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Curr Neurobiol 2016; 7 (2): 62-73 ISSN 0975-9042 Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish. Florian Ulrich1#, Charlotte Grove2#, Jesús Torres-Vázquez1 and Robert Baker2 Department of Developmental Genetics, Skirball Institute of Molecular Medicine, 540 1st Avenue, New York City, New York 10016, USA. 2 Department of Neuroscience and Physiology, New York University Medical Center, 550 1st Avenue, New York City, New York 10016, USA. # Florian Ulrich and Charlotte Grove are co-first authors. 1 Abstract We investigated the contribution of blood vessel formation and neuronal excitability to the development of functional neural circuitry in larval zebrafish by analyzing oculomotor performance in response to visual and vestibular stimuli. To address the dependence of neuronal function on the presence of blood vessels, we compared wild type embryos to reck and cloche mutants that lacked intracerebral blood vessels. To test how neuronal excitability impacts neuronal development and intracerebral vascularization, we blocked neural activity using Tetraodotoxin (TTX) and Tricaine. In reck mutants, we found both slow phase horizontal tracking and fast phase resets with only a slightly reduced amplitude and bandwidth. Spontaneous saccades, eye position holding and vestibular gravitoinertial induced eye rotation were also present. All of these behaviors except for visual tracking were observed in cloche mutants that lacked any head vasculature. Thus, numerous oculomotor neuronal circuits spanning the forebrain, midbrain and hindbrain compartments, ending in motor innervations of the eye muscles, were correctly formed and generated appropriate oculomotor behaviors without blood vessels. However, our observations indicate that beginning at approximately six days, circulation was required for sustained behavioral performance. We further found that blocking neuronal excitability with either TTX or Tricaine up to 4-5 days post fertilization did not noticeably interfere with intracerebral blood vessel formation in wild type larvae. After removal of drug treatments, the oculomotor behaviors returned within hours. Thus, development of neuronal circuits that drive oculomotor performance does not require neuronal spiking or activity. Together these findings demonstrate that neither vascularization nor neuronal excitability are essential for the formation of numerous oculomotor nuclei with intricately designed connectivity and signal processing. We conclude that a genetic blueprint specifies early larval structural and physiological features, and this developmental strategy may be viewed as a unique adaptation required for early survival. Keywords: Larval zebrafish, Neuronal activity, Vascularization, Oculomotor, Excitability, Neuronal development, TTX. Accepted July 25, 2016 Introduction In nearly all adult vertebrates, the vasculature is of key importance for supplying nutrients and oxygen to the brain. Throughout the nervous system, neurons and blood vessels align closely to form the neurovascular unit [1]. Little is known, however, about the causal role of the vasculature during the embryonic establishment of neuronal pathways and functional activity. In addition, little information exists 62 about the interplay between neuronal excitability and embryonic development of the neurovascular unit. Several studies have shown that endothelial cells can stimulate the formation of neurons [2-4] and that neurons can instruct blood vessel development [5,6]. Moreover, neuronal activity can regulate vascular tonus in mouse brain slices, suggesting that the supply of nutrients and oxygen to the brain is tightly controlled by local energy demands in the brain [7]. Curr Neurobiol 2016 Volume 7 Issue 2 Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish. Shortly after the end of gastrulation in zebrafish endothelial precursors from the anterior lateral plate coalesce into rostral and midbrain organizing centers [8,9]. The latter centers gives rise to the angiogenic sprouts forming primordial hindbrain channels which then provide the endothelial cells migrating into the center of each rhombomere to form the hindbrain central arteries as illustrated in Figures 1A and 1C. Interestingly, most of the neuronal subgroups and circuits that are essential for oculomotor performance (as shown schematically in Supplementary Figure 1) lie close to blood vessels in the fore-, mid- and hindbrain compartments. Nevertheless, we recently found that the small central arteries growing into rhombomere centers were not required for either neuronal development or maintenance in the developing zebrafish hindbrain (Figures 1B and 1D) [9]. Hence, we hypothesized that endothelial sprouting and neuronal activity might also be independent. Accordingly, we tested whether neuronal activity depended on the presence of endothelial cells in reck and cloche mutants that are devoid of intracerebral blood vessels by utilizing the well established neuronal circuitry and signal processing underlying oculomotor behavior [10,11]. Conversely, to explore whether neuronal activity was required for endothelial sprouting or the emergence of oculomotor behavior, we blocked neuronal excitability and synaptic transmission for several days with either tetraodotoxin (TTX) or Tricaine. Larval zebrafish exhibit many robust individual oculomotor behaviors employing clearly defined sets of neurons that A WT can