Octopaminergic descending neurons in Drosophila: Connectivity, tonic activity and relation to locomotion.

Projection neurons that communicate between different brain regions and local neurons that shape computation within a brain region form the majority of all neurons in the brain. Another important class of neurons is neuromodulatory neurons; these neurons are in much smaller numbers than projection/local neurons but have a large influence on computations in the brain. Neuromodulatory neurons are classified by the neurotransmitters they carry, such as dopamine and serotonin. Much of our knowledge of the effect of neuromodulators comes from experiments in which either a large population of neuromodulatory neurons or the entire population is perturbed. Alternatively, a given neuromodulator is exogenously applied. While these experiments are informative of the general role of the neurotransmitter, one limitation of these experiments is that the role of individual neuromodulatory neurons remains unknown. In this study, we investigate the role of a class of octopaminergic (octopamine is the invertebrate equivalent of norepinephrine) neurons in Drosophila or fruit fly. Neuromodulation in Drosophila work along similar principles as humans; and the smaller number of neuromodulatory neurons allow us to assess the role of individual neurons. This study focuses on a subpopulation of octopaminergic descending neurons (OA-DNs) whose cell bodies are in the brain and project to the thoracic ganglia. Using in-vivo whole-cell patch-clamp recordings and anatomical analyses that allow us to compare light microscopy data to the electron microscopic volumes available in the fly, we find that neurons within each cluster have similar physiological properties, including their relation to locomotion. However, neurons in the same cluster with similar anatomy have very different connectivity. Our data is consistent with the hypothesis that each OA-DN is recruited individually and has a unique function within the fly's brain.

Neurons underlying aggressive actions that are shared by both males and females in Drosophila.

Aggression involves both sexually monomorphic and dimorphic actions. How the brain implements these two types of actions is poorly understood. We found that a set of neurons, which we call CL062, previously shown to mediate male aggression also mediate female aggression. These neurons elicit aggression acutely and without the presence of a target. Although the same set of actions is elicited in males and females, the overall behavior is sexually dimorphic. The CL062 neurons do not express fruitless , a gene required for sexual dimorphism in flies, and expressed by most other neurons important for controlling fly aggression. Connectomic analysis suggests that these neurons have limited connections with fruitless expressing neurons that have been shown to be important for aggression, and signal to different descending neurons. Thus, CL062 is part of a monomorphic circuit for aggression that functions parallel to the known dimorphic circuits.

Sensorimotor transformation underlying odor-modulated locomotion in walking Drosophila.

Most real-world behaviors - such as odor-guided locomotion - are performed with incomplete information. Activity in olfactory receptor neuron (ORN) classes provides information about odor identity but not the location of its source. In this study, we investigate the sensorimotor transformation that relates ORN activation to locomotion changes in Drosophila by optogenetically activating different combinations of ORN classes and measuring the resulting changes in locomotion. Three features describe this sensorimotor transformation: First, locomotion depends on both the instantaneous firing frequency (f) and its change (df); the two together serve as a short-term memory that allows the fly to adapt its motor program to sensory context automatically. Second, the mapping between (f, df) and locomotor parameters such as speed or curvature is distinct for each pattern of activated ORNs. Finally, the sensorimotor mapping changes with time after odor exposure, allowing information integration over a longer timescale.

Behavioral algorithms and neural mechanisms underlying odor-modulated locomotion in insects.

Odors released from mates and resources such as a host and food are often the first sensory signals that an animal can detect. Changes in locomotion in response to odors are an important mechanism by which animals access resources important to their survival. Odor-modulated changes in locomotion in insects constitute a whole suite of flexible behaviors that allow insects to close in on these resources from long distances and perform local searches to locate and subsequently assess them. Here, we review changes in odor-mediated locomotion across many insect species. We emphasize that changes in locomotion induced by odors are diverse. In particular, the olfactory stimulus is sporadic at long distances and becomes more continuous at short distances. This distance-dependent change in temporal profile produces a corresponding change in an insect's locomotory strategy. We also discuss the neural circuits underlying odor modulation of locomotion.

