Search strategy is regulated by somatostatin signaling and deep brain photoreceptors in zebrafish
© Burgess et al. 2017
Received: 13 December 2016
Accepted: 22 December 2016
Published: 26 January 2017
Animals use sensory cues to efficiently locate resources, but when sensory information is insufficient, they may rely on internally coded search strategies. Despite the importance of search behavior, there is limited understanding of the underlying neural mechanisms in vertebrates.
Here, we report that loss of illumination initiates sophisticated light-search behavior in larval zebrafish. Using three-dimensional tracking, we show that at the onset of darkness larvae swim in a helical trajectory that is spatially restricted in the horizontal plane, before gradually transitioning to an outward movement profile. Local and outward swim patterns display characteristic features of area-restricted and roaming search strategies, differentially enhancing phototaxis to nearby and remote sources of light. Retinal signaling is only required to initiate area-restricted search, implying that photoreceptors within the brain drive the transition to the roaming search state. Supporting this, orthopediaA mutant larvae manifest impaired transition to roaming search, a phenotype which is recapitulated by loss of the non-visual opsin opn4a and somatostatin signaling.
These findings define distinct neuronal pathways for area-restricted and roaming search behaviors and clarify how internal drives promote goal-directed activity.
KeywordsZebrafish Search Motivation Goal-directed behavior Non-visual photoreceptor CRISPR Orthopedia Somatostatin sst1.1 Melanopsin opn4a
Essential resources tend to be clustered, rather than uniformly distributed in the natural environment. Accordingly, animals use a range of behavioral strategies to search for resource rich areas . Search behaviors have been characterized in invertebrate species [2–4], including in C. elegans, where detailed underlying neuronal circuits have been described [5–7]. Although sophisticated search behaviors under natural conditions have also been catalogued in vertebrates, surprisingly few studies have exploited vertebrate models to probe genetic factors and neuronal connections that initiate and maintain active search states [8–11].
To date, the powerful repertoire of neurogenetic tools available in zebrafish have been primarily applied to decoding neural mechanisms for acute behavioral responses to sensory stimuli [12, 13]. For instance, larval zebrafish show phototaxis toward restricted areas of illumination in dark environments [14, 15], maneuver within thermal and chemical gradients [16, 17], and actively oppose water currents by swimming against whole-field visual motion. Thus, larvae use sensory information in several modalities to actively navigate within the environment, initiating approach or avoidance behaviors as required. However, much less is known about whether larvae efficiently locate resources that are not in the immediate vicinity. Search behavior requires the maintenance of an internal state that appropriately regulates motor activity, and potentially also modulates sensory thresholds to facilitate the discovery of desirable resources [18, 19]. Short-term internal states such as arousal and hunger, and movement profiles consistent with exploratory behavior are present in larval stage zebrafish [10, 11, 20–22], raising the possibility that autogenic search behavior has also developed.
One promising avenue to investigate search behavior is the hyperactive response of zebrafish larvae to a complete loss of illumination [23–25]. At the onset of darkness, zebrafish larvae become hyperactive for 5–10 min, before settling into a low-activity sleep-like state. The hyperactive state is referred to as the visual motor response, or dark photokinesis . Hyperactivity propels larvae out of dark regions – slower movement in the light causes larvae to aggregate in illuminated regions, an example of classical photokinesis behavior [26, 27].
Here, we investigated whether the locomotor hyperactivity that occurs during dark photokinesis is randomly directed or rather shows spatial patterning that might facilitate light-search behavior. We found that larval swim trajectories during the first few minutes of photokinesis possess characteristic features of a widely utilized animal foraging strategy known as area-restricted search [2, 3, 8, 28]. Area-restricted search is a strategy for temporarily restricting movement to the region where a specific resource was last detected. Typically, area-restricted search is a transient behavior that transitions to a roaming search strategy if the resource is not quickly located . Similarly, we show that after 2–3 min of locally restricted movement, larval swim paths transition to an outward trajectory, consistent with a roaming strategy for light sources that are outside the initial visual range. We demonstrate that the distinct swimming patterns associated with area-restricted and roaming search states respectively increase the ability of larvae to find local and remote light sources. Using high speed recording, we reveal the swim motor patterns associated with area-restricted search and roaming, and show that area-restricted search is generated by utilizing an efficient helical swim pattern composed of same direction turns. Finally, we establish that initiating area-restricted search requires retinal input while the deep brain photoreceptor opn4a and otpa-specified somatostatin releasing neurons control the transition from intensive local search to a roaming state.
