CHAPTER 10

Descending neuron for freezing behavior in Drosophila melanogaster

Shigehiro Namiki

Abstract Freezing is a defensive behavior characterized by immobility, which makes it difficult to be detected by predators. Flies show freezing behavior in response to visual stimulus like looming, which is relevant to the attack of predators. Brain control of freezing behavior is largely unknown. Here I introduce the analysis of the neuronal mechanism, using a set of driver lines for a population of descending neurons, which is a bottleneck of the nervous system, connecting the brain and ventral nerve cord in Drosophila. A pair of descending neurons has been identified as a critical component for the behavior. I also discuss the connectivity of the neurons.

S. Namiki

Research Center for Advanced Science and Technology, The University of Tokyo,

Meguro 153-8904, Tokyo, Japan

e-mail: namiki@rcast.u-tokyo.ac.jp

10.1 Tonic immobility and freezing

Defense behavior are behavioral actions that aim to minimize the chances of being predated. When face with the threat, insects can show difference types of defensive behavior. One of these is tonic immobility (TI), where insects show a motionless posture lasting from seconds to hours. TI is triggered by physical contact or close proximity of a predator (Humphreys and Ruxton 2018). Although sensory and motor mechanisms have been investigated (Chapter 5), neuronal mechanisms are largely unknown.

Another kind of defensive behavior is freezing, where insects show a cessation of all movements except those associated with respiration and vision (Misslin 2003). Because movement acts as a releasing stimulus for predatory attack, freezing contribute to prevent detection from predator (Eilam 2005). Freezing behavior have been observed in a wide variety of insects such as cricket (Adamo et al. 2013), moth (Chapter 4). Locust also show freezing behavior in response to vibration stimuli on the platform (Friedel 1999).

Freezing behavior should be distinguished with TI. Whereas freezing occurs much earlier in the sequence of a predatory attack, TI occurs at later stage of defensive behavior. Unlike the case of TI, freezing behavior is not necessary to associate with specific posture and usually keep the posture when detect the presence of predator. In addition, responsiveness to the external sensory stimuli is reduced in TI, whereas functionally unchanged in freezing behavior.

Both TI and freezing behavior are a different option in defensive behavior. In spite of the differences, freezing share several behavioral features with TI. Both behavior affect muscle activity throughout the body. Both types of behavior can be driven by the brain (e.g. visual stimulus of predator), and the brain control of the behaviors are mediated by a population of descending neurons, which connect the brain and body ganglia. Indeed, studies using surgical experiment suggest the importance of the brain in maintaining TI in some species.

Although TI is not reported in Drosophila, some female flies show thanatosis, a form of TI to prevent males from mating them in the fly of Asilidae, Efferia vapipes (Dennis and Lavigne 1976). Candidate neuronal pathways for TI is totally unknown but those for freezing behavior has been reported recently. In this chapter, I first describe the descending neural command for freezing behavior identified in crayfish. Second, I describe recent work about the finding of a descending neuronal component relevant for executing freezing behavior in Drosophila (Zacarias et al. 2018).

10.2 Defensive behavior and candidate command for freezing in crayfish

Freezing behavior has been observed in many crustacean (Pereyra et al. 1999; Oliva et al. 2007). Juvenile crayfish exhibits either escape or freezing in response to predatory attack. In the case of escape, crayfishes exhibit tail-flip movement and go away from predator. Liden et al. 2010 examine behavioral output of crayfishes in response to approaching shadow (Liden et al. 2010). The authors present a shadow on the white paper and capture the crayfish movement in a narrow tunnel. They also measure the filed potentials generated during crayfish response. The escape and freezing are exclusive to all type of stimulus tested in the study and the occurrence varies depending on the approaching speed of the shadow. Animals exhibit fewer escape and more freezing in response to fast-approaching shadow. By contrast, animals exhibit more escape and less freezing in response to slow-approaching shadow. The variability of behavioral output is explained by cost-benefit relationship. Whereas tail-flipping is effective strategy for the escape response (Herberholz et al. 2004), the energetic cost is high (Webb 1979). When predator approaches at the velocities that makes escape impossible, animals show freezing along with reducing the cost.

The neuronal circuits for escape response have been identified in crayfish thus far. For example, it is known that tail-flipping is driven by a pair of medial giant interneurons (MG) (Liden and Herberholz 2008). The MG is activated when moving visual stimuli or tactile input. Single action potential in the MG is sufficient to activate the escape response.

