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  • 5 Deep turns and the dynamics of reorientation in escape response and free crawling of C. elegans Abstract — In the previous chapter, we developed an automated tracking algo- rithm for C. elegans 2D motor behavior that solves self-occluding body shapes. Here, we apply that algorithm to study the dynamics of deep turning during two important behaviors: the escape response, and abrupt reorientations in freely crawling worms. During the escape response, we find that the worm steers sharply away from the noxious stimulus by 180◦ on average, in a tightly controlled way. Additional reorientations before and after the turn broaden the distribution of reorientation angles, and can be linked to known underlying neural and molec- ular mechanisms. During free crawling, we find that abrupt turns, appearing in previous literature as ‘omega turns’, are in fact differentiated into two distinct classes. Despite a dramatically different visual appearance, both statically and dynamically, the two turns share similar kinematics in posture space; only the amplitude of a pulse in the third postural eigenmode is a dominant distinguish- ing feature. The two classes of turns reorient the worm towards different sides: omega turns ventrally; delta turns dorsally, by over-turning through the ventral side. We show that the two turns occur independently, but with approximately equal rates that remain equal during adaptation. Taken together, these obser- vations suggest a shared underlying neural infrastructure, and a more diverse navigational repertoire than previously thought.

    See the footnote at the start of Chapter 4, p. 97.


  • 5 Deep turns and dynamics of reorientation in C. elegans

    5.1 Introduction

    In the previous chapter, we have developed a tracking algorithm for the full 2D motor behavior of C. elegans. This algorithm is capable of tracking not just ‘simple’ postures, but also self-occluding ones. This enables us to now take a closer, quantitative look at behaviors of the worm that feature such self-occluding body shapes, and that have thus far not been fully resolved. In the first part of this chapter, we focus on the escape response of

    C. elegans. This sequence of orchestrated behavioral motifs is evoked by a noxious stimulus (in our case a localized heating of the worm’s head), and turns the worm away from the stimulus. The ‘omega turn’, a sharp reorientation featuring self-overlapping body shapes, is the crucial part of the sequence, and can now for the first time be resolved. Studying this highly stereotyped behavior also helps us to identify quantitative patterns in the data, which will guide our analysis in the second part. In the second part, we analyze free crawling experiments. Extensive

    research has been done on the strategies used by the worm to localize food and favorable habitats, but the sharp turns that occur during this behavior have never been fully investigated. Here, we take the first steps towards resolving these turns.

    5.2 The C. elegans escape response

    5.2.1 Introduction to the escape response

    C. elegans is capable of responding to a range of potentially harmful con- ditions. Its sensory neurons, most of which are connected to the outside world through non-motile cilia [1] [2, Ch. 3.1], can detect noxious stimuli of a chemical, mechanical, optical, or thermal nature [3]. Upon encounter- ing a sufficiently strong stimulus, the worm executes a stereotyped escape response: a behavioral sequence that steers the worm away from the ob- served threat.


  • 5.2 The C. elegans escape response

















    forward locomotion

    forward locomotion

    backward locomotion

    RIM head

    movement locomotory system


    SMD / RIV

    deep head swing


    extra-synaptic tyramine

    sensory neurons

    (i) (ii) (iii) (iv) (v)

    Figure 5.1 | Schematic overview of the C. elegans escape response (based on ref. 4). Top row shows worm body shapes extracted from tracking data: (i) forward locomotion and exploratory head motions; (ii) infrared laser stimulus; (iii) reversal phase; (iv) omega turn; (v) resumption of forward locomotion in opposite direction. Diagram below shows sequence of events in the C. elegans nervous system. Time flows from left to right. RIM, SMD, and RIV designate C. elegans neurons. Green plus signs indicate stimulation; red minus signs inhibition. Inset shows the neural network implicated in the escape response [4, 5]. Rectangles: sensory neurons; hexagons: command neurons; circles: motor neurons. Synaptic connections are indicated by triangles, gap junctions by bars, and extra-synaptic diffusion of tyramine by a dashed arrow. Green connections are stimulatory, red ones inhibitory. Details have been omitted; see ref. 4 for a more in-depth overview.