Helical swimming follows loss of illumination
We next separately analyzed the X–Y planar and vertical components of the response. Taking the vertical component first, we confirmed a previous observation that larvae tend to swim near the top of the water column, and after loss of illumination, swim downwards . However when tested during prolonged loss of illumination, we noted that the ‘diving response’ was transient, lasting for around 1 minute before larvae slowly returned to their baseline position in the water column (time to half recovery = 210 s after light extinction; Fig. 1d). The mean rate of descent following the loss of illumination was 0.75 ± 0.08 mm/s (n = 54), two orders of magnitude slower than the velocity of burst swims (100 mm/s) , which are a characteristic feature of escape movements in larvae. Downward swimming is therefore unlikely to be part of a defensive response. Thus, in the vertical dimension, the response to loss of illumination has two phases: a slow downwards swim, followed by a gradual return to the water surface.
Local movement shows characteristic features of area-restricted search behavior
Spatially restricted movement forms part of a behavioral strategy that is used by many animals, known as area-restricted search. Area-restricted search occurs during foraging and mate-search behavior in insects, birds and mammals [2, 8, 28], and during navigation to a shelter or nesting site [30–32]. Characteristic features of area-restricted search are (1) a reduction in travel distance, (2) an increase in movements that cause a change in orientation, (3) an increase in movement path complexity, and (4) a directional bias to movement trajectories [1, 4, 33]. Collectively, these changes allow efficient sampling of the local environment . Similarly, for the first 2–3 min after loss of illumination, in addition to reduced displacement, we found an elevated rate of re-orientation (Meander, Fig. 2c) and greater path complexity (Fractal Dimension, Fig. 2d; see Additional file 2: Figure S1 for illustrative examples).
As for other short-term internal states, search behaviors are typically time-limited and reversible . If a resource is not located within the local environment, a roaming search strategy is often initiated, which is characterized by an outward movement profile [36, 37]. Similarly, spatially restricted movement by larvae persisted only for about 2 min after loss of illumination, after which movement parameters transitioned toward baseline levels. Intriguingly though, two measures, namely displacement and the rate of reorientation, did not match baseline levels even after 10 min (Fig. 2b, c). Principal component analysis confirmed that trajectories in the first minute after loss of illumination were distinct from baseline movements (Additional file 4: Figure S3a), whereas trajectories after 10 min sustained dark did not form a separate group but distributed with either the baseline or first-minute dark response trajectories. Thus, the apparent gradual change in mean trajectory parameters after loss of illumination may in fact be due to changes in the frequency with which larvae switch between discrete episodes of spatially restricted movement, and a movement profile similar to baseline (Additional file 4: Figure S3b–e). Nevertheless, episodes of spatially restricted behavior declined in frequency after loss of illumination, confirming that this response is time-limited (Additional file 4: Figure S3f).
Local and outward movement patterns differentially facilitate light-search behavior
We next employed a concealed target assay to determine whether outward movement promotes discovery of light that is not in the immediate environment. Here, we used real-time tracking of larval position to project a light spot on the opposite side of a central barrier. The spot was illuminated either 1 s or 3–5 min after loss of full-field illumination to test larvae in the local and outward movement phases, respectively. In trials without a central barrier, larvae rapidly navigated to the light spot regardless of the duration of time spent in the dark (Fig. 5e). Conversely, in the presence of the barrier, larvae only initiated phototaxis if they first swam to the edge of the barrier (Fig. 5f). During control trials (no light spot illuminated) larvae infrequently circumnavigated the barrier (Fig 5g, No spot). Similarly, when the spot was illuminated 1 s after loss of full-field illumination, larvae did not reach the edge of the barrier and therefore did not initiate phototaxis (Fig. 5g; T0). In contrast, most larvae tested 3–5 min after loss of full-field illumination swam to the edge of the barrier, then quickly navigated to the light spot (Fig. 5g; T4). The difference in final proximity to the light spot was significant (ANOVA, main effect of time in dark F1,38 = 36.3, P < 0.001), demonstrating that larvae locate remote light sources more rapidly during the outward movement phase.