Although neuronal mechanisms for the freezing remains unclear, a candidate neuron for freezing behavior has been reported (Bowerman and Larimer 1974). A movement-suppressing command fiber called the ‘Statue fibre’, has been reported in descending system in crayfish Procambarus clarkii. In the study, crayfish is secured by a branchiostegite to the end of a rigid object and one side of circumoesophageal connective is de-sheathed and small bundles of fibres are stripped from the connective and suspended on the electrodes for stimulation. The position of the axon running the connective is relative conserved and researchers can create a cross-sectional map of axon for individual neurons (Wiersma 1958) (Fig. 10.1A). This allows to record from a target neuron via electrodes. The animal is positioned over the outer edge of a freely rotating horizontal walking wheel. Nerve bundle is stimulated with electric pulses and the behavior is filed by camera. Activation of this unit terminate the ongoing activity by freezing irrespective of position (Fig. 10.1B). The position is maintained for the stimulus duration, which is reminiscent to naturally occurred freezing response.

10.3 Defensive behavior to looming object in flies

When visual stimuli such as looming presented, flies show defensive behavior including jumping, running (increasing speed) and freezing. Flies show a sequence of defensive behavior in response to a naturalistic threatening stimulus, a looming object (Hammond and O’Shea 2007). Looming-stimulus elicits sequence of actions: freezing, posture adjustment, wing elevation and jump (Card 2012) (Fig. 10.2). The sequence of each action is flexible, suggesting the escape behavior is not fixed action pattern.

Neuronal circuits for jumping behavior have been investigated (Trimarchi and Schneiderman 1995; Card and Dickinson 2008b; von Reyn et al. 2014). Simple visually elicited jump response is mediated by the giant fiber neuron (Allen et al. 2006). Single action potential of the giant fiber is sufficient to trigger the full sequence of jump response. However, the action of the giant fiber does not induce other behavioral modules: postural adjustment and wing elevation. Other neuronal pathways must be involved but such neuronal components have not yet been anatomically identified thus far. Escape response which does not require the giant fiber activation is reported (Holmqvist 1994). Another study electrophysiologically characterize the unit which are responsive to the looming stimulus (Fotowat et al. 2009). These studies indicate the presence of parallel neuronal pathway for controlling escape behavior.

In addition, flies also exhibit freezing behavior (also referred to as “pause”) in response to the visual stimuli such as moving shadow (Gibson et al. 2015) and looming (Card and Dickinson 2008a). Moving shadow is a stimulus like a predator (e.g. dragonfly) flying around the fly and looming is a stimulus like a predator approaching the fly. To the looming stimuli, flies display stereotyped responses beginning with an initial freezing period lasting less than a second before escape behaviors are initiated (Card 2012). Freezing behavior in Drosophila is also reported in response to translational motion of a small fly-sized robot moving in the same plane as the fly (Zabala et al. 2012). In contrast to escape behavior, the neuronal components for freezing behavior are less investigated.

10.4 DNp09: Descending neuron for freezing

Because visual signal is process at the optic lobe in the brain, looming-stimulus triggered freezing behavior must be controlled by the brain. A population of descending neurons (DNs) is good target to investigate the neuronal components for freezing behavior (Fig. 10.3). DNs connect the brain and ventral nerve cord (VNC) and hence is a bottleneck of the information flow in the central nervous system. However, no systematic data for individual neuron morphology has been available in any insects until recently. Namiki et al. 2018 identify ~100 types of DNs in female Drosophila (Namiki et al. 2018) and create a set of driver lines which selectively labels individual DNs by use of split-GAL4 technique (Luan et al. 2006). The authors performs photoactivation at the neck connective and selectively labels descending and ascending neurons. The number of labeled cell bodies, as an estimate of the total number of DNs is counted ~350 at maximum. Among these, a population of DNs arising from optic glomeruli has been identified. For example, the giant fiber (also termed as DNp01) receives input from specific set of visual glomeruli: lobula columnar neuron type 4 (LC4), lobula plate/lobula columnar neuron type 2 (von Reyn et al. 2017; Klapoetke et al. 2017). Seven DNs arborizing the same glomerulus to the giant fiber are identified (Namiki et al. 2018).

A candidate DN for freezing behavior has been identified recent work in Drosophila. Using the driver lines for DNs established, Zacarias et al. 2018 examines silencing neuronal activity with Inwardly rectifying K+ channels (Zacarias et al. 2018). The movement of unrestrained flies are monitored in the experiment (Fig. 10.4A). Through the screening, the authors find that silencing a specific DN, called the DNp09, significantly reduce the probability of freezing behavior in response to the looming stimuli. Furthermore, DNp09-silencing increase the probability of jumping than in control flies, suggesting the presence of inhibitory mechanism onto the jump pathway. Optogenetic activation of DNp09 with channelrhodopsin induce freezing (Fig. 10.4B). The effect persists during the activation. Running at the initial phase is often observed, suggesting DNp09 is also involved in locomotion. The probability of DNp09-activation induced freezing deduce as walking speed increase, indicating this behavioral choice, freezing/running is dependent of the walking speed (Zacarias et al. 2018). It would be interesting to study he neuronal mechanism to drive distinct behavior by the same DNs.

