A behavioral and modeling study of control algorithms underlying the translational optomotor response in larval zebrafish with implications for neural circuit function.

The optomotor response (OMR) is central to the locomotory behavior in diverse animal species including insects, fish and mammals. Furthermore, the study of the OMR in larval zebrafish has become a key model system for investigating the neural basis of sensorimotor control. However, a comprehensive understanding of the underlying control algorithms is still outstanding. In fish it is often assumed that the OMR, by reducing average optic flow across the retina, serves to stabilize position with respect to the ground. Yet the degree to which this is achieved, and how it could emerge from the intermittent burst dynamics of larval zebrafish swimming, are unclear. Here, we combine detailed computational modeling with a new approach to free-swimming experiments in which we control the amount of visual feedback produced by a given motor effort by varying the height of the larva above a moving grid stimulus. We develop an account of underlying feedback control mechanisms that describes both the bout initiation process and the control of swim speed during bouts. We observe that the degree to which fish stabilize their position is only partial and height-dependent, raising questions about its function. We find the relative speed profile during bouts follows a fixed temporal pattern independent of absolute bout speed, suggesting that bout speed and bout termination are not separately controlled. We also find that the reverse optic flow, experienced when the fish is swimming faster than the stimulus, plays a minimal role in control of the OMR despite carrying most of the sensory information about self-movement. These results shed new light on the underlying dynamics of the OMR in larval zebrafish and will be crucial for future work aimed at identifying the neural basis of this behavior.

µSPIM Toolset: A software platform for selective plane illumination microscopy.

BACKGROUND: Selective Plane Illumination Microscopy (SPIM) is a fluorescence imaging technique that allows volumetric imaging at high spatio-temporal resolution to monitor neural activity in live organisms such as larval zebrafish. A major challenge in the construction of a custom SPIM microscope using a scanned laser beam is the control and synchronization of the various hardware components. NEW METHOD: We present an open-source software, µSPIM Toolset, built around the widely adopted MicroManager platform, that provides control and acquisition functionality for a SPIM. A key advantage of µSPIM Toolset is a series of calibration procedures that optimize acquisition for a given set-up, making it relatively independent of the optical design of the microscope or the hardware used to build it. RESULTS: µSPIM Toolset allows imaging of calcium activity throughout the brain of larval zebrafish at rates of 100 planes per second with single cell resolution. COMPARISON WITH EXISTING METHODS: Several designs of SPIM have been published but are focused on imaging of developmental processes using a slower setup with a moving stage and therefore have limited use for functional imaging. In comparison, µSPIM Toolset uses a scanned beam to allow imaging at higher acquisition frequencies while minimizing disturbance of the sample. CONCLUSIONS: The µSPIM Toolset provides a flexible solution for the control of SPIM microscopes and demonstrated its utility for brain-wide imaging of neural activity in larval zebrafish.

Motor Behavior Selectively Inhibits Hair Cells Activated by Forward Motion in the Lateral Line of Zebrafish.

How do sensory systems disambiguate events in the external world from signals generated by the animal's own motor actions? One strategy is to use an "efference copy" of the motor command to inhibit the sensory input caused by active behavior [1]. But does inhibition of self-generated inputs also block transmission of external stimuli? We investigated this question in the lateral line, a sensory system that allows fish and amphibians to detect water currents and that contributes to behaviors such as rheotaxis [2] and predator avoidance [3, 4]. This mechanical sense begins in hair cells grouped into neuromasts dotted along the animal's body [5]. Each neuromast contains two populations of hair cells, activated by deflection in either the anterior or posterior direction [6], as well as efferent fibers that are active during motor behavior to suppress afferents projecting to the brain [7-12]. To test how far the efference copy signal modulates responses to external stimuli, we imaged neural and synaptic activity in larval zebrafish during fictive swimming. We find that efferents transmit a precise copy of the motor signal and a single spike in the motor nerve can be associated with ∼50% inhibition of glutamate release. The efference copy signal acted with high selectivity on hair cells polarized to be activated by posterior deflections, as would occur during forward motion. During swimming, therefore, "push-pull" encoding of stimulus direction by afferents of opposite polarity is disrupted while still allowing a subset of hair cells to detect stimuli originating in the external world.