Mechanisms of Variability Underlying Odor-Guided Locomotion.

Changes in locomotion mediated by odors (odor-guided locomotion) are an important mechanism by which animals discover resources important to their survival. Odor-guided locomotion, like most other behaviors, is highly variable. Variability in behavior can arise at many nodes along the circuit that performs sensorimotor transformation. We review these sources of variability in the context of the Drosophila olfactory system. While these sources of variability are important, using a model for locomotion, we show that another important contributor to behavioral variability is the stochastic nature of decision-making during locomotion as well as the persistence of these decisions: Flies choose the speed and curvature stochastically from a distribution and locomote with the same speed and curvature for extended periods. This stochasticity in locomotion will result in variability in behavior even if there is no noise in sensorimotor transformation. Overall, the noise in sensorimotor transformation is amplified by mechanisms of locomotion making odor-guided locomotion in flies highly variable.

Drosophila uses a tripod gait across all walking speeds, and the geometry of the tripod is important for speed control.

Changes in walking speed are characterized by changes in both the animal's gait and the mechanics of its interaction with the ground. Here we study these changes in walking Drosophila. We measured the fly's center of mass movement with high spatial resolution and the position of its footprints. Flies predominantly employ a modified tripod gait that only changes marginally with speed. The mechanics of a tripod gait can be approximated with a simple model - angular and radial spring-loaded inverted pendulum (ARSLIP) - which is characterized by two springs of an effective leg that become stiffer as the speed increases. Surprisingly, the change in the stiffness of the spring is mediated by the change in tripod shape rather than a change in stiffness of individual legs. The effect of tripod shape on mechanics can also explain the large variation in kinematics among insects, and ARSLIP can model these variations.

Mechanisms underlying attraction to odors in walking Drosophila.

Mechanisms that control movements range from navigational mechanisms, in which the animal employs directional cues to reach a specific destination, to search movements during which there are little or no environmental cues. Even though most real-world movements result from an interplay between these mechanisms, an experimental system and theoretical framework for the study of interplay of these mechanisms is not available. Here, we rectify this deficit. We create a new method to stimulate the olfactory system in Drosophila or fruit flies. As flies explore a circular arena, their olfactory receptor neuron (ORNs) are optogenetically activated within a central region making this region attractive to the flies without emitting any clear directional signals outside this central region. In the absence of ORN activation, the fly's locomotion can be described by a random walk model where a fly's movement is described by its speed and turn-rate (or kinematics). Upon optogenetic stimulation, the fly's behavior changes dramatically in two respects. First, there are large kinematic changes. Second, there are more turns at the border between light-zone and no-light-zone and these turns have an inward bias. Surprisingly, there is no increase in turn-rate, rather a large decrease in speed that makes it appear that the flies are turning at the border. Similarly, the inward bias of the turns is a result of the increase in turn angle. These two mechanisms entirely account for the change in a fly's locomotion. No complex mechanisms such as path-integration or a careful evaluation of gradients are necessary.

Spring-loaded inverted pendulum goes through two contraction-extension cycles during the single-support phase of walking.

Despite the overall complexity of legged locomotion, the motion of the center of mass (COM) itself is relatively simple, and can be qualitatively described by simple mechanical models. In particular, walking can be qualitatively modeled by a simple model in which each leg is described by a spring-loaded inverted pendulum (SLIP). However, SLIP has many limitations and is unlikely to serve as a quantitative model. As a first step to obtaining a quantitative model for walking, we explored the ability of SLIP to model the single-support phase of walking, and found that SLIP has two limitations. First, it predicts larger horizontal ground reaction forces (GRFs) than empirically observed. A new model - angular and radial spring-loaded inverted pendulum (ARSLIP) - can overcome this deficit. Second, although the leg spring (surprisingly) goes through contraction-extension-contraction-extensions (CECEs) during the single-support phase of walking and can produce the characteristic M-shaped vertical GRFs, modeling the single-support phase requires active elements. Despite these limitations, SLIP as a model provides important insights. It shows that the CECE cycling lengthens the stance duration allowing the COM to travel passively for longer, and decreases the velocity redirection between the beginning and end of a step.