be used as a read out for neuronal development and function [12] (Figure 2A, Supplementary Figures 1A-1D). Optokinetic tracking performance begins in the forebrain pretectum with a visual slip velocity signal that directly reaches the motoneurons (Supplementary Figures 1A and 1B, Direct). A pretectal pathway through the vestibular nucleus converts the signal to eye velocity before reaching the ocular motoneurons (Supplementary Figures 1A and 1B, Indirect). The vestibular neurons also project to a hindbrain neural integrator (VPNI) located in the caudal most hindbrain rhombomere (r8) that converts the signal to eye position (Supplementary Figures 1A and 1B). Importantly, this hindbrain neural integrator is separately recruited during horizontal fast phases as well as during saccades for eye position holding (Supplementary Figures 1C and 1D). In addition, larval zebrafish have a novel vestibular gravitoinertial system generating compensatory torsional eye rotations in response to either upward or downward head motion [13]. Utricular signaling alone, independent of the semicircular canals, connects with specific subsets of tangential neurons in r5 projecting to inferior rectus/ superior oblique, inferior oblique/superior rectus motor nuclei and extraocular muscles (Supplementary Figures 1E and 1F). These straightforward, but highly detailed sets of central oculomotor connections and signal processing units therefore provides a rich repertoire of neuronal subgroups and circuits to analyze any perturbation in embryonic development. Accordingly, we measured components of the slower B Neurons reck-/Neurons Vessels r8 Vessels r7 r6 r5 Vessels r8 r4 r3 r2 r1 5 dpf P C A r5 r4 r3 r2 r1 P 5 dpf D WT RSN r7 r6 clo-/RSN Vessels MN MN r8 r8 r7 3 dpf r6 A r5 r4 r3 r2 r1 P A 3 dpf r7 r6 r5 r4 r3 r2 r1 Vessels Absent P A Figure 1. Vasculature in wild type (WT), cloche and reck mutants. (A-B) Maximum intensity confocal projections of a 5 dpf WT (A) embryo and a reck (B) mutant sibling lacking central arteries in the hindbrain. Green, pan-neuronal ngn:GFP and red, pan-endothelial kdrl:cherry. (C-D) Maximum intensity confocal projections of a WT embryo (C) and a cloche mutant (D) sibling lacking complete hindbrain vasculature, at 3 dpf. Red, pan-endothelial kdrl:cherry and green, retrograde labeling of reticulospinal neurons (RSNs). Lateral orientation is shown in A-D with anterior to the right and dorsal to the top Abbreviations: r, rhombomere; WT, Wild Type; RSN, Reticulospinal Neuron; MN, Mauthner Neuron; P and A, Posterior and Anterior Curr Neurobiol 2016 Volume 7 Issue 2 63 Ulrich/Grove/Torres-Vázquez/Baker A Midbrain Hindbrain SC H SR SO T Vestibular LR MR Trochlear Oculomotor Integrator r8 B r7 Neurons r6 r5 LR IR IO MR r1-3 r4 Temporal WT Vessels nX Pretectal Burst Internuclear Abducens C reck-/- D clo-/- Nasal nIV nVII nV r8 Vessels 4 dpf r7 r6 r5 r4 r3 r2 r1 P 5 dpf nft-/- A 4 dpf Figure 2. Schematic overlay of neuronal groups with vasculature. (A) Rhombomeric location of the major oculomotor neurons from the pretectum to hindbrain that mediate horizontal, vertical and torsional eye movements. (B) Lateral confocal view of a 4 dpf wild type embryo labeled with the pan-endothelial transgene kdrl:cherry and hindbrain motoneurons identified by the marker Islet:GFP. (C-D) Photographs of 5 dpf reck and 4 dpf cloche larvae with eye movements illustrated in Figure 3 Abbreviations: H, Horizontal; T, Tangential; LR, Lateral Rectus; SR, Superior Rectus; MR, Medial Rectus, IR, Inferior Rectus; IO, Inferior Oblique; SO, Superior Oblique; r, rhombomere; SC, Spinal Cord; nIV, trochlear nucleus; nV, trigeminal nucleus; nVII, facial nucleus (visual tracking) and fast (resetting) phases of horizontal optokinetic reflexes (OKR) as well as spontaneous scanning saccades and eye position holding in the presence and absence of intracerebral vessels in wild type vs. reck or cloche mutant larvae within a 4-6 dpf window. We found both directional OKR tracking and fast phase resets including vestibular gravitoinertial compensatory eye rotations in all reck mutants tested. All of these behaviors except for visual tracking were observed in cloche mutants. While response robustness was variable and less than that in wild type, our findings indicate that neuronal circuits mediating oculomotor performance were still correctly formed and exhibited activity without blood vessels. Interestingly, sustained blocking of neuronal excitability and synaptic activity from embryonic onset using TTX or Tricaine did not interfere with neuronal development, since oculomotor behavioral patterns could be found with a near normal profile after a short recovery from the drug treatments. Moreover, blocking neuronal activity did not noticeably interfere with intracerebral blood vessel formation. Our findings, therefore, emphasize the remarkable extent to which neurons differentiate, form precise connections and acquire functional oculomotor activity independent of the presence of either vasculature or excitability. 