Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish.

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

Convergent evolution of the lateral line system in Apogonidae (Teleostei: Percomorpha) determined from innervation.

The lateral line system and its innervation were examined in two species of the family Apogonidae (Cercamia eremia [Apogoninae] and Pseudamia gelatinosa [Pseudamiinae]). Both species were characterized by numerous superficial neuromasts (SNs; total 2,717 in C. eremia; 9,650 in P. gelatinosa), including rows on the dorsal and ventral halves of the trunk, associated with one (in C. eremia) and three (in P. gelatinosa) reduced trunk canals. The pattern of SN innervation clearly demonstrated that the overall pattern of SN distribution had evolved convergently in the two species. In C. eremia, SN rows over the entire trunk were innervated by elongated branches of the dorsal longitudinal collector nerve (DLCN) anteriorly and lateral ramus posteriorly. In P. gelatinosa, the innervation pattern of the DLCN was mirrored on the ventral half of the trunk (ventral longitudinal collector nerve: VLCN). Elongated branches of the DLCN and VLCN innervated SN rows on the dorsal and ventral halves of the trunk, respectively. The reduced trunk canal(s) apparently had no direct relationship with the increase of SNs, because these branches originated deep to the lateral line scales, none innervating canal neuromast (CN) homologues on the surface of the scales. In P. gelatinosa, a CN (or an SN row: CN homologue) occurred on every other one of their small lateral line scales, while congeners (P. hayashii and P. zonata) had an SN row (CN homologue) on every one of their large lateral line scales.

The peripheral nerves of Lepidobatrachus tadpoles (Anura, Ceratophryidae).

The peripheral nervous system of anuran larvae has traditionally been assumed to be largely invariant. Here, we describe the organization of cranial, spinal, and lateral line nerves at different larval stages of Lepidobatrachus spp. based on whole mounts. This is the first detailed description of cranial, spinal, and lateral lines innervation at premetamorphic stages of anuran larvae with notes on temporal variation. We distinguish three sources of morphological variation with respect to other anuran larvae: (a) the loss or reduction of some exclusively larval elements (i.e., the absence of the middle lateral line nerve); (b) spatial changes in the lateral line system (i.e., the supralabial arrangement of component of the anteroventral lateral line nerve); and (c) temporal changes in the disappearance of most of the lateral line system and in the premetamorphic repatterning of the spatial relationships of mandibularis ramus of the trigeminal (V) and hyomandibularis ramus of facial (VII). The innervation of limbs is achieved during late larval stages. Furthermore, comparisons among selected anurans reveal differences in tadpole brain morphology. The spatial and temporal variation found in the peripheral nerves of Lepidobatrachus larvae testifies to previously unappreciated variation in anuran larval morphology.

Mathematical Modeling and Analyses of Interspike-Intervals of Spontaneous Activity in Afferent Neurons of the Zebrafish Lateral Line.

Without stimuli, hair cells spontaneously release neurotransmitter leading to spontaneous generation of action potentials (spikes) in innervating afferent neurons. We analyzed spontaneous spike patterns recorded from the lateral line of zebrafish and found that distributions of interspike intervals (ISIs) either have an exponential shape or an "L" shape that is characterized by a sharp decay but wide tail. ISI data were fitted to renewal-process models that accounted for the neuron refractory periods and hair-cell synaptic release. Modeling the timing of synaptic release using a mixture of two exponential distributions yielded the best fit for our ISI data. Additionally, lateral line ISIs displayed positive serial correlation and appeared to exhibit switching between faster and slower modes of spike generation. This pattern contrasts with previous findings from the auditory system where ISIs tended to have negative serial correlation due to synaptic depletion. We propose that afferent neuron innervation with multiple and heterogenous hair-cells synapses, each influenced by changes in calcium domains, can serve as a mechanism for the random switching behavior. Overall, our analyses provide evidence of how physiological similarities and differences between synapses and innervation patterns in the auditory, vestibular, and lateral line systems can lead to variations in spontaneous activity.

Connectomics of the zebrafish's lateral-line neuromast reveals wiring and miswiring in a simple microcircuit.

The lateral-line neuromast of the zebrafish displays a restricted, consistent pattern of innervation that facilitates the comparison of microcircuits across individuals, developmental stages, and genotypes. We used serial blockface scanning electron microscopy to determine from multiple specimens the neuromast connectome, a comprehensive set of connections between hair cells and afferent and efferent nerve fibers. This analysis delineated a complex but consistent wiring pattern with three striking characteristics: each nerve terminal is highly specific in receiving innervation from hair cells of a single directional sensitivity; the innervation is redundant; and the terminals manifest a hierarchy of dominance. Mutation of the canonical planar-cell-polarity gene vangl2, which decouples the asymmetric phenotypes of sibling hair-cell pairs, results in randomly positioned, randomly oriented sibling cells that nonetheless retain specific wiring. Because larvae that overexpress Notch exhibit uniformly oriented, uniformly innervating hair-cell siblings, wiring specificity is mediated by the Notch signaling pathway.

Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse.

In vivo analysis of axonal transport in zebrafish.

Intracellular transport of proteins and organelles in neurons plays an essential role in nervous system development and maintenance. Axon outgrowth, synapse formation, and synapse function, among other physiological processes, require active transport of these cargos between the neuronal soma and axon terminals. Abnormalities in this axonal transport are associated with a number of neurodevelopmental and neurodegenerative disorders, such as Charcot-Marie-Tooth disease, Alzheimer disease, and amyotrophic lateral sclerosis. Despite its importance for nervous system development and health, methods for visualizing axonal transport in an intact vertebrate have been lacking. Using the advantages of the zebrafish system, we have developed a straightforward approach to visualize axonal transport of various cargos and motor proteins in intact zebrafish embryos and larvae. Here, we describe this approach in detail and discuss how it can be applied to address questions related to cargo-specific transport regulation and its effects on axon morphology and function in the developing and mature nervous system.

Dopamine Modulates the Activity of Sensory Hair Cells.

The senses of hearing and balance are subject to modulation by efferent signaling, including the release of dopamine (DA). How DA influences the activity of the auditory and vestibular systems and its site of action are not well understood. Here we show that dopaminergic efferent fibers innervate the acousticolateralis epithelium of the zebrafish during development but do not directly form synapses with hair cells. However, a member of the D1-like receptor family, D1b, tightly localizes to ribbon synapses in inner ear and lateral-line hair cells. To assess modulation of hair-cell activity, we reversibly activated or inhibited D1-like receptors (D1Rs) in lateral-line hair cells. In extracellular recordings from hair cells, we observed that D1R agonist SKF-38393 increased microphonic potentials, whereas D1R antagonist SCH-23390 decreased microphonic potentials. Using ratiometric calcium imaging, we found that increased D1R activity resulted in larger calcium transients in hair cells. The increase of intracellular calcium requires Cav1.3a channels, as a Cav1 calcium channel antagonist, isradipine, blocked the increase in calcium transients elicited by the agonist SKF-38393. Collectively, our results suggest that DA is released in a paracrine fashion and acts at ribbon synapses, likely enhancing the activity of presynaptic Cav1.3a channels and thereby increasing neurotransmission. SIGNIFICANCE STATEMENT: The neurotransmitter dopamine acts in a paracrine fashion (diffusion over a short distance) in several tissues and bodily organs, influencing and regulating their activity. The cellular target and mechanism of the action of dopamine in mechanosensory organs, such as the inner ear and lateral-line organ, is not clearly understood. Here we demonstrate that dopamine receptors are present in sensory hair cells at synaptic sites that are required for signaling to the brain. When nearby neurons release dopamine, activation of the dopamine receptors increases the activity of these mechanosensitive cells. The mechanism of dopamine activation requires voltage-gated calcium channels that are also present at hair-cell synapses.

Superficial neuromasts facilitate non-visual feeding by larval striped bass (Morone saxatilis).

To investigate whether mechanoreception is used in non-visual feeding in larval striped bass (Morone saxatilis), the ontogeny of superficial neuromasts along the lateral line was described using the vital stain FM1-43FX and fluorescent microscopy. The number of neuromasts visible along one flank increased from 11 at first feeding [5 to 7 days post-hatch (dph)] to >150 by the juvenile stage (27 dph). A neomycin dose response (0, 1, 2 and 5 mmol l(-1)) was evaluated for neuromast ablation of bass aged 10, 13, 17 and 20 dph. Using these same age groups, the ability of bass to catch Artemia salina prey in both dark and light tank-based feeding trials was compared between larvae with neuromasts ablated using neomycin (5 mmol l(-1)) and controls. Neomycin significantly reduced the incidence of feeding in the light and dark. Among larvae that fed, those in the dark treated with neomycin caught fewer Artemia (~5 prey h(-1); P<0.05) than controls (16 prey h(-1) at 10 dph; 72 prey h(-1) at 20 dph). In the light, by contrast, neomycin treatment had no significant effect on prey capture by larvae age 13 to 20 dph, but did inhibit feeding of 10 dph larvae. Verification that neomycin was specifically ablating the hair cells of superficial neuromasts and not affecting either neuromast innervation, olfactory pits, or taste cells was achieved by a combination of staining with FM1-43FX and immunocytochemistry for tubulin and the calcium binding proteins, S100 and calretinin.

Innervation is required for sense organ development in the lateral line system of adult zebrafish.