Statistical structure of locomotion and its modulation by odors.

Most behaviors such as making tea are not stereotypical but have an obvious structure. However, analytical methods to objectively extract structure from non-stereotyped behaviors are immature. In this study, we analyze the locomotion of fruit flies and show that this non-stereotyped behavior is well-described by a Hierarchical Hidden Markov Model (HHMM). HHMM shows that a fly's locomotion can be decomposed into a few locomotor features, and odors modulate locomotion by altering the time a fly spends performing different locomotor features. Importantly, although all flies in our dataset use the same set of locomotor features, individual flies vary considerably in how often they employ a given locomotor feature, and how this usage is modulated by odor. This variation is so large that the behavior of individual flies is best understood as being grouped into at least three to five distinct clusters, rather than variations around an average fly.

A simple extension of inverted pendulum template to explain features of slow walking✰.

Locomotion involves complex interactions between an organism and its environment. Despite these complex interactions, many characteristics of the motion of an animal's center of mass (COM) can be modeled using simple mechanical models such as inverted pendulum (IP) and spring-loaded inverted pendulum (SLIP) which employ a single effective leg to model an animal's COM. However, because these models are simple, they also have many limitations. We show that one limitation of IP and SLIP and many other simple mechanical models of locomotion is that they cannot model many observed features of locomotion at slow speeds. This limitation is due to the fact that the gravitational force is too strong, and, if unopposed, compels the animal to complete its stance in a relatively short time. We propose a new model, AS-IP (Angular Spring modulated Inverted Pendulum), in which the body is attached to the leg using springs which resist the leg's movement away from the vertical plane, and thus provides a means to model forces that effectively counter gravity. We show that AS-IP provides a mechanism by which an animal can tune its stance duration, and provide evidence that AS-IP is an excellent model for the motion of a fly's COM. More generally, we conclude that combining AS-IP with SLIP will greatly expand our ability to model legged locomotion over a range of speeds.

Organization of descending neurons in Drosophila melanogaster.

Neural processing in the brain controls behavior through descending neurons (DNs) - neurons which carry signals from the brain to the spinal cord (or thoracic ganglia in insects). Because DNs arise from multiple circuits in the brain, the numerical simplicity and availability of genetic tools make Drosophila a tractable model for understanding descending motor control. As a first step towards a comprehensive study of descending motor control, here we estimate the number and distribution of DNs in the Drosophila brain. We labeled DNs by backfilling them with dextran dye applied to the neck connective and estimated that there are ~1100 DNs distributed in 6 clusters in Drosophila. To assess the distribution of DNs by neurotransmitters, we labeled DNs in flies in which neurons expressing the major neurotransmitters were also labeled. We found DNs belonging to every neurotransmitter class we tested: acetylcholine, GABA, glutamate, serotonin, dopamine and octopamine. Both the major excitatory neurotransmitter (acetylcholine) and the major inhibitory neurotransmitter (GABA) are employed equally; this stands in contrast to vertebrate DNs which are predominantly excitatory. By comparing the distribution of DNs in Drosophila to those reported previously in other insects, we conclude that the organization of DNs in insects is highly conserved.

Odor-identity dependent motor programs underlie behavioral responses to odors.