64 Methods Zebrafish Lines and Maintenance We handled and maintained adult, larval and embryonic zebrafish under standard laboratory conditions following NYU’s IACUC/IBC-approved protocols. For wildtype specimens, we used EK [14] TL, Wik, AB and mixed genetic backgrounds [15]. For vasculature mutant lines we used recky72 [8] and cloches5 [16]. Recky72 is a recessive null allele of a reversion-inducing cysteine-rich protein with Kazal motifs, a gene encoding a membrane-anchored tumor suppressor glycoprotein required for promoting canonical Wnt signaling that enables cerebrovascular development. Reck is both expressed and required in endothelial cells for intracerebral vascularization and proper expression of molecular markers associated with blood-brain barrier. Reck mutants display a brainspecific vascularization deficit (Figure 1B). Cloche is an early acting zebrafish gene that is required by both the endothelial and hematopoietic lineages; however, the gene affected has not been identified. Cloche (clo) mutants display a severe deficit in the specification of both the endothelial and hematopoietic head lineages (Figure 1D). Curr Neurobiol 2016 Volume 7 Issue 2 Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish. To enhance the vasculature in vivo, we took advantage of the following transgenic endothelial reporters: Tg(kdrl:RFP) s896 [17] and Tg(kdrl:EGFP)la116 [18]. To enhance neurons, we used Tg(neurog1:eGFP)w61 and Tg(isl1:GFP)rw0 lines [19]. These transgenic reporters were used in wild type lines (see above) and mutants: recky72 [8] and cloches5 [16]. For visualizing reticulospinal neurons, we inserted a Rhodamine-Dextran crystal into the hindbrain/spinal cord at the level of myotome 5 as described in [9]. Drug Treatments For Tetraodotoxin (TTX) applications, we anesthetized embryos at 24–28 hpf in 0.016% Tricaine in embryo medium and mounted them vertically by carefully inserting their tails into individual holes in an agarose plate. Thus, the heads were sticking out with the hindbrain facing in dorsal direction. We then injected 1 nL of 1 mM TTX in Danieau’s solution into the hindbrain ventricle, washed the Tricaine out and kept the specimen in embryo medium at 28.5ºC until measuring neuronal function or confocal imaging. For sustained Tricaine treatments, we kept embryos in 0.016% Tricaine @ 28.5ºC from 18 hpf to 96 hpf. We refreshed the medium once a day to avoid bacterial growth. Paralysis induced by either TTX or Tricaine was assessed by lack of response to dish taps and tail touches with an eyelash tool. In addition, paralyzed embryos did not show any vestibular righting reflex and often remained upside-down with yolk facing upwards. Recovery from these drugs was established by upright posture, tail motion and escape-like responses soon followed by swim-like movements. Fixing and Staining of Embryos To allow for an easier handling during confocal imaging, we fixed the embryos overnight in 4% PFA in PBS at 4ºC. After washing several times with PBT (PBS+0.1% Triton X-100), we blocked the embryos for 1h at room temperature in blocking solution (1% BSA in PBT) and stained the embryos overnight at 4ºC with anti-RFP (1:500, Becton Dickinson 632496) or anti-GFP antibodies (1:200, Invitrogen, A-11122) in blocking solution. We washed the embryos 4x for 15 minutes in Blocking Solution and then stained them with secondary antibodies: donkey-rabbit Alexa 488 (A-21206) or donkey-rabbit 546 (A-10040), each at 1:2,000 in blocking buffer overnight at 4ºC. We then washed the embryos 4x for 15 minutes in PBT at room temperature and kept them at 4ºC in PBT until imaging. If embryos were labeled with both RFP and GFP, we only stained against RFP, since GFP gave a strong signal on its own even after fixation. Confocal Imaging We mounted the embryos in 1% agarose/PBS and acquired images of the vasculature and neuroepithelium using a Leica SP5 confocal microscope with a 40x dipping/water immersion objective (NA=0.8). Bidirectional scans were performed at 200 lines/s in 1024 × 1024 pixel windows; Curr Neurobiol 2016 Volume 7 Issue 2 z-stacks were taken at 1 µm z intervals. Subsequently, the image stacks were turned into Maximum Intensity Projections and further edited using ImageJ and Adobe Photoshop. Experimental set-up and eye movement behaviors Larval fish, 4-6 dpf, were embedded in low melting temperature agarose (2.0%, Sigma Type VII-A). The block of agarose was pinned to a Sylgard 182 (Dow Corning, Midland, MI) disk and excess agarose removed from around the eyes and head rostral to the swim bladder enabling unhindered tracking of either a horizontal or torsional visual stimulus (Supplementary Figures 2B and 2C). The disk with the mounted larva was lowered into a 19 mm diameter or 25 mm square transparent glass specimen chamber for horizontal OKR or pitch tilt response, respectively, containing ~ 2.5 ml of 30% Danieau’s solution. A round cover glass slip held in position by surface tension served as the chamber ceiling. While the larvae remained stationary on the vestibular turntable a rotating visual surround stimulus was produced by a servo controlled motor (Supplementary Figures 2D and 2E) [20]. Transparent acrylic drums of different sizes, nested concentrically around the fish chamber provided rotating and stationary patterned stimuli, alternating black and white stripes at 15.5º frequency. The rotating drum was illuminated through the table shaft by visible light LEDs. Stimulus waveforms controlling drum motions were produced with an Agilent (Palo Alto, CA) 33120A phase-linked function generator. Both sinusoidal and velocity step waveforms were available from 0.125 to 2 Hz and at up to ± 20º in amplitude. Torsional eye rotation measurements were made in a custom-built tilting apparatus consisting of a rotating platform driven by a DC motor [13]. Motor command signals were generated with a custom code written in LabView and