Summary The neural circuit mechanisms underlying emotion states remain poorly understood. Drosophila offers powerful genetic approaches for dissecting neural circuit function, but whether flies exhibit emotion-like behaviors has not been clear. We recently proposed that model organisms may express internal states displaying “emotion primitives,” which are general characteristics common to different emotions, rather than specific anthropomorphic emotions such as “fear” or “anxiety”. These emotion primitives include scalability, persistence, valence and generalization to multiple contexts. Here we have applied this approach to determine whether flies' defensive responses to shadows are purely reflexive, or may express underlying emotion states. We describe a new behavioral assay in which flies confined in an enclosed arena are repeatedly exposed to an overhead translational shadow. Repetitive shadows promoted graded (scalable) and persistent increases in locomotor velocity and hopping, and occasional freezing. The shadow also dispersed feeding flies from a food resource, suggesting both negative valence and context generalization. Strikingly, there was a significant delay before the flies returned to the food following shadow-induced dispersal, suggestive of a slowly decaying internal defensive state. The length of this delay was increased when more shadows were delivered for initial dispersal. These responses can be mathematically modeled by assuming an internal state that behaves as a leaky integrator of shadow exposure. Our results suggest that flies' responses to repetitive shadow stimuli express an internal state exhibiting canonical emotion primitives, possibly analogous to “fear” in mammals. The mechanistic basis of this state can now be investigated in a genetically tractable insect species.

Highlights d Serotonergic neurons in the fly VNC extensively innervate the leg neuropils d Activating these neurons causes flies to walk slower but maintain coordination d Silencing these neurons causes flies to walk faster in many contexts d Silencing these neurons alters how flies respond to being startled

16To navigate complex environments, animals must generate highly robust, yet flexible, locomotor 17 behaviors. For example, walking speed must be tailored to the needs of a particular 18 environment: Not only must animals choose the correct speed and gait, they must also rapidly 19 adapt to changing conditions, and respond to sudden and surprising new stimuli. 20Neuromodulators, particularly the small biogenic amine neurotransmitters, allow motor circuits 21 to rapidly alter their output by changing their functional connectivity. Here we show that the 22 serotonergic system in the vinegar fly, Drosophila melanogaster, can modulate walking speed in 23 a variety of contexts and in response to sudden changes in the environment. These multifaceted 24 roles of serotonin in locomotion are differentially mediated by a family of serotonergic receptors 25 with distinct activities and expression patterns. 27walk forwards, backwards, and upside down, navigate complex terrains, and rapidly recover 31 after injury [1][2][3][4][5][6][7][8][9]. To achieve this wide range of behaviors, insects regulate their global walking 32 speed and kinematic parameters, allowing them to modify stereotyped gaits as needed [3,5-33 7,9,10]. Because overlapping sets of motor neurons and muscles are recruited for all of these 34 behaviors, animals must be able to rapidly modulate the circuit dynamics that control locomotor 35 parameters [11-13] (reviewed in [14]). 36As with limbed vertebrates, most insects use multi-jointed legs to walk [8,10,[15][16][17]. Locomotor 37 circuits that orchestrate these complex gaits are located in the ventral nerve cord (VNC), a 38 functional analogue of the vertebrate spinal cord that includes three pairs of thoracic 39 neuromeres (T1, T2, and T3) that coordinate the movements of the three pairs of thoracic legs 40 [1][2][3][4][5][6][7][8][9][18][19][20]. The insect VNC receives descending commands from the brain and sends motor 41 output instructions via motor neurons to peripheral musculature [3,[5][6][7]9,10,19]. Leg motor 42 neuron dendrites innervate the leg neuropils within the VNC and their axons exit the VNC to 43 synapse onto muscles in the appendages [11][12][13]21,22]. Sensory neurons, which convey 44 proprioceptive and tactile information, project axons from the appendages to the VNC by these 45 same fiber tracts, where they arborize in the leg neuropils [14,23,24] ( Figure 1A). Notably, the 46 VNC is capable of executing coordinated leg motor behaviors, such as walking and grooming, 48 drive the coordinated flexion and extension of each leg joint and, therefore, walking gaits 49 [8,10,17,20,26]. 50Numerous studies have shown that sensory input from the legs are required for robust 51 and stereotyped locomotor patterns, regulating both the timing and magnitude of locomotor 52 activity and also facilitating coordination between legs [6,8,10,12,[27][28][29]. However, sensory 53 feedback cannot be the only means for tuning locomotion: mutation of proprioceptive receptors 54 or even deafferenting limbs d...