During the concealed target assay, larvae rarely swam continuously beside the barrier, but often appeared to contact it then move away. This was surprising because several studies have argued that zebrafish larvae perform thigmotaxis (touch-seeking) behavior [34, 41]. However, supporting experiments used an arena with square or concave walls where it is difficult to distinguish true thigmotaxis from mere wall-following due to outward swim trajectories. In fact, under baseline conditions, larvae tended to swim in close contact with the walls of a concave arena but did not show a significant preference to swim in contact with a convex barrier (Additional file 7: Figure S5a–c), indicating that continuous contact with the perimeter of a chamber is a trivial consequence of outward movement trajectories. In contrast, after loss of illumination, larvae tended to avoid the edges of the concave arena (repeated measures ANOVA main effect of illumination on wall proximity F1,14 = 49.4, P < 0.001; Additional file 7: Figure S5d, e). Local and outward movement phases are thus associated with wall-avoidance behavior.
Retinal signaling is required to initiate the local movement response
Orthopedia-specified somatostatin-releasing neurons and melanopsin are necessary for search strategy transition
We previously proposed that the hyperactivity manifest by larvae in the minutes after loss of illumination promotes aggregation in illuminated regions via photokinesis. Photokinesis is a simple behavioral strategy for navigation that uses rapid non-directional movement in dark areas and slower movement in illuminated regions. However, we now demonstrate that the locomotor activity after loss of illumination is not undirected. Rather, movement trajectories are highly spatially structured and change during the first few minutes in darkness. For the first 1–2 min after loss of illumination, larvae turn intensively to the same side in the horizontal plane, while gradually swimming downwards, resulting in a helical trajectory. After 3 min, larvae continue to execute re-orienting movements at high frequency, but no longer continuously in the same direction; swim trajectories thus propel larvae out of the local environment.
We considered various interpretations of these movement profiles. For instance, light extinction may trigger an aversive or flight response. Indeed, larvae perform a vigorous O-bend response in the first second after loss of illumination ; however, because swim travel distance thereafter diminishes below baseline levels, local and outward swim profiles are inconsistent with escape behavior. Alternatively, the high fractal dimension and slow descent during the local movement phase might suggest a disorganized or confused behavior, but this is contraindicated by the highly-accurate light-orientation behavior, efficient phototaxis, stable same-side turning and wall-avoidance that also occur during this period. Finally, these movement profiles are inconsistent with behavioral sleep, which includes reduced sensory thresholds and substantial reductions in locomotor activity .
Rather, we interpret the local and outward movement phases as sequential light-seeking responses. Initially, larvae execute an area-restricted light-search behavior, which if unsuccessful, is followed by a remote light-search behavior. Multiple lines of evidence support this view. First, swim trajectories during the local movement phase (reduced travel distance, increased meander, fractal dimension and directionality) are similar to characteristic features of area-restricted search. Similar three-dimensional helical foraging trajectories have been documented in Drosophila and microzooplankton and proposed to yield an optimal search strategy when searching for sparsely distributed food sources [48, 49]. Second, changes to swim profiles were time-limited and rapidly reversed upon restoring illumination, consistent with other motivated states. Third, as for other short-term internal states [20, 21], larvae manifest changes in responsiveness to sensory cues, namely increased sensitivity to a light-spot during the local movement phase, and greater wall avoidance during both local and outward movement phases. Finally, functional tests of light-seeking behavior support this interpretation – larvae more rapidly reached a nearby patch of light during the local movement phase, and conversely located distant illuminated regions only when tested during the outward movement phase. These changes fulfill operational criteria for defining motivated states , in that loss of light produces a change in behavior that (1) is maintained internally, (2) is reversible, and (3) facilitates a specific objective. We therefore propose that the swim profiles seen after loss of illumination constitute internally driven search behaviors that enable larvae to efficiently navigate back into illuminated regions. During active phototaxis, movements that re-orient larvae away from a light-spot are rapidly corrected by contraversive turns [15, 35]. However, in the natural environment, a more extensive area-restricted search may be triggered after larvae swim into a small patch of thick foliage or if light is obscured by falling debris. In contrast, sustained remote search activity may be deployed after water currents sweep larvae into large regions of dense undergrowth.