  • 5 Deep turns and dynamics of reorientation in C. elegans

    Fig. 5.1 (top) shows the timeline of the C. elegans escape response. Dur- ing normal locomotion on an agar surface (i), the worm moves by propa- gating a sinusoidal wave through the body. This snake-like motion is typ- ically accompanied by exploratory head movements. In our experiments, we elicit an escape response by applying a localized thermal stimulus to the worm’s head, using an infrared laser pulse (ii) (this causes a 0.5 ◦C temperature increase at the head over 100 ms) [6]. The stimulus causes the worm to abruptly pause, and then back up (iii). During this reversal phase, head movements are suppressed, which has been suggested to in- crease the worm’s chances of escaping from the ‘snare traps’ of predatory fungi [7]. After roughly 5 to 10 seconds of reversing, the worm executes an omega turn: a sharp reorientation manoeuvre, during which the worm’s body briefly resembles the shape of the Greek letter Ω (iv) [5]. The turn reorients the worm away from the stimulus, and allows it to resume forward locomotion in the opposite direction (v).

    Some aspects of the escape response sequence have been teased apart at the cellular, molecular, and genetic level. This has been greatly facilitated by the availability of a genetic toolbox for C. elegans [8] as well as the worm’s stereotyped development [9]. The latter means that each individ- ual worm has the exact same body plan, in which each of its roughly 1 000 somatic cells can be named, and each cell develops from a fully predictable lineage, starting at the fertilized egg [9]. Genetic changes can be easily in- troduced into the worm’s genome, and can often be made to target specific cells [8]. As an example, using these techniques, the nociceptive ASH neu- rons have been found to express ion channel proteins that are implicated in osmo- and mechanosensation [10]. This makes the ASH neurons mul- timodal, and downstream signaling therefore relies on signal multiplexing [11]. As the connectivity of the C. elegans nervous system is largely known [1], it has even been possible to partly trace the neural network that drives the escape response (Fig. 5.1, inset) [5, 4].

    The escape response is classically studied with a ‘gentle anterior touch’: a touch to the head of the worm using a human hair [12] (typically, an eye-


  • 5.2 The C. elegans escape response

    brow hair). In this scenario, a qualitative description of the mechanisms that orchestrate the different phases of the escape response has emerged from experiments (Fig. 5.1b) [4, 13], and a key element is the neurotrans- mitter tyramine, released by the RIM interneurons. Tyramine acts through multiple pathways, one of which is extra-synaptic diffusion and binding to G-protein coupled receptors (GPCRs) in distant cells. As this diffusion process has a timescale of seconds (see Methods) — much slower than the millisecond timescale associated with the initial synapse-mediated reversal response [14] — it creates a time separation between the reversal and the subsequent omega turn.

    At the moment the worm is touched, the signaling cascade starts with the sensory neurons ALM and AVM registering the touch (Fig. 5.1, dia- gram and inset) [4]. These neurons initiate backward locomotion (through the AVD and AVA command neurons), while inhibiting forward locomo- tion (through the PVC and AVB command neurons). AVA, in turn, acti- vates the RIM interneurons, which release the neurotransmitter tyramine. Donnelly et al. show that tyramine further inhibits forward locomotion (via AVB), thus prolonging the reversal. Through fast-acting ion chan- nels (LGC-55) in neck muscles and head motor neurons, it additionally suppresses head movements. Crucially, tyramine also diffuses out of the synaptic cleft (‘synaptic spillover’), and activates GPCRs (SER-2) else- where in the worm’s body. SER-2 activation by diffusing tyramine sets up an asymmetry in the worm’s locomotory system: it disinhibits the ven- tral body wall muscles (through suppression of the VD inhibitory motor neuron). After the omega turn has been initiated by a steep ventral head swing (controlled by the SMD neuron [5]), the resulting hypercontraction of the ventral body wall muscles during the body wave produces the char- acteristic omega turn.

    The construction and validation of such a description relies on classic methods from biology. Typically, a perturbation is introduced into the worm’s locomotory system, either through genetic knockouts or cell abla- tions [15]. To assess the impact on the worm’s behavioral phenotype, one


  • 5 Deep turns and dynamics o