations of bouton-like synaptic structures. To this aim, we pan-neuronally expressed a membrane-bound form of the calcium probe protein, CD4::GCaMP6f in Drosophila larvae. With the probe, we succeeded in visualizing a calcium signal in the neuropil. We obtained the baseline of the signal in the spatial- and temporal-dimension by an iteration method, which enabled the alignment of the neuropil at a single bouton scale and attenuation of the sample deformation and stage drift during the imaging. To group subsets of pixels into bouton-like structures, we introduced a proximity matrix and optimized the clustering configuration by the Markov Chain Monte Carlo method with Simulated Annealing algorithm, which is established in statistical physics. We applied this procedure to the neuropil in the isolated central nervous system of Drosophila larvae and succeeded in extracting individual bouton-like structures and relating them to population neural activity for larval fictive motion. These results demonstrate the usefulness and significance of the neuropil calcium imaging and its decomposition to study the spatiotemporal patterns during the operation of neural processing.

Funder: Howard Hughes Medical Institute, Typical patterned movements in animals are achieved through combinations of contraction and delayed relaxation of groups of muscles. However, how intersegmentally coordinated patterns of muscular relaxation are regulated by the neural circuits remains poorly understood. Here, we identify Canon, a class of higher-order premotor interneurons, that regulates muscular relaxation during backward locomotion of Drosophila larvae. Canon neurons are cholinergic interneurons present in each abdominal neuromere and show wave-like activity during fictive backward locomotion. Optogenetic activation of Canon neurons induces relaxation of body wall muscles, whereas inhibition of these neurons disrupts timely muscle relaxation. Canon neurons provide excitatory outputs to inhibitory premotor interneurons. Canon neurons also connect with each other to form an intersegmental circuit and regulate their own wave-like activities. Thus, our results demonstrate how coordinated muscle relaxation can be realized by an intersegmental circuit that regulates its own patterned activity and sequentially terminates motor activities along the anterior-posterior axis.

Mapping the wired connectivity of a nervous system is a prerequisite for full understanding of function. In this respect, such endeavours can be likened to genome sequencing projects. These projects similarly produce impressive amounts of data which, whilst a technical tour-de-force, remain under-utilised without validation. Validation of neuron synaptic connectivity requires electrophysiology which has the necessary temporal and spatial resolution to map synaptic connectivity. However, this technique is not common and requires extensive equipment and training to master, particularly when applied to the small CNS of the Drosophila larva. Thus, validation of connectivity in this CNS has been more reliant on behavioural analyses and, in particular, activity imaging using the calcium-sensor GCaMP. Whilst both techniques are powerful, they each have significant limitations for this purpose. Here we use electrophysiology to validate an array of driver lines reported to label specific premotor interneurons that the Drosophila connectome project suggests are monosynaptically connected to an identified motoneuron termed the anterior corner cell (aCC). Our results validate this proposition for four selected lines. Thus, in addition to validating the connectome with respect to these four premotor interneurons, our study highlights the need to functionally validate driver lines prior to use.

Animal locomotion requires spatiotemporally coordinated contraction of muscles throughout the body. Here, we investigate how contractions of antagonistic groups of muscles are intersegmentally coordinated during bidirectional crawling of Drosophila larvae. We identify two pairs of higher-order premotor excitatory interneurons present in each abdominal neuromere that intersegmentally provide feedback to the adjacent neuromere during motor propagation. The two feedback neuron pairs are differentially active during either forward or backward locomotion but commonly target a group of premotor interneurons that together provide excitatory inputs to transverse muscles and inhibitory inputs to the antagonistic longitudinal muscles. Inhibition of either feedback neuron pair compromises contraction of transverse muscles in a direction-specific manner. Our results suggest that the intersegmental feedback neurons coordinate contraction of synergistic muscles by acting as delay circuits representing the phase lag between segments. The identified circuit architecture also shows how bidirectional motor networks could be economically embedded in the nervous system., Locomotion involves the coordinated contraction of antagonistic muscles. Here, the authors report that in Drosophila larvae a pair of higher-order feedback neurons temporally regulates the intersegmental coordination of contraction of synergistic muscles enabling bidirectional movement.

The way in which the central nervous system (CNS) governs animal movement is complex and difficult to solve solely by the analyses of muscle movement patterns. We tackle this problem by observing the activity of a large population of neurons in the CNS of larval Drosophila. We focused on two major behaviors of the larvae, forward and backward locomotion, and analyzed the neuronal activity related to these behaviors during fictive locomotion that spontaneously occurs in the isolated CNS. We expressed genetically-encoded calcium indicator, GCaMP, and a nuclear marker in all neurons and used digital scanned light-sheet microscopy to record neural activities in the entire ventral nerve cord at a fast frame rate. We developed image processing tools that automatically detect the cell position based on the nuclear staining and allocate the activity signals to each detected cell. We also applied a machine learning-based method that we developed recently to assign motor status in each time frame. Based on these methods, we find cells whose activity is biased to forward versus backward locomotion and vice versa. In particular, we identified a group of neurons near the boundary of subesophageal zone (SEZ) and thoracic neuromeres, which are strongly active during an early phase of backward but not forward fictive locomotion. Our experimental procedure and computational pipeline enable systematic identification of neurons to show characteristic motor activities in larval Drosophila.