Locomotor responses to loss of illumination are driven by both retinal signaling and otpa-specified deep brain photoreceptors . Our results now reveal that retinal signaling and deep brain photoreceptors differentially trigger local and roaming search states; retinal signaling is required for local movement, whereas otpa-dependent sst1.1-expressing neurons and opn4a-expressing deep-brain photoreceptors sustain roaming search behavior. However, search strategy transition was less impaired in sst1.1 and opn4a mutants than in otpa mutants. Possible reasons are the presence of supernumerary oxytocin expressing neurons in otpa mutants , and the presence of 32 nonvisual opsins in the zebrafish , raising the potential for uncharacterized additional photoreceptive neurons in the otpa expression domain. Nevertheless, our data suggests that after loss of illumination, retinal signaling activates an intense area-restricted search, after which sst1.1- and opn4a-dependent pathways gradually drive the transition to a roaming search state. Indeed, somatostatin signaling regulates other motivated behaviors, tuning response thresholds during reward [51, 52] and defensive  behaviors in mammals. In our current model, otpa neurons provide a relatively weak drive, which predominates only when the local search state declines. This idea is supported by the finding that enucleated larvae acutely increase re-orientation rates immediately after the loss of illumination. Somatostatin and opn4a may therefore continuously drive the roaming search state in darkness, an impulse which is normally masked by retinal-driven local search. Conversely, in the absence of otpa circuitry, retinal signaling is unimpeded by remote search drive, prolonging the local response.
Light-search behavior is likely impelled by dwindling nutritional resources. By 6 dpf, yolk supplies are almost depleted and larvae actively pursue prey. Predation requires visual input; larvae are almost completely unable to locate food in darkness . Accordingly, larvae are hard-wired to seek and remain in illuminated regions, demonstrating vigorous phototaxis behavior toward restricted areas of light. During phototaxis, sensory information signals through retinal ON and OFF pathways to trigger forward swims and turns, respectively . Our new results reveal that, in the absence of directional light cues, loss of illumination triggers sequential internally driven light-search strategies involving, first, an area-restricted search for light in the immediate vicinity, followed – if unsuccessful – by a roaming search for light in distant regions.
An unresolved question concerns the “baseline” state of larvae. After 1–2 min of local search, larvae continue executing turn movements at a high rate, but no longer strongly biased to one side, thereby causing outward swimming. Although functionally, outward swimming enabled larvae to locate remote light sources more quickly than during the local movement phase, principle component analysis indicated that outward movement parameters clustered either with local search or baseline swim profiles. Thus, the outward swimming phase, while occasionally interrupted by episodes of local search behavior, showed similar trajectory characteristics to baseline activity under full-field illumination. Nevertheless, wall-avoidance and meander measurements revealed differences between baseline and outward swimming, indicating distinct behavioral states. A strong possibility is that “baseline” behavior under full-field illumination is actually a food-search state; unfed 6 dpf larvae, their yolk supplies exhausted, may already be in a roaming search for food, an idea which is consistent with recent work analyzing movement under constant illumination  and changes in sensory responsiveness after feeding .
Unexpectedly, net locomotor activity decreased after a loss of illumination. Reduced activity contrasts sharply with many studies on the visual motor response, including from our laboratory, reporting that loss of illumination triggers 5–10 min of hyperactivity [26, 55, 56]. We speculate that chamber size accounts for the discrepancy. In most previous work, movement has been constrained using small wells in which larvae constantly encounter the edge of the arena. However, we found that after loss of illumination, larvae avoid barriers, suggesting that in small arenas such as multi-well plates, where the perimeter is frequently encountered, wall avoidance may trigger increased activity. The rationale for wall avoidance after loss of illumination remains unclear, possibly reflecting a strategy to reduce collisions in darkness, or alternatively facilitating exploration by moving larvae away from barriers that prevent them from detecting distant light sources.