All animals use olfactory information to perform tasks essential to their survival. Odors typically activate multiple olfactory receptor neuron (ORN) classes and are therefore represented by the patterns of active ORNs. How the patterns of active ORN classes are decoded to drive behavior is under intense investigation. In this study, using Drosophila as a model system, we investigate the logic by which odors modulate locomotion. We designed a novel behavioral arena in which we could examine a fly's locomotion under precisely controlled stimulus condition. In this arena, in response to similarly attractive odors, flies modulate their locomotion differently implying that odors have a more diverse effect on locomotion than was anticipated. Three features underlie odor-guided locomotion: First, in response to odors, flies modulate a surprisingly large number of motor parameters. Second, similarly attractive odors elicit changes in different motor programs. Third, different ORN classes modulate different subset of motor parameters.

Activity in descending dopaminergic neurons represents but is not required for leg movements in the fruit fly Drosophila.

Modulatory descending neurons (DNs) that link the brain to body motor circuits, including dopaminergic DNs (DA-DNs), are thought to contribute to the flexible control of behavior. Dopamine elicits locomotor-like outputs and influences neuronal excitability in isolated body motor circuits over tens of seconds to minutes, but it remains unknown how and over what time scale DA-DN activity relates to movement in behaving animals. To address this question, we identified DA-DNs in the Drosophila brain and developed an electrophysiological preparation to record and manipulate the activity of these cells during behavior. We find that DA-DN spike rates are rapidly modulated during a subset of leg movements and scale with the total speed of ongoing leg movements, whether occurring spontaneously or in response to stimuli. However, activating DA-DNs does not elicit leg movements in intact flies, nor do acute bidirectional manipulations of DA-DN activity affect the probability or speed of leg movements over a time scale of seconds to minutes. Our findings indicate that in the context of intact descending control, changes in DA-DN activity are not sufficient to influence ongoing leg movements and open the door to studies investigating how these cells interact with other descending and local neuromodulatory inputs to influence body motor output.

Olfactory modulation of flight in Drosophila is sensitive, selective and rapid.

Freely flying Drosophila melanogaster respond to odors by increasing their flight speed and turning upwind. Both these flight behaviors can be recapitulated in a tethered fly, which permits the odor stimulus to be precisely controlled. In this study, we investigated the relationship between these behaviors and odor-evoked activity in primary sensory neurons. First, we verified that these behaviors are abolished by mutations that silence olfactory receptor neurons (ORNs). We also found that antennal mechanosensors in Johnston's organ are required to guide upwind turns. Flight responses to an odor depend on the identity of the ORNs that are active, meaning that these behaviors involve odor discrimination and not just odor detection. Flight modulation can begin rapidly (within about 85 ms) after the onset of olfactory transduction. Moreover, just a handful of spikes in a single ORN type is sufficient to trigger these behaviors. Finally, we found that the upwind turn is triggered independently from the increase in wingbeat frequency, implying that ORN signals diverge to activate two independent and parallel motor commands. Together, our results show that odor-evoked flight modulations are rapid and sensitive responses to specific patterns of sensory neuron activity. This makes these behaviors a useful paradigm for studying the relationship between sensory neuron activity and behavioral decision-making in a simple and genetically tractable organism.

Signaling by olfactory receptor neurons near threshold.

An important contributing factor for the high sensitivity of sensory systems is the exquisite sensitivity of the sensory receptor cells. We report here the signaling threshold of the olfactory receptor neuron (ORN). We first obtained a best estimate of the size of the physiological electrical response successfully triggered by a single odorant-binding event on a frog ORN, which was ∼0.034 pA and had an associated transduction domain spanning only a tiny fraction of the length of an ORN cilium. We also estimated the receptor-current threshold for an ORN to fire action potentials in response to an odorant pulse, which was ∼1.2 pA. Thus, it takes about 35 odorant-binding events successfully triggering transduction during a brief odorant pulse in order for an ORN to signal to the brain.

Divisive normalization in olfactory population codes.