Finding food sources is essential for survival. Insects detect nutrients with external taste receptor neurons. Drosophila possesses multiple taste organs that are distributed throughout its body. However, the role of different taste organs in feeding remains poorly understood. By blocking subsets of sweet taste receptor neurons, we show that receptor neurons in the legs are required for immediate sugar choice. Furthermore, we identify two anatomically distinct classes of sweet taste receptor neurons in the leg. The axonal projections of one class terminate in the thoracic ganglia, whereas the other projects directly to the brain. These two classes are functionally distinct: the brain-projecting neurons are involved in feeding initiation, whereas the thoracic ganglia-projecting neurons play a role in sugar-dependent suppression of locomotion. Distinct receptor neurons for the same taste quality may coordinate early appetitive responses, taking advantage of the legs as the first appendages to contact food., Locating food sources is essential for the survival of animals. Here, the authors identify two functionally and anatomically distinct classes of sweet taste receptor neurons in Drosophila legs, involved in feeding initiation and sugar-dependent suppression of locomotion.

Animals adaptively respond to a tactile stimulus by choosing an ethologically relevant behavior depending on the body location of the stimuli. Here, we investigate how somatosensory inputs on different body segments are linked to distinct motor outputs in Drosophila larvae. Larvae escape by backward locomotion when touched on the head, while they crawl forward when touched on the tail. We identify a class of segmentally repeated second-order somatosensory interneurons, that we named Wave, whose activation in anterior and posterior segments elicit backward and forward locomotion, respectively. Anterior and posterior Wave neurons extend their dendrites in opposite directions to receive somatosensory inputs from the head and tail, respectively. Downstream of anterior Wave neurons we identify premotor circuits including the neuron A03a5, which together with Wave is necessary for the backward locomotion touch response. Thus, Wave neurons match their receptive field to appropriate motor programs by participating in different circuits in different segments.

Animals adaptively respond to a tactile stimulus by choosing an ethologically relevant behavior depending on the location of the stimuli. Here, we investigate how somatosensory inputs on different body segments are linked to distinct motor outputs in Drosophila larvae. Larvae escape by backward locomotion when touched on the head, while they crawl forward when touched on the tail. We identify a class of segmentally repeated second-order somatosensory interneurons, that we named Wave, whose activation in anterior and posterior segments elicit backward and forward locomotion, respectively. Anterior and posterior Wave neurons extend their dendrites in opposite directions to receive somatosensory inputs from the head and tail, respectively. Downstream of anterior Wave neurons, we identify premotor circuits including the neuron A03a5, which together with Wave, is necessary for the backward locomotion touch response. Thus, Wave neurons match their receptive field to appropriate motor programs by participating in different circuits in different segments.

Serotonin (5-HT) is known to modulate motor outputs in a variety of animal behaviors. However, the downstream neural pathways of 5-HT remain poorly understood. We studied the role of 5-HT in directional change, or turning, behavior of fruit fly (Drosophila melanogaster) larvae. We analyzed light- and touch-induced turning and found that turning is a combination of three components: bending, retreating, and rearing. Serotonin transmission suppresses rearing; when we inhibited 5-HT neurons with Shibire or Kir2.1, rearing increased without affecting the occurrence of bending or retreating. Increased rearing in the absence of 5-HT transmission often results in slower or failed turning, indicating that suppression of rearing by 5-HT is critical for successful turning. We identified a class of abdominal neurons called the abdominal LK neurons (ABLKs), which express the 5-HT1B receptor and the neuropeptide leucokinin, as downstream targets of 5-HT that are involved in the control of turning. Increased rearing was observed when neural transmission or leucokinin synthesis was inhibited in these cells. Forced activation of ABLKs also increased rearing, suggesting that an appropriate level of ABLK activity is critical for the control of turning. Calcium imaging revealed that ABLKs show periodic activation with an interval of ∼15 s. The activity level of ABLKs increased and decreased in response to a 5-HT agonist and antagonist, respectively. Our results suggest that 5-HT modulates larval turning by regulating the activity level of downstream ABLK neurons and secretion of the neuropeptide leucokinin.