platform position was measured via an attached tachometer. A high speed camera (Pike IEEE 1394b, Allied Vision Technologies) mounted to the rotation axis recorded right eye rotation. Compensatory vestibuloocular reflexes were produced in response to a pitch tilt sinusoid waveform at 0.125 Hz and a maximum amplitude of ± 20°. Eye Movement Measurements A three-axis micromanipulator on the mounting stage for the specimen holder allowed the head/eyes to be centered for focusing of the overhead CCD camera. Horizontal and torsional eye positions in 4-6 dpf larvae were sampled at 100 Hz and 60 Hz, respectively. Infrared CCD cameras (Supplementary Figure 2F) recorded the weakly pigmented eyes and threshold masks (Supplementary Figure 2G) were utilized to locate the major axes of both the body and eyes (Supplementary Figure 2I). Video images collected by the CCD camera were processed in real time to extract horizontal eye position. Eye velocity was obtained by 65 Ulrich/Grove/Torres-Vázquez/Baker digitally differentiating filtered eye position records and OKR gain calculated as drum velocity/eye velocity [20]. Eye position changes generated by torsional stimuli were extracted by comparing recorded images with initial 0º reference images. Saccades, optokinetic and torsional responses, were recorded and analyzed with a custom code written in LabView (National Instruments, Austin, TX) and MATLAB (MathWorks, Natick, MA) [13,21]. Results Larval wild type 4-6 dpf zebrafish exhibit robust oculomotor performance in response to vertical axis visual (Supplementary Figure 3) and/or head pitch/roll vestibular stimuli [13] with the neuronal subgroups and connections extending from the forebrain pretectum to the hindbrain vestibular, reticular, internuclear and motoneurons as illustrated in the Supplementray Figures 1A-F vignettes. To address whether the establishment of neuronal circuits and the ensuing function depends on the presence of blood vessels, we measured the slow (tracking) and fast (resetting) phases of the horizontal optokinetic reflexes (OKR) in 20 reck mutant larvae from 7 separate experimental series (Figures 3 and 4). We also recorded spontaneous scanning saccades and eye position Spontaneous Scanning reck-/- 5dpf Light A 20° Saccade 0° Eye Position Stable N B Right Eye Left Eye Dark N T 20° 20s T 20° Eye Position Leak Saccade 0° 20° C Spontaneous Scanning clo-/- 4dpf Unstable 20° 0° Stable Leak 20s 20° Figure 3. Spontaneous horizontal scanning eye movements in reck and cloche mutants. (A-B) Scanning behaviors of a 5 dpf reck embryos shown either in light (A) or dark (B). Records are from the right (red) and left (blue) eyes with nasal (N) and temporal (T) directions as indicated. Arrows mark saccades and eye position holding that is stable in A or leaky in B. (C) Scanning eye movements in a 4 dpf cloche mutant. Arrows illustrate stable, unstable and leaky eye position holding A Optokinetic StepTracking reck-/- 5dpf Eye Position Slow Phase 20° 0° 0.125 Hz B Right Eye Eye Velocity N T Left Eye T N Drum Rotation Fast Phase Direct 20° 20°/s 0°/s Indirect 8s 20°/s Figure 4. Optokinetic tracking in reck mutants. (A-B) Eye movements showing eye position (A) and eye velocity (B) performance in response to ± 20°/s drum rotation at 0.125 Hz. Arrows mark the slow and fast phases in A and the direct and indirect components in B. Records are from the right (red) and left (blue) eyes with nasal (N) and temporal (T) directions as indicated. For both the RE and LE the direct and indirect gains were 0.4 that were reduced by 33% from a wild type gain of 0.6 (from Supplementary Figure 3) 66 Curr Neurobiol 2016 Volume 7 Issue 2 Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish. holding patterns in the same reck larvae and an additional 13 cloche larvae. To test vestibular performance, we measured torsional eye rotation in another 12 reck and 13 cloche mutants in response to nose-up/nose-down pitch (Figure 5). Spontaneous Horizontal Scanning Eye Movements in reck and cloche Mutants Horizontal saccadic scanning and eye position holding at 5 dpf was well developed in both wild type larva (Supplementary Figure 3A) and reck mutants (Figure 3A). Conjugate saccadic movement of both the right and left eyes occurred in their respective nasal and temporal directions. Sustained eye position holding was observed primarily due to the visual slip feedback provided by the retinal-pretectal-hindbrain loop (Supplementary Figures 1A and 1B). In short, this oculomotor performance in light demonstrates the presence and functional integrity of nearly every neuronal group and connectivity illustrated in Figure 2A and Supplementary Figures 1A and 1B, respectively. We also observed scanning saccadic behavior in the absence A of visual feedback (Figure 3B). In the dark, eye position in reck mutants decayed with time constants ranging from 5-10 s similar to, but on average a few seconds less than that of a normal wild type larva (Supplementary Figure 3A). In cloche mutants, scanning saccades were conjugate and of reasonable peak velocity for both eyes in either the temporal or nasal direction (Figure 3C). Since none of the cloche mutants tested responded to any visual tracking stimuli, eye position holding was entirely due to integrator function which was found to be quite variable, performing as either stable, leaky or unstable as labeled in Figure 3C. This highly variable eye position holding behavior contrasts to the symmetrical leak