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 GCaMP¹, 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 feeding⁶ (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER⁷ with a calcium imaging technique⁸ 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 Ca²⁺ 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.     

Serotonin and Central Mechanisms Underlying Motor Control

The review considers in a historical aspect the published data on the role of serotonin in brain activity, as well as on the structure and organization of neuronal projections of serotonergic nuclei. In addition, information on the facilitatory and inhibitory effects of serotonin on neurons of various brain regions under both in vivo and in vitro conditions is presented. General characteristics of the main types of central serotonin receptors are also given. It is emphasized that such receptors form a heterogeneous group, and this is the reason for the diversity of the effects when agonists and antagonists are applied. Regularities characteristic of changes in the activity of serotonergic system over the sleep-wakefulness cycle are also analyzed in this review; data on the involvement of serotonin in motor control are cited. Possible reasons for the complexity and multiplicity of the effects evoked by serotonin at different levels of the CNS and within various neuronal structures in the course of motor behavior are discussed.     

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.Download video file.^{(188M, mp4)}     

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker^1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain^3,4 and ventral nerve cord of embryonic^5,6, larval^7,8,9,10, and adult Drosophila^11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN5¹⁵), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.     

Immunocytochemical Localization of TASK-3 Channels in Rat Motor Neurons

Motor neurons are large cholinergic neurons located in the brain stem and spinal cord. In recent years, a functional role for TASK channels in cellular excitability and vulnerability to anesthetics of motor neurons has been described. Using a polyclonal monospecific antibody against the tandem pore domain K⁺ channel (K2P channel) TWIK-related acid-sensitive K⁺ channel (TASK-3), we analyzed the expression of the TASK-3 protein in motor systems of the rat CNS. Immunocytochemical staining showed strong TASK-3 expression in motor neurons of the facial, trigeminal, ambiguus, and hypoglossal nuclei. Oculomotor nuclei (including trochlear and abducens nucleus) were also strongly positive for TASK-3. The parasympathetic Edinger-Westphal nucleus and dorsal vagal nucleus showed significant, but weaker expression compared with somato- and branchiomotoric neurons. In addition, motor neurons in the anterior horn of the spinal cord were also strongly labeled for TASK-3 immunoreactivity. Based on morphological criteria, TASK-3 was found in the somatodendritic compartment of motor neurons. Cellular staining using methyl green and immunofluorescence double-labeling with anti-vesicular acetylcholine transporter (anti-vAChT) indicated ubiquitous TASK-3 expression in motor neurons, whereas in other brain regions TASK-3 showed a widespread but not ubiquitous expression. In situ hybridization using a TASK-3 specific riboprobe verified the expression of TASK-3 in motor neurons at the mRNA level.     

A Plastic Visual Pathway Regulates Cooperative Behavior in Drosophila Larvae

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Correlated Conductance Parameters in Leech Heart Motor Neurons Contribute to Motor Pattern Formation

Neurons can have widely differing intrinsic membrane properties, in particular the density of specific conductances, but how these contribute to characteristic neuronal activity or pattern formation is not well understood. To explore the relationship between conductances, and in particular how they influence the activity of motor neurons in the well characterized leech heartbeat system, we developed a new multi-compartmental Hodgkin-Huxley style leech heart motor neuron model. To do so, we evolved a population of model instances, which differed in the density of specific conductances, capable of achieving specific output activity targets given an associated input pattern. We then examined the sensitivity of measures of output activity to conductances and how the model instances responded to hyperpolarizing current injections. We found that the strengths of many conductances, including those with differing dynamics, had strong partial correlations and that these relationships appeared to be linked by their influence on heart motor neuron activity. Conductances that had positive correlations opposed one another and had the opposite effects on activity metrics when perturbed whereas conductances that had negative correlations could compensate for one another and had similar effects on activity metrics.     

Feeding Motor Patterns in Anurans: Insights from Biomechanical Modeling

During feeding in anurans, the mouth opens while the tongue,which is attached to the mandible at the front of the mouth,rotates forward. Due to the relative simplicity of its anatomyand the complexity of its motion, tongue protraction in frogspresents an ideal system for exploring the neural control ofmultijoint movements. In this study, we used a forward dynamic,rigid body model with four segments and two muscles to investigateopen loop control of tongue protraction in the Australian white-lippedtree frog, Litoria caerulea. Model parameters include the massdistribution, initial position and initial angular velocityof each segment and the anatomy and physiology of each muscle.Model variables include the level of muscle activation at eachtime step and impulsive torques to open and close the mouth.The model gives X,Y coordinates of each segment and joint anglesat each time step as output. The model was tested using scaled,normalized EMG signals and impulsive joint torques to predictthe paths of the lower jaw tip and tongue tip. Predicted pathswere compared to experimentally observed paths using Pearsonproduct-moment correlation coefficients. Simulations demonstratethat the genioglossus muscles likely play a minor role, if any,in determining the trajectory of the tongue in most anurans.Most of the force for tongue protraction comes from angularmomentum transferred to the tongue by the opening jaws. In anurans,tongue protraction is dynamically stable and will occur as longas the musculoskeletal elements are in the correct initial position.     