No evidence for a magnetite-based magnetoreceptor in the lagena of pigeons.

It is well established that an array of avian species sense the Earth's magnetic field and use this information for orientation and navigation. While the existence of a magnetic sense can no longer be disputed, the underlying cellular and biophysical basis remains unknown. It has been proposed that pigeons exploit a magnetoreceptor based on magnetite crystals (Fe3O4) that are located within the lagena [1], a sensory epithelium of the inner ear. It has been hypothesised that these magnetic crystals form a bed of otoconia that stimulate hair cells transducing magnetic information into a neuronal impulse. We performed a systematic high-sensitivity screen for iron in the pigeon lagena using synchrotron X-ray fluorescence microscopy coupled with the analysis of serial sections by transmission electron microscopy. We find no evidence for extracellular magnetic otoconia or intracellular magnetite crystals, suggesting that if an inner ear magnetic sensor does exist it relies on a different biophysical mechanism.

The Transfer Characteristics of Hair Cells Encoding Mechanical Stimuli in the Lateral Line of Zebrafish.

Hair cells transmit mechanical information by converting deflection of the hair bundle into synaptic release of glutamate. We have investigated this process in the lateral line of larval zebrafish (male and female) to understand how stimuli are encoded within a neuromast. Using multiphoton microscopy in vivo, we imaged synaptic release of glutamate using the reporter iGluSnFR as well as deflections of the cupula. We found that the neuromast is composed of a functionally diverse population of hair cells. Half the hair cells signaled cupula motion in both directions from rest, either by increasing glutamate release in response to a deflection in the positive direction or by reducing release in the negative direction. The relationship between cupula deflection and glutamate release demonstrated maximum sensitivity at displacements of just ∼40 nm in the positive direction. The remaining hair cells only signaled motion in one direction and were less sensitive, extending the operating range of the neuromast beyond 1 µm. Adaptation of the synaptic output was also heterogeneous, with some hair cells generating sustained glutamate release in response to a steady deflection of the cupula and others generating transient outputs. Finally, a distinct signal encoded a return of the cupula to rest: a large and transient burst of glutamate release from hair cells unresponsive to the initial stimulus. A population of hair cells with these different sensitivities, operating ranges, and adaptive properties will allow the neuromast to encode weak stimuli while maintaining the dynamic range to signal the amplitude and duration of stronger deflections.SIGNIFICANCE STATEMENT Hair cells transmit information about mechanical stimuli by converting very small deflections of their hair bundle into changes in the release of the neurotransmitter glutamate. We have measured this input/output relation in the live fish using a fluorescent protein and find that different hair cells vary in their mechanical sensitivity and the time course of their response. These variations will allow the fish to sense the timing and duration of both very weak stimuli (∼40 nm deflections) and strong stimuli (∼1 µm), underlying the ability of the fish to avoid predators and maintain its body position in flowing water.

No evidence for intracellular magnetite in putative vertebrate magnetoreceptors identified by magnetic screening.

The cellular basis of the magnetic sense remains an unsolved scientific mystery. One theory that aims to explain how animals detect the magnetic field is the magnetite hypothesis. It argues that intracellular crystals of the iron oxide magnetite (Fe3O4) are coupled to mechanosensitive channels that elicit neuronal activity in specialized sensory cells. Attempts to find these primary sensors have largely relied on the Prussian Blue stain that labels cells rich in ferric iron. This method has proved problematic as it has led investigators to conflate iron-rich macrophages with magnetoreceptors. An alternative approach developed by Eder et al. [Eder SH, et al. (2012) Proc Natl Acad Sci USA 109(30):12022-12027] is to identify candidate magnetoreceptive cells based on their magnetic moment. Here, we explore the utility of this method by undertaking a screen for magnetic cells in the pigeon. We report the identification of a small number of cells (1 in 476,000) with large magnetic moments (8-106 fAm(2)) from various tissues. The development of single-cell correlative light and electron microscopy (CLEM) coupled with electron energy loss spectroscopy (EELS) and energy-filtered transmission electron microscopy (EFTEM) permitted subcellular analysis of magnetic cells. This revealed the presence of extracellular structures composed of iron, titanium, and chromium accounting for the magnetic properties of these cells. Application of single-cell CLEM to magnetic cells from the trout failed to identify any intracellular structures consistent with biogenically derived magnetite. Our work illustrates the need for new methods to test the magnetite hypothesis of magnetosensation.