in both the right and left oculomotor integrators in reck mutants (Figure 3B) and wild type (Supplementary Figure 3). Simultaneous stable (LE) and leaky (RE) eye position holding in Figure 3C could occur for the same movement direction, because the integrator circuits are separate for the left and right side as shown in Supplementary Figures 1C and 1D. In conclusion, all oculomotor subnuclei and connections for spontaneous scanning and eye position holding were present and functional without intracerebral vasculature in Torsion in reck-/- 4 dpf 30° Nose-up tilt SR-IO RE 0° SO-IR Nose-down tilt B Torsion in clo-/- 4 dpf 20s 30° 30° Nose-up tilt SR-IO RE 0° SO-IR Nose-down tilt 30° Figure 5. Torsional eye movements in reck and cloche mutants. (A-B) Torsional eye rotations in response to a 30° sinusoidal nose-up and nose-down tilt at 0.1 Hz in reck (A) and cloche (B) mutants. The compensatory direction of eye rotation and the responding eye muscle pairs are indicated as color coded in Figure 2. Records are from the right (red) eye with VOR gains in reck and cloche mutants of 0.36 and 0.25, respectively that were reduced by 10% and 38% from a wild type gain of 0.4 [13] Curr Neurobiol 2016 Volume 7 Issue 2 67 Ulrich/Grove/Torres-Vázquez/Baker reck mutants or even without any head vasculature at all in cloche mutants. Optokinetic Eye Movements in reck Mutants By 5 dpf, binocular visual tracking of full field targets is well developed in wild type zebrafish larvae with eye velocity proportional to, but less than, stimulus velocity (Gain=0.60, Supplementary Figure 3) [20]. Optokinetic performance in reck mutants was comparable as shown by the eye position records in Figure 4A (alternating slow and fast phases) and the matching step-like eye velocity records in Figure 4B. Each 4 second eye velocity period reflects neuronal activity in two distinct brainstem circuits that can be separated into an early (direct) and sustained (indirect) component (Figure 4B) [12,22]. Notably, the indirect component of eye velocity reflects the functional status of horizontal vestibular neurons and their connectivity with the abducens internuclear, motor and integrator neurons (Supplementary Figures 1A and 1B) [12]. For both the RE and LE in reck mutants the direct and indirect gains were 0.4, lower by 33% from a wild type gain of 0.6 (from Supplementary Figure 3). The use of optokinetic sinusoidal tracking in reck mutants (Supplementary Figure 4A) supports and extends the step tracking findings by showing that both eye velocity phase and gain are maintained over a wide frequency bandwidth. For example, at 0.125 Hz both RE and LE OKR gains were 0.43 and, compared to a wild type gain of 0.6, reduced by 28%. At two higher frequencies, 0.5 Hz and 1.0 Hz, OKR gains were reduced by 45% and 55%, respectively (Supplementary Figures 4B and 4C). Phases at 0.125 Hz and 0.5 Hz were ~ 0° and at 1.0 Hz -20° that were not significantly different than wild type [23]. In summary, visual performance from the retina to the pretectum through to the hindbrain oculomotor nuclei and circuitry was similar to, but of less amplitude than that observed in wild type larva with intact vasculature. Vestibular Behavior in reck and cloche Mutants In order to test the status of hindbrain circuitry wiring that exists from the vestibular sensory periphery to the individual midbrain oculomotor subnuclei shown in Figure 2A, both reck and cloche mutants were presented with a sinusoidal nose-up/nose-down stimulus as diagrammed in Supplementary Figures 1E and 1F. Both mutants exhibited eye position changes compensatory to the direction of head tilt (e.g. for nose-up tilt clockwise rotation of the right eye). This response pattern demonstrated that individual tangential subgroups in rhombomere 5 established correct connections with their respective oculomotor targets as shown for the right eye in Figure 5 (e.g. SO-IR in the nose down case and SR-IO in the nose-up). For comparable head tilts compensatory eye position amplitudes in reck mutants were nearly the same as in normal wild type larva [13], but noticeably less in cloche mutants (Figures 5A and 5B, n=6). Records from the right 68 (red) eye showed vestibulocular (VOR) gains in reck and cloche mutants of 0.36 and 0.25, respectively that in turn were lower than wild type gain by 10% and 38%. In addition, eye velocity was step-like rather than sinusoidal in cloche mutants (Figures 5B) suggesting somewhat compromised circuit performance perhaps reflective of the metabolic state. Not unexpectedly, behavioral performance could be greatly diminished after 15-30 min of continuous testing, and in some cases to zero, consistent with the avascular head condition. Together these observations provide a direct demonstration that in either the absence of intracerebral (reck) or complete head vasculature (cloche), all the essential neurons and circuits are formed from the forebrain through the hindbrain, including the pathways and function of orbital eye muscles. Collectively these observations (Figures 3-5) show that oculomotor function can be, and often is, nearly equivalent to that of wild type larva, particularly with 4-5 dpf reck mutants. These findings support the hypothesis that a hard-wired genetic blueprint gives rise to a functional neural network in the absence of vascular patterning cues and the accompanying metabolic resources. Role of Neuronal Excitability in Neuronal Function and Vascular Development Subsequent