Sensory Feedback Mechanism Underlying Entrainment of Central Pattern Generator to Mechanical Resonance

Rhythmic body motions observed in animal locomotion are known to be controlled by neuronal circuits called central pattern generators (CPGs). It appears that CPGs are energy efficient controllers that cooperate with biomechanical and environmental constraints through sensory feedback. In particular, the CPGs tend to induce rhythmic motion of the body at a natural frequency, i.e., the CPGs are entrained to a mechanical resonance by sensory feedback. The objective of this paper is to uncover the mechanism of entrainment resulting from the dynamic interaction of the CPG and mechanical system. We first develop multiple CPG models for the reciprocal inhibition oscillator (RIO) and examine through numerical experiments whether they can be entrained to a simple pendulum. This comparative study identifies the neuronal properties essential for the entrainment. We then analyze the simplest model that captures the essential dynamics via the method of harmonic balance. It is shown that robust entrainment results from a strong, positive-feedback coupling of a lightly damped mechanical system and the RIO consisting of neurons with the complete adaptation property     

Contribution of Drosophila TRPA1-Expressing Neurons to Circadian Locomotor Activity Patterns

In both vertebrates and invertebrates, Transient Receptor Potential (TRP) channels are expressed in sensory neurons and mediate environmental stimuli such as light, sound, temperature, and taste. Some of these channels, however, are expressed only in the brain and their functions remain incompletely understood. Using the GAL4/UAS binary system with a line in which the GAL4 had been knocked into the trpA1 locus in Drosophila, we recently reported new insights into TRPA1 localization and function, including its expression in approximately 15% of all circadian neurons. TRPA1 is expressed in lateral posterior neurons (LPNs), which are known to be highly sensitive to entrainment by temperature cycles. Here, I used the bacterial sodium channel, NaChBac, to examine the effects of altering the electrical properties of trpA1 neurons on circadian rhythms. My results indicate that circadian activity of the flies in the morning, daytime, and evening was affected in a temperature-dependent manner following TRPA1 neuronal activation. Remarkably, TRPA1 neuron activation in flies kept at 18°C impacted the morning peak of circadian activity even though TRPA1 is not expressed in morning cells. Taken together, these results suggest that the activation of TRPA1-expressing neurons may differentially coordinate light/dark circadian entrainment, depending on the temperature.     

The Evolution of Feeding Motor Patterns in Lizards: Modulatory Complexity and Possible Constraints

Previous research indicated that the evolution of feeding motorpatterns across major taxonomic groups might have occurred withoutlarge modifications of the control of the jaw and hyolingualmuscles. However, the proposal of this evolutionary scheme washampered by the lack of data for some key taxa such as lizards.Recent data on jaw and hyolingual feeding motor patterns ofa number of lizard families suggest extensive variability withinand among species. Although most lizards respond to changesin the structural properties of food items by modulating theactivation of the jaw and hyolingual muscles, some food specialistsmight have lost this ability. Whereas the overall similarityin motor patterns across different lineages of lizards is largefor the hyolingual muscles, jaw muscle activation patterns seemto be more flexible. Nevertheless, all data suggest that boththe jaw and hyolingual system are complexly integrated. Theelimination of feedback pathways from the hyolingual systemthrough nerve transection experiments clearly shows that feedingcycles are largely shaped by feedback interactions. Yet, novelmotor patterns including unilateral control seem to have emergedin the evolution from lizards to snakes.     

Localization and Central Projections of Primary Afferent Neurons That Innervate the Temporomandibular Joint in Cats

Primary afferent neurons that innervate the temporomandibular joint (TMJ) in cats were labeled by injecting a 2-5% solution of wheatgerm agglutinin bound to horseradish peroxidase into the joint capsule and capsular tissues in 14 cats and processing the brain stem and trigeminal ganglia using the tetramethylbenzidine method described by Mesulam (1978). The perikarya of ganglion cells that innervate the TMJ ranged in diameter from 15 to 109 μm and were primarily located in the posterolateral portion of the trigeminal ganglion. The central processes of these neurons entered the brain stem in middle pons and were distributed to all portions of the sensory trigeminal nuclei. However, the majority of labeled fibers and greatest density of terminal labeling were observed in the dorsal part of the main sensory nucleus and the subnucleus oralis of the spinal trigeminal nucleus. Very few labeled fibers were observed in the spinal tract of the trigeminal nerve below the obex. However, evidence for axon terminals was consistently observed in laminae I, II, and III of the medullary dorsal horn. These findings concur with physiological evidence showing that information from the TMJ influences neurons in rostral (Kawamura et al, 1967) and in caudal (Broton et al, 1985) portions of the trigeminal sensory nuclei.     