Larval stage zebrafish larvae maintain distinct behavioral states, including arousal and sleep [10, 20–22]. Our new findings reveal that internal drives in larvae can additionally produce sophisticated motivated behaviors such as search activity. It is likely that larvae exhibit additional types of autogenic search behavior provoked by exposure to, or loss of stimuli in other sensory modalities. The finding that non-retinal photoreception by melanopsin and somatostatin signaling play a selective role in driving remote light-search is consistent with the idea that non-visual photoreceptors are intimately linked to motivational state control [57–59], and provides the opportunity to dissect the ancient evolutionary pathways that allow organisms to adapt to short-term challenges and opportunities in the dynamic natural environment.
Zebrafish were maintained on a Tubingen long fin strain background. Larvae were raised at 28 °C in E3 medium, under Light:Dark cycle (14:10 h) and maintained at a maximum density of 30 per 6-cm Petri dish. otpa m866 homozygous mutant and cousin wildtype pairs (for control experiments) were genotyped as previously described . To test the effect of DA neuron loss we used the previously established mi174 mutant, which has a global loss or dysfunction of dopaminergic neurons, including Orthopedia-specified DA neurons [60, 61]. Homozygous mutant mi174 larvae (null for trpM7) were identified by the hypopigmentation phenotype and normally pigmented siblings from the same clutches were used for controls  (kind gift from Jim Hudspeth lab). Enucleations were performed on 4 dpf larvae that were anaesthetized in Evans solution with tricaine. Using a Sylgard-coated dish with a groove to steady the larva, both eyes were removed with sharp forceps. After enucleation, larvae were allowed to recover in fresh Evans solution (134 mM NaCl, 2.9 mM KCl, 2.1 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose and 10 mM Hepes pH 7.8). After approximately 24 h larvae were transferred to E3 media. Control larvae were treated with tricaine and incubated in Evans solution in parallel with enucleated larvae. Enucleation produced characteristic melanophore expansion and a complete loss of high angular O-bend responses in response to a dark flash , confirming that larvae were functionally blind (not shown). All in vivo experimental protocols were approved by the NICHD animal care and use committee.
Tests were performed on larvae at 6 or 7 dpf. Behavioral assays were monitored using infrared illumination (CMVision Supplies, 850 nm) positioned below the chamber, with IR-longpass filters on the camera lens to exclude visible light. For white-light illumination we used LEDs positioned above the chamber unless otherwise specified. Prior to behavioral recording larvae were adapted for a minimum of 30 min to the same light intensity as in the testing arena. Light intensities were measured using a radiometer (International Light Technologies). Testing arenas were maintained at 28 °C. Image acquisition and illumination were controlled using DAQtimer event control software . We added new motion trigger code to DAQtimer to detect when a larva entered a specified ROI. For kinematic analysis, we recorded video at 1000 frames per s (fps) using a Fastcam 1024 (Tech Imaging). Tracking was performed offline using Flote tracking software, which also measured swim kinematics and classified locomotor maneuvers . For trajectory analysis (all recordings at less than 1000 fps) images were captured with a μEye IDS-1545LE-M CMOS camera (1st Vision, Andover, MA), tracked in real-time using DAQtimer , and analyzed offline using custom scripts written in IDL (Exelis, Boulder, CO).
3D trajectory analysis
Single larvae were placed in an 85 × 85 × 85 mm clear acrylic chamber filled to a depth of 75 mm, illuminated at 50 μW/cm2. Images were captured at 20 Hz with the recording window adjusted to simultaneously record the X–Y plane and the vertical Z position of the larvae using a mirror placed adjacent to the chamber at a 45° angle. Each larva was adapted to the arena for at least 5 min and recordings were started when the larva was centrally positioned in the arena. Tracking was performed offline and horizontal and vertical displacement analyzed in 30-s time windows.
Groups of five larvae were placed in a 40 × 45 × 100 mm (length × width × height) clear acrylic chamber filled to a height of 80 mm, illuminated at 200 μW/cm2. Larvae were adapted to the arena for 10 min before testing. Recordings were captured at 2 Hz and included 300 s of full-field illumination (baseline) followed by 1700 s in darkness. The mean position of the larvae was analyzed in 2.5 s bins. We measured descent speed using individual larvae in the same container during the period from 1.6 to 6.0 s after loss of illumination to capture the linear portion of the response (i.e. after initiation of the downward swim but before larvae reached the bottom of the arena).