In many regions of the visual system, the activity of a neuron is normalized by the activity of other neurons in the same region. Here we show that a similar normalization occurs during olfactory processing in the Drosophila antennal lobe. We exploit the orderly anatomy of this circuit to independently manipulate feedforward and lateral input to second-order projection neurons (PNs). Lateral inhibition increases the level of feedforward input needed to drive PNs to saturation, and this normalization scales with the total activity of the olfactory receptor neuron (ORN) population. Increasing total ORN activity also makes PN responses more transient. Strikingly, a model with just two variables (feedforward and total ORN activity) accurately predicts PN odor responses. Finally, we show that discrimination by a linear decoder is facilitated by two complementary transformations: the saturating transformation intrinsic to each processing channel boosts weak signals, while normalization helps equalize responses to different stimuli.

Functional properties of synaptic transmission in primary sense organs.

Sensory receptors transduce physical stimuli in the environment into neural signals that are interpreted by the brain. Although considerable attention has been given to how the sensitivity and dynamic range of sensory receptors is established, peripheral synaptic interactions improve the fidelity with which receptor output is transferred to the brain. For instance, synapses in the retina, cochlea, and primary olfactory system use mechanisms that fine-tune the responsiveness of postsynaptic neurons and the dynamics of exocytosis; these permit microcircuit interactions to encode efficiently the output of sensory receptors with the fidelity and dynamic range necessary to extract the salient features of the physical stimuli. The continuous matching of presynaptic and postsynaptic responsiveness highlight how the primary sensory organs have been optimized and can be modulated to resolve sparse sensory signals and to encode the entire range of receptor output.

Sensory processing in the Drosophila antennal lobe increases reliability and separability of ensemble odor representations.

Here we describe several fundamental principles of olfactory processing in the Drosophila melanogaster antennal lobe (the analog of the vertebrate olfactory bulb), through the systematic analysis of input and output spike trains of seven identified glomeruli. Repeated presentations of the same odor elicit more reproducible responses in second-order projection neurons (PNs) than in their presynaptic olfactory receptor neurons (ORNs). PN responses rise and accommodate rapidly, emphasizing odor onset. Furthermore, weak ORN inputs are amplified in the PN layer but strong inputs are not. This nonlinear transformation broadens PN tuning and produces more uniform distances between odor representations in PN coding space. In addition, portions of the odor response profile of a PN are not systematically related to their direct ORN inputs, which probably indicates the presence of lateral connections between glomeruli. Finally, we show that a linear discriminator classifies odors more accurately using PN spike trains than using an equivalent number of ORN spike trains.

Excitatory interactions between olfactory processing channels in the Drosophila antennal lobe.

Each odorant receptor gene defines a unique type of olfactory receptor neuron (ORN) and a corresponding type of second-order neuron. Because each odor can activate multiple ORN types, information must ultimately be integrated across these processing channels to form a unified percept. Here, we show that, in Drosophila, integration begins at the level of second-order projection neurons (PNs). We genetically silence all the ORNs that normally express a particular odorant receptor and find that PNs postsynaptic to the silent glomerulus receive substantial lateral excitatory input from other glomeruli. Genetically confining odor-evoked ORN input to just one glomerulus reveals that most PNs postsynaptic to other glomeruli receive indirect excitatory input from the single ORN type that is active. Lateral connections between identified glomeruli vary in strength, and this pattern of connections is stereotyped across flies. Thus, a dense network of lateral connections distributes odor-evoked excitation between channels in the first brain region of the olfactory processing stream.

Elementary response of olfactory receptor neurons to odorants.

Signaling by heterotrimeric GTP-binding proteins (G proteins) drives numerous cellular processes. The number of G protein molecules activated by a single membrane receptor is a determinant of signal amplification, although in most cases this parameter remains unknown. In retinal rod photoreceptors, a long-lived photoisomerized rhodopsin molecule activates many G protein molecules (transducins), yielding substantial amplification and a large elementary (single-photon) response, before rhodopsin activity is terminated. Here we report that the elementary response in olfactory transduction is extremely small. A ligand-bound odorant receptor has a low probability of activating even one G protein molecule because the odorant dwell-time is very brief. Thus, signal amplification in olfactory transduction appears fundamentally different from that of phototransduction.