to the previous observations demonstrating that neuronal activity did not depend on the presence of endothelial cells in reck or cloche mutants, we explored the converse paradigm of whether either endothelial sprouting or oculomotor behavior depended on neuronal excitability and/or activity dependent behaviors. In the first set of experiments, we injected at ~ 24 h 1 nL of a 1 mM solution of Tetraodotoxin (TTX) into the hindbrain ventricle. Oculomotor behaviors were recorded at 4-6 dpf between 4 to 7 h after the recovery from complete paralysis with the appearance of spontaneous and escape-like body movements, but not yet full righting and/or swimming reflexes. Comparison of wild type (Figure 6A, WT) with TTX treated larvae (Figure 6B at 6 dpf) revealed that both the vasculature and neuronal subgroups were not noticeably different. Surprisingly, in all TTX larvae examined (n=12), both spontaneous scanning and visual tracking behaviors were present and not too different from untreated wild type larva (Figure 7 and Supplementary Figure 3). The frequency of spontaneous scanning saccades was often slightly reduced, but eye position holding was stable (Figure 7A), indicative of the presence of a visual slip feedback from retina through the pretectum. In the dark, decay in eye position was similar to that observed in wild type larvae (Figure 7B and Supplementary Figure 3B) suggesting nearly normal operation of the oculomotor integrator network (VPNI in Supplementary Figures 1C and 1D [12,24,25]. These observations were complemented by records showing optokinetic tracking and fast phase resets to be quite typical with, in this case, Curr Neurobiol 2016 Volume 7 Issue 2 Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish. A B WT Neurons Vessels nVII nV nX r8 r7 r6 r5 TTX Neurons nX nIV nVII r6 r3 r2 r1 5 dpf P r5 r4 r3 r2 r1 5 dpf A nIV nV r8 r7 r4 Vessels P A Figure 6. Vasculature in WT and TTX treated larvae. (A-B) Maximum intensity confocal projections of a WT (A) embryo and after 5 days in TTX (B) showing neurons and hindbrain central arteries. In both cases, green shows Islet-GFP and red, pan-endothelial kdrl:cherry. Lateral orientation is shown in A-B with anterior to the right and dorsal to the top. Abbreviations are as in Figure 1 Spontaneous Scanning TTX 6dpf A B Light N Right Eye Left Eye T T N Stable Saccade Saccade 0° Leak C Eye Position 20° Dark 20s 20° Optokinetic Step Tracking TTX 6dpf 20° Slow Phase 0° D Eye Velocity Drum Rotation Fast Phase 8s Direct 20° 20°/s 0°/s Indirect 20°/s Figure 7. Spontaneous and optokinetic behavior after TTX. (A-B) Scanning behavior of a 6 dpf embryo shown in light (A) and dark (B). Arrows mark saccades and eye position holding (stable in A or leak in B). (C-D) Optokinetic step tracking performance in response to ± 20°/s drum rotation at 0.125 Hz. Eye position is illustrated in C and eye velocity in D. Arrows mark the slow and fast phases in C and the direct and indirect components in D. Records are from the right (red) and left (blue) eyes with nasal (N) and temporal (T) directions as indicated. RE and LE direct OKR gains were 0.56 and 0.52 that were reduced by 10% and 13% from a wild type gain of 0.6. RE and LE indirect OKR gains were 0.51 and 0.52 that were reduced by 17% and 13%, respectively from a wild type gain of 0.6 (from Supplementary Figure 3) the left eye direct component slightly better than the right eye (Figures 7C and 7D). RE and LE direct OKR gains were 0.56 and 0.52 that were lower from wild type gain of 0.6 by 10% and 13%, respectively. However, RE and LE indirect OKR gains were similar at 0.51 and 0.52 and lower from wild type gain of 0.6 by 17% and 13%, respectively (from Supplementary Figure 3). For treatment with Tricaine, wild type zebrafish were raised from 18 h to 4-5 days in 0.016% Tricaine solutions. All spontaneous and/or evoked movements, save for cardiovascular function, were absent until the larvae were removed from the Tricaine. Righting and swimming reflexes were often quite slow to recover (similar to the TTX treatments), however, larval escape responses often were regained within minutes; we thus tested oculomotor behaviors within 1-4 h after removal from Tricaine. Curr Neurobiol 2016 Volume 7 Issue 2 Somewhat unexpectedly, and despite severe edema, 14 of the 17 tested Tricaine treated larvae exhibited spontaneous scanning saccadic behavior with reasonably high velocity saccades, but clearly with a diminished frequency (Figures 8A and 8B). A pretectal visual feedback loop appeared to be present as shown by the stable right eye position holding in the nasal direction (RE, Stable), but not for the other direction (RE, temporal leak). However, on comparison with scanning in the dark (Figure 8B), these behaviors much more likely reflected the individual neural integrator (VPNI) functions for the nasal and temporal directions (as illustrated by the circuits in Supplementary Figures 1C and 1D). Optokinetic tracking showed that a direct retinal-pretectal connection to motoneurons was intact as can be seen in the step-like changes in eye position (Figure 8C) and the 69 Ulrich/Grove/Torres-Vázquez/Baker A Spontaneous Scanning Tricaine 5dpf B Light Stable Stable Saccade Dark 10° Saccade 0° Right Eye C N Leak Left Eye 10° Leak N T Eye Position T Optokinetic Step Tracking Tricaine 5dpf 20s 10° Fast Phase 0° D Eye Velocity Drum Rotation Slow Phase 4s 10° Direct 10°/s 0°/s Indirect 10°/s Figure 8. Spontaneous and optokinetic