10.5 Optic glomeruli

The neuroanatomy of DNp09 is shown in Figure 3. DNp09 has wide field innervation in the brain, including ventral protocerebrum (Fig. 10.5A&B). The cell body is located on the posterior brain surface. The innervation with smooth appearance are located in wedge, inferior clamp, gorget, and ventral protocerebrum (VLP). The innervation with blebby appearance are superior/inferior posterior slope and gnathal ganglia (GNG) and the terminal exhibit anti-synaptotagmin immunoreactivity. The VLP is an array of optic glomeruli, each of which receive input from the lobula complex. Optic glomeruli process ethologically relevant feature of the visual object. DNp09 arborize one of the glomeruli which receive the input of lobula columnar neuron type 9 (LC9), whose function has not yet been characterized. The functional connectivity between LC9 and DNp09 has been confirmed by combination of optogenetics and calcium imaging (Bidaye et al. 2019). Wu et al. 2016 shows that optogenetic activation of visual projection neuron population LC6 also drive long-mode escape jump (Wu et al. 2016). Large-scale behavioral screening study combined with machine learning observes increasing jump-response for GAL4 lines labeling LC9 and LC10, as well as LC6 (Robie et al. 2017). The wedge is connected with the antenno-mechanosensory motor center receiving input from Johnston’s organ (Kamikouchi et al. 2006; Lai et al. 2012). Because flies also show freezing behavior in response to vibration stimulus (Howard et al. 2019), DNp09 innervation in the wedge may mediate vibration-sensitive freezing behavior. The function of other protocerebral regions, inferior clamp and gorget are unknown.

Also, the optic glomerulus LC11 is a candidate population for presynaptic partner of DNp09. A class of lobula columnar cells that is relevant to freezing behavior is identified recently. LC11 is known to respond small moving object and silencing the LC11 does not affect object avoidance behavior (Keleş and Frye 2017). Tanaka and Clark investigate the function of LC11 by use of silencing, imaging and behavior on the track ball. In calcium imaging, LC11 neurons respond to moving small object. Silencing LC11 reduced the probability of stopping behavior , whereas optogenetic activation of LC11 briefly increased stopping probability. These suggest that LC11 is sufficient to trigger the freezing behavior. Functional connectivity from LC11 on DNp09 has been confirmed (Bidaye et al. 2019). Summary connection scheme of DNp09 is shown in Figure 10.6

10.6 Axonal projection of freezing descending neuron

DNp09 axon descends through the ipsilateral neck connective and projects into leg neuropils in the VNC (Fig. 10.5C). Dense innervation with blebby appearance are present all regions: foreleg, middleleg and hindleg neuropils. These blebby terminals exhibit anti-synaptotagmin immunoreactivity. In addition, DNp09 also projects to the region called the tectulum, a bundle of descending and ascending neurons running central zone of the VNC (Power 1948) and the function is unknown.

DN terminals in the leg neuropils could be sorted into two major types in Namiki et al. 2018: DNs projecting to the dorso-medial part of each neuropil (type-I) and DNs penetrating through the neuropil via pathway passing through core of leg neuropil called the oblique tract (type-II). For example, giant fiber and other three DNs (p02, p05 & p11) arising from the LC4 glomerulus project to leg neuropils and all of these DNs are classified as type-II (Namiki et al. 2018). Innervation pattern of DNp09 is classified as type-I and most of the blebby terminals are present in the medio-dorsal portion. The connectivity of type-I DNs with downstream neurons is still unclear.

Recent work suggests that the serotonergic system in the VNC helps to facilitate an intermediate and stimulus-independent pause response when flies are startled (Howard et al. 2019). The work performs silencing experiment using tryptophan hydroxylase (TRH)-Gal4 line. Although the individual neuron morphology in the Gal4-lines has not yet been analyzed, the line may contain the candidate downstream neurons of DNp09.

In addition, DNp09 also have presynaptic terminals in the GNG, which contains the largest number of DNs projecting to the leg neuropil (Namiki et al. 2018). There is a possibility for the information flow of freezing behavior: DNp09-GNG-Leg neuropils.

10.7 Neuromodulation of the startle responses

Howard et al. 2019 investigated the effect of neuromodulation on walking and startle response using neurogenetics in Drosophila (Howard et al. 2019). Gal4 line which labels serotonergic system in entire nervous system is used. The line was crossed with teashirt-GAL80 (Rubinstein et al. 2010), so that the expression in the brain is suppressed. The neurons in the leg neuropils were densely labeled, suggesting contribution of leg motion. This technique enables manipulation of neuronal activity which is specific to the VNC. Among neuromodulators examined, they found the activation of serotonergic system in caused speed change in flies. When serotonergic system was activated, walking became slow down, whereas the system was genetically inhibited, walking became faster. Also, silencing serotonergic system altered how flies respond to being startled, taking longer time to exhibit escape response. These results suggest that serotonergic system in the VNC serves a role for freezing response. However, flies are still able to show startle response, suggesting that there is another pathway for freezing behavior. Also, serotonin effect on the startle response are observed both to visual and vibration stimuli, neurons for leg control are shared by both systems.