While much evidence suggests that postsynaptic dynamism contributes to the formation of synapses, few studies have addressed its possible role in target selection. Do postsynaptic motile structures seek specific synaptic partner cells, as does the presynaptic growth cone? Here we studied the dynamics of myopodia, postsynaptic filopodia in Drosophila muscles, and the role of Capricious (CAPS) during the process of synaptic matchmaking. CAPS is a target recognition molecule with an extracellular domain containing leucine-rich repeat sequences. It is expressed in specific subsets of embryonic/larval body wall muscles, including muscle 12 (M12). We provide evidence that implicates the tips of myopodia as loci of initial neuromuscular recognition: (1) CAPS, expressed as a GFP-fusion protein in M12, accumulated at the tips of myopodia; and (2) simultaneous live imaging of presynaptic motoneurons and postsynaptic myopodia revealed that initial neuromuscular contacts occur at the tips of myopodia. The live imaging also showed that individual postsynaptic myopodia appear to be able to discriminate partner and non-partner presynaptic cells: whereas many myopodial contacts with the partner motoneurons are stabilized to form synapses, those with non-partner neurons are retracted. In caps mutants, or in double mutants lacking both CAPS and the closely related protein Tartan, we observed fewer contacts between myopodia of M12 and the presynaptic growth cones during the process of initial neuromuscular interaction. The nascent synaptic sites of M12 were also reduced. These results provide evidence for the sensing function of postsynaptic filopodia, and implicate Caps-mediated recognition at the tips of myopodia in synaptic matching.

Halorhodopsin (NpHR), a light-driven microbial chloride pump, enables silencing of neuronal function with superb temporal and spatial resolution. Here, we generated a transgenic line of Drosophila that drives expression of NpHR under control of the Gal4/UAS system. Then, we used it to dissect the functional properties of neural circuits that regulate larval peristalsis, a continuous wave of muscular contraction from posterior to anterior segments. We first demonstrate the effectiveness of NpHR by showing that global and continuous NpHR-mediated optical inhibition of motor neurons or sensory feedback neurons induce the same behavioral responses in crawling larvae to those elicited when the function of these neurons are inhibited by Shibire(ts), namely complete paralyses or slowed locomotion, respectively. We then applied transient and/or focused light stimuli to inhibit the activity of motor neurons in a more temporally and spatially restricted manner and studied the effects of the optical inhibition on peristalsis. When a brief light stimulus (1-10 sec) was applied to a crawling larva, the wave of muscular contraction stopped transiently but resumed from the halted position when the light was turned off. Similarly, when a focused light stimulus was applied to inhibit motor neurons in one or a few segments which were about to be activated in a dissected larva undergoing fictive locomotion, the propagation of muscular constriction paused during the light stimulus but resumed from the halted position when the inhibition (>5 sec) was removed. These results suggest that (1) Firing of motor neurons at the forefront of the wave is required for the wave to proceed to more anterior segments, and (2) The information about the phase of the wave, namely which segment is active at a given time, can be memorized in the neural circuits for several seconds.

Despite extensive investigations into the mechanisms of aerobic respiration in mitochondria, the spontaneous metabolic activity of individual cells within a whole animal has not been observed in real time. Consequently, little is known about whether and how the level of mitochondrial energy metabolism is regulated in a cell during development of intact systems. Here we studied the dynamics of postsynaptic oxidative metabolism by monitoring the redox state of mitochondrial flavoproteins, an established indicator of energy metabolism, at the developing Drosophila neuromuscular junction. We detected transient and spatially synchronized flavoprotein autofluorescence signals in postsynaptic muscle cells. These signals were dependent on the energy substrates and coupled to changes in mitochondrial membrane potential and Ca2+ concentration. Notably, the rate of autofluorescence signals increased during synapse formation through contact with the motoneuronal axon. This rate was also influenced by the magnitude of synaptic inputs. Thus, presynaptic cells tightly regulate postsynaptic energy metabolism presumably to maintain an energetic balance during neuromuscular synaptogenesis. Our results suggest that flavoprotein autofluorescence imaging should allow us to begin assessing the progress of synapse formation from a metabolic perspective.

Neuronal circuits are formed according to a genetically predetermined program and then reconstructed in an experience-dependent manner. While the existence of experience-dependent plasticity has been demonstrated for the visual and other sensory systems, it remains unknown whether this is also the case for motor systems. Here we examined the effects of eliminating sensory inputs on the development of peristaltic movements in Drosophila embryos and larvae. The peristalsis is initially slow and uncoordinated, but gradually develops into a mature pattern during late embryonic stages. We tested whether inhibiting the transmission of specific sensory neurons during this period would have lasting effects on the properties of the sensorimotor circuits. We applied Shibire-mediated inhibition for six hours during embryonic development (15–21 h after egg laying [AEL]) and studied its effects on peristalsis in the mature second- and third-instar larvae. We found that inhibition of chordotonal organs, but not multidendritic neurons, led to a lasting decrease in the speed of larval locomotion. To narrow down the sensitive period, we applied shorter inhibition at various embryonic and larval stages and found that two-hour inhibition during 16–20 h AEL, but not at earlier or later stages, was sufficient to cause the effect. These results suggest that neural activity mediated by specific sensory neurons is involved in the maturation of sensorimotor circuits in Drosophila and that there is a critical period for this plastic change. Consistent with a role of chordotonal neurons in sensory feedback, these neurons were activated during larval peristalsis and acute inhibition of their activity decreased the speed of larval locomotion.