2D trajectory analysis
Single larvae were placed in a 200 × 200 mm chamber (5 mm depth, illumination 50 μW/cm2). After a 10 min adaptation period, recordings were initiated once the larva entered a 120 × 120 mm motion trigger ROI in the center of the arena, after which the larva was tracked at 10 Hz under constant illumination for 10 min. The motion trigger ROI was then restored. Once the trigger was again activated, illumination was turned off and the larva tracked for a second 10-min period. For each larva we then used 30-s track segments to measure displacement, distance, meander, fractal dimension, and trajectory bias. Displacement: the straight-line distance between the starting and ending point for each 30 s interval. Distance: total travel distance, calculated as the total of the straight-line distances covered in each of the thirty 1 s line segments. Meander: a measure of the rate of reorientation , calculated using the total of the absolute values of angles formed by all trajectory changes during the 30 s interval, divided by the distance. Fractal dimension: a measure of path complexity  frequently used in studies of search behavior [28, 49, 65], which we implemented using the box-counting method . In brief, the fractal dimension was estimated as the slope of the line fitting the log ratio of the number of box units that cover part of the trajectory versus box size. Trajectory bias: a measure of the directional (left or right) bias of the trajectory, calculated using the absolute value of the total of the angles formed by all trajectory changes during the 30-s interval. Leftward and rightward trajectory changes produced negative and positive angles, respectively; thus, these changes summed to near zero when larvae swam in predominantly straight lines. As tracking was not reliable when larvae were adjacent to the wall of this arena, we excluded segments that included less than 10 s of data. Principal component analysis was performed on measures of displacement, meander, fractal dimension, and trajectory bias after standardization to unit variance and mean-subtraction.
Covert phototaxis assay
To measure how quickly larvae swam to a nearby light source, we used a 58 × 58 mm chamber (depth 5 mm), illuminated overhead at 50 μW/cm2. Single larvae were placed in the arena, adapted for 2 min and tracked in real-time. After the larva swam into a 20 × 20 mm motion trigger ROI in the center of the arena, full-field illumination was extinguished. After either 3 s or 3 min in darkness, we established a second motion trigger in the center of the arena. Once this was triggered we used the position and orientation of the larva (from real-time tracking) to project a stationary 14-mm diameter light spot of 23 μW/cm2 intensity at a position 7 mm directly behind the tail of the fish (AAXA P2 Pico Projector). We recorded the response of the larva, and the position of the light-spot using a second camera with an IR-blocking filter at 60 Hz, then measured offline the time for the larva to enter the perimeter of the spot. During “no target” control experiments the light spot was flashed on for 50 ms, then extinguished, so that we could calculate the time taken for the larva to swim into an equivalent target region by chance. Larvae reached the target spot significantly more quickly when it remained illuminated, confirming that efficient phototaxis occurred (ctrl vs. phototaxis: for T0, t56.6 = 2.8, P = 0.007; for T4, t36 = 2.5, P = 0.016).
Concealed target assay
The efficiency of locating a light source that was not immediately visible was tested using a 200 × 200 mm chamber (5 mm depth, illumination 50 μW/cm2). Two LEDs were positioned beneath the chamber on opposite sides, each with an iris to produce a 25-mm light spot on a diffuser immediately below the chamber. We first confirmed that larvae performed robust phototaxis to a light spot illuminated on the opposite side of the chamber (Additional file 4: Figure S3). Then, to prevent larvae from directly detecting the position of a light spot on the opposite side, we added a 140-mm opaque barrier into the middle of the arena. After 10 min adaptation to the arena, we established a rectangular motion trigger ROI in the center of the arena (20 mm away from the ends of the barrier, encompassing regions on both sides of the barrier). After the trigger was activated, full-field illumination was turned off. Next, either 1 s or 3–5 min after the onset of darkness, a light-spot was activated on the side of the barrier opposite to the position of the larva. The larva was then tracked for a 2-min period during which we measured the closest position of each larva to the center of the light spot.