Central Projections of Gustatory Receptor Neurons in the Medial and the Lateral Sensilla Styloconica of Helicoverpa armigera Larvae

Food selection behavior of lepidopteran larvae is predominantly governed by the activation of taste neurons present in two sensilla styloconica located on the galea of the maxilla. In this study, we present the ultrastructure of the sensilla styloconica and the central projection pattern of their associated receptor neurons in larvae of the heliothine moth, Helicoverpa armigera. By means of light microscopy and scanning electron microscopy, the previous findings of two morphologically fairly similar sensilla comprising a socketed conic tip inserted into a large peg were confirmed. However, the peg size of the medial sensillum was found to be significantly bigger than that of the lateral sensillum. The sensory neurons derived from each sensillum styloconicum were mapped separately using anterograde staining experiments combined with confocal laser-scanning microscopy. For determining the afferents’ target regions relative to each other, we reconstructed the labeled axons and placed them into a common reference framework. The sensory axons from both sensilla projected via the ipsilateral maxillary nerve to the suboesophageal ganglion and further through the ipsilateral circumoesophageal connective to the brain. In the suboesophageal ganglion, the sensory projections targeted two areas of the ipsilateral maxillary neuropil, one located in the ventrolateral neuromere and the other adjacent to the neuromere midline. In the brain, the axon terminals targeted the dorso-anterior area of the ipsilateral tritocerebrum. As confirmed by the three-dimensional reconstructions, the target regions of the neural projections originating from each of the two sensilla styloconica were identical.     

Influence of Seasonality on Activity Patterns, Feeding Behavior, Ranging, and Grouping Patterns in Taï Chimpanzees

Lowland rain forest chimpanzees (Pan troglodytes verus) of the Taï National Park, Ivory Coast, responded to the minor dry season (July and August) of 1988 in a predictable manner by spending more time feeding, feeding more frequently on lower quality food items, reducing day range and party size, and spending more time solitarily and less time in mixed groups than during the rainy season. These behaviors are consistent with a response to scarce resources. My findings do not support Boesch's (1991, 1996) hypothesis of bisexually bonded chimpanzees. Females spent 45% of time alone and associated with males in mixed parties only 18% of their time. This major discrepancy in our results probably stems from differences in the time of year when our studies were conducted, the year in which my study was conducted (potentially scarcer resources than on average), and methodological differences: focal animal sampling of males and females equally. Although Boesch (1991, 1996) and Steiner (1996) have demonstrated that Taï parties are usually larger and more mixed, Taï chimpanzee social structure—party size and composition—during this study closely resembles that found at other study sites.     

Spindle-F Is the Central Mediator of Ik2 Kinase-Dependent Dendrite Pruning in Drosophila Sensory Neurons

During development, certain Drosophila sensory neurons undergo dendrite pruning that selectively eliminates their dendrites but leaves the axons intact. How these neurons regulate pruning activity in the dendrites remains unknown. Here, we identify a coiled-coil protein Spindle-F (Spn-F) that is required for dendrite pruning in Drosophila sensory neurons. Spn-F acts downstream of IKK-related kinase Ik2 in the same pathway for dendrite pruning. Spn-F exhibits a punctate pattern in larval neurons, whereas these Spn-F puncta become redistributed in pupal neurons, a step that is essential for dendrite pruning. The redistribution of Spn-F from puncta in pupal neurons requires the phosphorylation of Spn-F by Ik2 kinase to decrease Spn-F self-association, and depends on the function of microtubule motor dynein complex. Spn-F is a key component to link Ik2 kinase to dynein motor complex, and the formation of Ik2/Spn-F/dynein complex is critical for Spn-F redistribution and for dendrite pruning. Our findings reveal a novel regulatory mechanism for dendrite pruning achieved by temporal activation of Ik2 kinase and dynein-mediated redistribution of Ik2/Spn-F complex in neurons.     

Potency of Transgenic Effectors for Neurogenetic Manipulation in Drosophila Larvae

Genetic manipulations of neuronal activity are a cornerstone of studies aimed to identify the functional impact of defined neurons for animal behavior. With its small nervous system, rapid life cycle, and genetic amenability, the fruit fly Drosophila melanogaster provides an attractive model system to study neuronal circuit function. In the past two decades, a large repertoire of elegant genetic tools has been developed to manipulate and study neural circuits in the fruit fly. Current techniques allow genetic ablation, constitutive silencing, or hyperactivation of neuronal activity and also include conditional thermogenetic or optogenetic activation or inhibition. As for all genetic techniques, the choice of the proper transgenic tool is essential for behavioral studies. Potency and impact of effectors may vary in distinct neuron types or distinct types of behavior. We here systematically test genetic effectors for their potency to alter the behavior of Drosophila larvae, using two distinct behavioral paradigms: general locomotor activity and directed, visually guided navigation. Our results show largely similar but not equal effects with different effector lines in both assays. Interestingly, differences in the magnitude of induced behavioral alterations between different effector lines remain largely consistent between the two behavioral assays. The observed potencies of the effector lines in aminergic and cholinergic neurons assessed here may help researchers to choose the best-suited genetic tools to dissect neuronal networks underlying the behavior of larval fruit flies.