behavior after Tricaine. (A-B) Scanning behavior of a 5 dpf embryo shown in either light (A) or in dark (B). Arrows mark saccades and eye position holding showing either stable (RE) or leak (LE) in A and the same pattern in B. (C-D) Optokinetic step tracking performance in response to ± 20°/s drum rotation at 0.125 Hz. Eye position is illustrated in C and eye velocity in D. Arrows mark the slow and fast phases in C and the direct and indirect components in D. Records are from the right (red) and left (blue) eyes with nasal (N) and temporal (T) directions as indicated. Both RE and LE direct OKR gains were 0.4 and reduced by 33% from wild type gain of 0.6 (from Supplementary Figure 3). RE and LE indirect OKR gains were 0.15 and 0.1, respectively and were reduced by 75% and 83% from a wild type gain of 0.6 (from Supplementary Figure 4) accompanying direct component of eye velocity (Figure 8D, Direct). Both RE and LE direct OKR gains were 0.4, reduced by 33% from wild type gain of 0.6. Also, the saccadic reset circuitry was functional as indicated by the low velocity fast phases (Figure 8C, Fast Phase). However, in all the Tricaine raised larva, the indirect component of step velocity was severely compromised (Figure 8D, Indirect), clearly indicating the pretectal to vestibular connectivity was affected (Supplementary Figures 1A and 1B). RE and LE indirect OKR gains were 0.15 and 0.1 and lower than wild type gains by 75% and 83%, respectively. In summary, at 4-5 days, after removal of drug treatments that prevented neuronal spiking and synaptic activity, oculomotor behavioral performance quickly returned within hours, but much more robustly for the TTX, rather than Tricaine, treated larvae. Discussion In this study we tested the hypothesis that the complex circuitry that gives rise to robust horizontal eye tracking, saccadic and torsional responses at 4-6 dpf is able to form and become functional in the absence of either metabolic support or neuronal activity. We found that reck and cloche mutants, each lacking intracerebral vessels and thus metabolic support for neuronal development, exhibited all oculomotor behaviors. Wild type larvae in which neuronal activity was blocked from 1 to 5 days also exhibited oculomotor responses comparable to untreated larvae once the block was removed. Our findings clearly indicate that neural network developmental patterning and central processing is independent of vasculature and neural excitability. 70 Our study provides an assessment of functional neuronal development through the observation of horizontal and torsional oculomotor performance, which is dependent on identified and well documented types of neurons and circuits residing in the forebrain, midbrain, hindbrain and periphery [12,13]. The presence of spontaneous scanning saccades in the dark for both reck and cloche mutants (Figures 3B and 3C) demonstrated the presence of a pattern generator (r3-4) interconnected with burst (r5) and integrator (r8) neurons both targeting Abd Mns and Abd Int neurons (r5-6) (Supplementary Figures 1C and 1D). All reck and many of the illustrated cloche decay time constants were similar to those of the wild type larvae. The degree to which larvae were able to maintain eye position and counteract opposing forces pulling the eye back in the original position is dependent, in part, on the maturity of the velocity to position neural network [24,26]. Reck mutants exhibited eye position holding with a network maturity on par with 5 dpf wild type larvae, albeit often with a slightly faster decay of eye position holding. Because the cloche mutants lacked visual performance and often exhibited considerable edema in the lateral orbital eye socket (Figure 2D), a wide variation of horizontal eye holding behavior was not unexpected. The eye socket edema likely compromised eye plant performance whose effect could not be separated from that of the integrator circuitry; however the presence of time constants far exceeding normal age wild type larvae suggested that the integrator circuitry was correctly established and operational. By contrast, the reck mutants did not experience any overt eye socket edema (Figure 2C) and thus the oculomotor integrator exhibited the Curr Neurobiol 2016 Volume 7 Issue 2 Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish. typical exponential eye holding position decay in dark, Figure 3B. Both reck and cloche mutants made compensatory eye movements in response to sinusoidal vertical pitch stimulus (Figures 5A and 5B), indicating the presence of a functioning three-neuron circuit spanning from the hindbrain to the midbrain in both mutants. The gain of the reck mutants in response to the pitch tilt stimulus nearly matched that of wild type only reduced by 10% from controls. In cloche mutants, after the beginning of the stimulus paradigm VOR gain was initially lower (Figure 5B, 38%) but diminished within 15-30 min likely reflecting the onset of metabolic influences on behavior. Two notable caveats exist to the conclusions that reck and often cloche mutant performance is comparable to that of wild type larvae. First, as seen in both the eye position and velocity records in Figure 4 and Supplementary Figure 3, there existed a ~ 4.0 Hz low amplitude (~ 2-4°/ s) back-ground rhythm that previously was attributed to a bandwidth limiting performance of the visual slip feedback circuitry [22]. In normal larvae, this oscillation is often present at ~ 1°s (Supplementary