10.6 Similarity between freezing in flies and fears in mammals

Whether flies have emotion like human remains to be unclear. There is no generally accepted definition for emotion. Anderson & Adolphs state the emotion as a type of internal brain state with central general properties than can exist independently of subjective, conscious feelings, which can only be studied in humans (Anderson and Adolphs 2014). In their definition, the emotion is decomposed into four building blocks: scalability, persistence, valence and generalization. To examine these characteristic, Gibson et al. 2015 create a new behavioral assay, where flies are confined in enclosed areas and repeatedly exposed to an overhead translational shadow (rotating paddle) (Gibson et al. 2015) (Fig. 10.7). Repeated presentation of shadows results in graded (scalable) and persistent increase of freezing behavior. The repetitive shadow also results in dispersal of feeding flies, suggesting the presence of negative valence and context generalization. The authors conclude that freezing behavior of flies in response to repetitive shadows express an internal state exhibiting emotion primitives, which may be analogous to fear in humans.

10.7 Future work

Identification of neurons involved in freezing behavior is helpful to understand the neuronal mechanism. However, most of neuronal components for pathway for freezing behavior is currently unknown. The missing link can be identified using fruitful genetic tool in Drosophila. Using driver lines which label DNp09 (Namiki et al. 2018), it is possible to identify potential upstream and downstream neurons with transsynaptic tracer (Feinberg et al. 2008; Talay et al. 2017). In addition, cell-type specific driver lines are being created for various brain regions (Aso et al. 2014; Wolff et al. 2015; Wu et al. 2016). For example, activation screening with optogenetic tools such as channelrhodopsin to search lines with freezing phenotype, in which unknown neurons for freezing behavior are present. Also, a synapse-resolution electronmicroscopic data is recently published for the use of anatomical approach (Maniates-Selvin et al. 2020). Searching neurons which have synaptic contact with DNp09 is another way to identify the pathway for freezing behavior.

Because fly’s freezing behavior share basic features with defensive behavior in mammals, suggesting the presence of shared neuronal mechanisms called motion primitive (Anderson and Adolphs 2014). Studies in fly will contribute to understand the neural mechanisms in mammalian brain.

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Figure legend

Fig.　10.1 Cessation of ongoing movement triggered by electrical stimulation.

(A) Schematics of cross-sectional map of the circumoesophageal connective. The position of stimulation electrode is shown (‘Stimulation’). The location of lateral giant (LG) and medial giant (MG) are shown. D, dorsal; M, medial.

(B) Four profile drawings of maintained position during electrical stimulation.

Images are modified from Bowerman and Larimer (1974).

Fig.　10.2 Behavioral sequence of fly to looming stimulus.

(A) When flies detects small moving object, they exhibit freezing behavior.

(B) When the object show expansion, flies show escape response.

Fig.　10.3 Descending neuron connecting the brain and thoracic motor center.

Fly’s body (left) and the structure of central nervous system are shown (right). Although the number of neurons in the brain and ventral nerve cord is the order of 5^th power, the number of neurons running through the neck connective is the order of 3^rd power.

Fig. 10.4 Silencing DNp09 disrupt freezing behavior in response to visual stimuli.

(A) Schematics of behavioral assay used in (Zacarias et al. 2018).

(B) Proportion of freezing in flies. Dashed lines represents ssonset of stimulus presentation. DNp09/+, Kir2.1/+, DNp09>Kir2.1 flies are shown. *** denotes p < 0.001.

Images are modified from Zacarias et al. 2018.

Fig. 10.5 Morphology of DNp09.

(A) Morphology of DNp09 and the brain. The neuron and neuropil are shown with magenta and gray.

(B) Three consecutive confocal stacks of the neuronal innervation in the brain. AVLP, anterior ventral protocerebrum; D, dorsal; EB, ellipsoid body; FB, fan-shaped body; GNG, gnathal ganglia; GOR, gorget; ICL, inferior clamp; IPS, inferior posterior slope; LC9, lobula columnar neuron type 9; M, medial.

(C) Axonal projection of DNp09. Coronal sections for prothoracic, mesothoracic and metathoracic segments are shown. D, dorsal; V, ventral.

Fig. 10.6 A schematics of connectivity for DNp09.

The optic lobe supplies visual input onto optic glomeruli (LC9 & LC11). The optic glomeruli supply the DNp09, which send information to the motor circuit in the VNC.

Fig. 10.7 Experimental setup used in Gibson et al. 2015.

Flies are in the petridish and the paddle show rotation, which provide moving stimulus to flies.