Turn bias assay
Groups of 15 larvae were recorded simultaneously in a 90-mm diameter arena (depth 4 mm, illumination 50 μW/cm2). After 5 min adaptation, we extinguished full-field illumination. Then, after either 2 s or 3 min, we induced phototaxis using a 9 mm diameter 20 μW/cm2 light spot illuminated on a diffuser below one side of the arena and recorded responses for 12 s with a high speed camera. Turn bias was the tendency of R-turn maneuvers to be made toward the light spot as previously described . Briefly, an index of –100 means all turns are directly away from the spot, and +100 means turns are executed toward the spot. For analysis, larvae were binned based on their orientation relative to the spot.
To determine maneuver pair frequency and LI we used a 40 × 40 mm chamber, illuminated at 200 μW/cm2. Single larvae were acclimated in the chamber for 2 min before a 15 × 15 mm motion trigger ROI in the center of the chamber was established. After the trigger was activated, we collected 10 s of data using a high speed camera, either maintaining illumination (baseline) or immediately after turning the light off. Larvae were tracked using Flote, then custom scripts were used to calculate the frequency and LI for each maneuver pair. To determine the initiation frequency of R-turns under sustained loss of illumination, experimental parameters were as described above with the following changes. Larvae were recorded in groups of five and acclimated for a period of 30 min after being placed into the recording arena. Initiation frequency is per 400 ms analysis window. To measure the R-turn LI during sustained darkness, single larva were recorded at the specified intervals over a 10-min time-period. The LI is the percentage of sequential maneuvers that are performed in the same direction, normalized between –100 and +100, such that random turning has a LI of 0, whereas for constant same-side turning the LI is +100 (as illustrated in Additional file 3: Figure S2). Only trials in which a larva executed at least three R-turns were used when calculating the LI.
Cloning free CRISPRs were designed and synthesized using methods as described . sgRNAs were designed using CRISPRscan . CRISPR sgRNA (120 pg), cas9 RNA (300 pg), and fluorescent protein RNA (30 pg) were co-injected into single cell wildtype embryos. Fluorescent protein RNA (either RFP or GFP) was used as a marker for successful injection and used to sort embryos for behavioral testing. Two-dimensional trajectory behavior and analysis, described above, was performed on successfully injected larvae. CRISPR efficacy was determined for every larva tested using CRISPR-STAT fluorescent PCR (Additional file 12: Figure S10) using an ABI 3100 Genetic Analyzer Avant . Additional file 13: Table S1 lists guide RNA sequences and genotyping primers. Custom software was used to measure percent of wildtype gene target degradation for semi-quantitative analysis of knockdown efficiency. Only larvae with bi-allelic conversion of the wildtype gene products were included for quantification – a maximum of 5% detectable wildtype PCR product was used as a threshold for inclusion into the data set. For control experiments, we injected a guide RNA targeting EGFP into in-crosses of a transgenic zebrafish line Tg(vglut2a:EGFP) which was derived from nns14Tg by Cre injection . Injected control larvae were processed as for experimental groups after assessing loss of EGFP PCR product.
Neuronal ablation was performed as previously described . Briefly, 1 dpf larvae from in-crosses of y227Tg, a line with pineal-specific expression of nitroreductase-tagged mCherry, were treated with 10 mM metronidazole in E3 media for 48 h with drug replacement after 24 h. After treatment, larvae were returned to fresh E3 media. Ablation efficacy was assessed by screening for mCherry fluorescence. Controls were wildtype siblings, also treated with 10 mM metronidazole.
Statistical analysis was performed using SPSS (IBM, Armonk, NY), IDL and Gnumeric (Available from: http://www.gnumeric.org/). Graphs show mean and standard error of the mean unless otherwise specified. Normality of data sets was determined using the Shapiro–Wilk test in SPSS. Significance for normally distributed data sets was calculated using the two-tailed Student t-test. For data not normally distributed, the Mann–Whitney U test was applied.
We would like to thank Benjamin Feldman (NICHD) for help with design and analysis of CRISPR experiments.
This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.
EH and HB conceived and designed the project. EH, YB and JS planned and executed experiments. HB wrote software. EH and HB wrote the manuscript. All authors read and approved the final manuscript.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All in vivo experimental protocols were approved by the NICHD animal care and use committee.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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