Figure 3). Under stressful circumstances, for example, large visual/ vestibular paradigm shifts, or as in the mutant cases likely, oxygen/nutrient deficits, a larger oscillation is quite evident from the onset of visuomotor testing. Predictably, prolonged optokinetic performance testing ranging from a few minutes to an hour eventually led to lower eye velocity gains, and in some cases, behavioral performance nearly disappeared (data not shown). We conclude that while there may be no essential role for vascularization in building the hindbrain oculomotor circuitry, there clearly is a role in maintaining circuitry robustness as would be required in a successful developmental transition to juvenile and adult stages. The Tricaine treated larvae did not exhibit the performance levels observed with the TTX treated animals that were almost indistinguishable from wild type larvae. In particular, behaviors dependent on temporal integration of input signals, i.e. eye position holding (compare Figures 7A and 7B to 8A and 8B) associated with spontaneous saccades and the indirect component of OKR (compare Figures 7C and 7D to 8C and 8D) were degraded and involve the vestibular and velocity position neural integrator (VPNI) networks (Supplementary Figures 1A1D). The susceptibility of these neuronal subtypes may be a function of the intrinsic and/or extrinsic synaptic conductances. TTX specifically blocks the family of Na+ channels, mostly at nM concentrations. In contrast, Tricaine, at the concentrations used in this study, has been shown to block both Na+ and Ca2+ channels in zebrafish [27-29]; furthermore, Tricaine and local anesthetics in general [30,31] not only block sodium channels, but potassium [32], nicotinic acetycholine receptor channels [33] and G protein receptor activity [34]. Even though integrative functions are compromised by Tricaine, the Curr Neurobiol 2016 Volume 7 Issue 2 presence of vestibular and neural integrator neurons and their associated connectivity is still evident as demonstrated during some stable eye position holding epochs (Figures 8A and 8B). Central signal processing was also evident for vestibular inputs to reach the burst pattern generator and trigger burst neuron activity responsible for generating fast phases and saccades (as illustrated in Supplementary Figures 1C and 1D). Velocity to position neural integrator neurons exhibit position and saccadic sensitivity [11]. The integrative property of the neural integrator neurons and associated network has been demonstrated in the adult goldfish [10,35] as well as the larval zebrafish [24,25]. TTX has minimal influence on the functional development of integration as demonstrated by spontaneous saccade and eye position holding behavior on par with that observed in wild type larvae in the dark (Figure 7B and Supplementary Figure 3B). These robust behaviors also indicate the presence of the burst pattern generator and associated functional connectivity with burst and vestibular neurons connected to Abd Mns and Abd Int neurons (Supplementary Figures 1C and 1D). While the overall performance of the Tricaine treated animals is degraded relative to the wild type and TTX treated animals, these larvae still generated saccades and exhibited eye position holding (Figures 8A and 8B). These results demonstrate the presence of a sufficient population of neurons exhibiting a correct connectivity to move the eyes appropriately. Although the slow and fast phases of OKR were also less than wild type controls (Figures 8C and 8D), eye velocity still reflected the directional change in drum velocity via the direct pretectal component to the MR and Abd Mns (Supplementary Figures 1A and 1B). However, the pretectal connection through the vestibular nucleus was not able to sustain the constant eye velocity tracking which quickly dropped by >80% (Figure 8D). Zebrafish larvae normally exhibit little eye velocity storage [20] and, therefore, are required to continuously convert slip velocity to eye velocity throughout the stimulus period (as in Supplementary Figure 3D). This constant velocity behavior was severely compromised after Tricaine, yet the vestibular slip velocity signal to the integrator network was adequate to produce a step of eye position as well as trigger fast phase resets. Overall the differences between the Tricaine and TTX treated larvae may be related to the broadly disrupted channel activity imposed by Tricaine compared to a more specific disruption of sodium channels caused by TTX. Overall in this study, we found that vascularization and neuronal excitability were not essential for the formation of numerous oculomotor nuclei displaying a substantial interconnectivity and signal processing. We, therefore, conclude that a genetic blueprint largely specifies early larval structural and physiological features; however, activity dependent feedback is clearly used to optimize an age-related oculomotor performance. 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Inhibition of mammalian Gq protein function by local anesthetics. Anesthesiology 2002; 97: 1451-1457. 35.Aksay E, Olasagasti I, Mensh BD, et al. Functional dissection of circuitry in a neural integrator. Nat Neurosci 2007; 10: 494-504. Correspondence to: Robert Baker, Department of Neuroscience and Physiology, New York University Medical Center, 550 First Avenue New York, NY 10016, USA. Tel: 914-645-2479 E-mail: [email protected] Curr Neurobiol 2016 Volume 7 Issue 2 73