An iron-rich organelle in the cuticular plate of avian hair cells.

Hair cells reside in specialized epithelia in the inner ear of vertebrates, mediating the detection of sound, motion, and gravity. The transduction of these stimuli into a neuronal impulse requires the deflection of stereocilia, which are stabilized by the actin-rich cuticular plate. Recent electrophysiological studies have implicated the vestibular system in pigeon magnetosensation. Here we report the discovery of a single iron-rich organelle that resides in the cuticular plate of cochlear and vestibular hair cells in the pigeon. Transmission electron microscopy, coupled with elemental analysis, has shown that this structure is composed of ferritin-like granules, is approximately 300-600 nm in diameter, is spherical, and in some instances is membrane-bound and/or organized in a paracrystalline array. This organelle is found in hair cells in a wide variety of avian species, but not in rodents or in humans. This structure may function as (1) a store of excess iron, (2) a stabilizer of stereocilia, or (3) a mediator of magnetic detection. Given the specific subcellular location, elemental composition, and evolutionary conservation, we propose that this structure is an integral component of the sensory apparatus in birds.

Clusters of iron-rich cells in the upper beak of pigeons are macrophages not magnetosensitive neurons.

Understanding the molecular and cellular mechanisms that mediate magnetosensation in vertebrates is a formidable scientific problem. One hypothesis is that magnetic information is transduced into neuronal impulses by using a magnetite-based magnetoreceptor. Previous studies claim to have identified a magnetic sense system in the pigeon, common to avian species, which consists of magnetite-containing trigeminal afferents located at six specific loci in the rostral subepidermis of the beak. These studies have been widely accepted in the field and heavily relied upon by both behavioural biologists and physicists. Here we show that clusters of iron-rich cells in the rostro-medial upper beak of the pigeon Columbia livia are macrophages, not magnetosensitive neurons. Our systematic characterization of the pigeon upper beak identified iron-rich cells in the stratum laxum of the subepidermis, the basal region of the respiratory epithelium and the apex of feather follicles. Using a three-dimensional blueprint of the pigeon beak created by magnetic resonance imaging and computed tomography, we mapped the location of iron-rich cells, revealing unexpected variation in their distribution and number--an observation that is inconsistent with a role in magnetic sensation. Ultrastructure analysis of these cells, which are not unique to the beak, showed that their subcellular architecture includes ferritin-like granules, siderosomes, haemosiderin and filopodia, characteristics of iron-rich macrophages. Our conclusion that these cells are macrophages and not magnetosensitive neurons is supported by immunohistological studies showing co-localization with the antigen-presenting molecule major histocompatibility complex class II. Our work necessitates a renewed search for the true magnetite-dependent magnetoreceptor in birds.

The nature of upright and inverted face representations: an adaptation-transfer study of configuration.

It is considered that whole-face processing of spatial structure may only be possible in upright faces, with only local feature processing in inverted faces. We asked whether this was due to impoverished representations of inverted faces. We performed two experiments. In the first, we divided faces into segments to create 'exploded' faces with disrupted second-order structures, and 'scrambled' faces with altered first-order relations; in the second we shifted features within intact facial outlines to create equivalent disruptions of spatial structure. In both we assessed the transfer of adaptation between faces with altered structure and intact faces. Scrambled adaptors did not adapt upright or inverted intact faces, indicating that a whole-face configuration is required at either orientation. Both upright and inverted faces showed a similar decline in aftereffect magnitude when adapting faces had altered second-order structure, implying that this structure is present in both upright and inverted face representations. We conclude that inverted faces are not represented simply as a collection of features, but have a whole-face configuration with second-order structure, similar to upright faces. Thus the qualitative impairments induced by inversion are not due to degraded inverted facial representations, but may reflect limitations in perceptual mechanisms.