Superficial mechanosensory organs (neuromasts) distributed over the head and body of fishes and amphibians form the "lateral line" system. During zebrafish adulthood, each neuromast of the body (posterior lateral line system, or PLL) produces "accessory" neuromasts that remain tightly clustered, thereby increasing the total number of PLL neuromasts by a factor of more than 10. This expansion is achieved by a budding process and is accompanied by branches of the afferent nerve that innervates the founder neuromast. Here we show that innervation is essential for the budding process, in complete contrast with the development of the embryonic PLL, where innervation is entirely dispensable. To obtain insight into the molecular mechanisms that underlie the budding process, we focused on the terminal system that develops at the posterior tip of the body and on the caudal fin. In this subset of PLL neuromasts, bud neuromasts form in a reproducible sequence over a few days, much faster than for other PLL neuromasts. We show that wingless/int (Wnt) signaling takes place during, and is required for, the budding process. We also show that the Wnt activator R-spondin is expressed by the axons that innervate budding neuromasts. We propose that the axon triggers Wnt signaling, which itself is involved in the proliferative phase that leads to bud formation. Finally, we show that innervation is required not only for budding, but also for long-term maintenance of all PLL neuromasts.

Patch-clamp recordings from lateral line neuromast hair cells of the living zebrafish.

Zebrafish are popular models for biological discovery. For investigators of the auditory and vestibular periphery, manipulations of hair cell and synaptic mechanisms have relied on inferences from extracellular recordings of physiological activity. We now provide data showing that hair cells and supporting cells of the lateral line can be directly patch-clamped, providing the first recordings of ionic channel activity, synaptic vesicle release, and gap junctional coupling in the neuromasts of living fish. Such capabilities will allow more detailed understanding of mechano-sensation of the zebrafish.

Morphological structure and peripheral innervation of the lateral line system in the Siberian sturgeon (Acipenser baerii).

Light and scanning electron microscopy (SEM) were used to study the epidermal lateral line system of the Siberian sturgeon (Acipenser baerii Brandt, 1869). This system consists of mechanoreceptive neuromasts, ampullae and the electroreceptive organ. The neuromasts are located in 5 pairs of cephalic and 1 pair of trunk canals and superficially in the middle and posterior pit lines that lie dorsomedially along the top of the skull immediately adjacent to the otic ampullae field. Both canal neuromasts and pit organ superficial neuromasts have opposite polarized hair cells that are parallel along the axis of the canal and pit line, respectively. However, they differ in both size and shape and in the density and length of the hair bundles. The ampullae are confined on the head, adjacent to the neuromast lines. The morphological structure of the ampullae in the Siberian sturgeon is similar to the ampullae in elasmobranchs and other primitive fish. Nevertheless, it has a relatively large mucus-filled ampulla, and a shorter and narrower canal leading to a small opening to the outer epidermal surface. We also present new information concerning the peripheral innervation of lateral line receptors in sturgeons. The receptors of the lateral line system are innervated by 2 pairs of cranial nerves: anterior and posterior lateral line nerves. The peripheral processes of the anterior lateral line nerve form superficial ophthalmic, buccal, otic and anteroventral rami. The peripheral processes of the posterior lateral line nerve form middle, supratemporal and lateral rami.

The paratympanic organ: a barometer and altimeter in the middle ear of birds?

A century has passed since the discovery of the paratympanic organ (PTO), a mechanoreceptive sense organ in the middle ear of birds and other tetrapods. This luminal organ contains a sensory epithelium with typical mechanosensory hair cells and may function as a barometer and altimeter. The organ is arguably the most neglected sense organ in living tetrapods. The PTO is believed to be homologous to a lateral line sense organ, the spiracular sense organ of nonteleostean fishes. Our review summarizes the current state of knowledge of the PTO and draws attention to the astounding lack of information about the unique and largely unexplored sensory modality of barometric perception.

No role for direct touch using the pectoral fins, as an information gathering strategy in a blind fish.

Blind Mexican cave fish (Astyanax fasciatus) lack a functional visual system and have been shown to sense their environment using a technique called hydrodynamic imaging, whereby nearby objects are detected by sensing distortions in the flow field of water around the body using the mechanosensory lateral line. This species has also been noted to touch obstacles, mainly with the pectoral fins, apparently using this tactile information alongside hydrodynamic imaging to sense their surroundings. This study aimed to determine the relative contributions of hydrodynamic and tactile information during wall following behaviour in blind Mexican cave fish. A wall was custom built with a 'netted' region in its centre, which provided very similar tactile information to a solid tank wall, but was undetectable using hydrodynamic imaging. The fish swam significantly closer to and collided more frequently with the netted region of this wall than the solid regions, indicating that the fish did not perceive the netted region as a solid obstacle despite being able to feel it as such with their pectoral fins. We conclude that the touching of objects with the pectoral fins may be an artefact of the intrinsic link between pectoral fin extensions and tail beating whilst swimming, and does not function to gather information. During wall following, hydrodynamic information appears to be used strongly in preference to tactile information in this non-visual system.

From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes.

The inner ear and the epibranchial ganglia constitute much of the sensory system in the caudal vertebrate head. The inner ear consists of mechanosensory hair cells, their neurons, and structures necessary for sound and balance sensation. The epibranchial ganglia are knots of neurons that innervate and relay sensory signals from several visceral organs and the taste buds. Their development was once thought to be independent, in line with their independent functions. However, recent studies indicate that both systems arise from a morphologically distinct common precursor domain: the posterior placodal area. This review summarises recent studies into the induction, morphogenesis and innervation of these systems and discusses lineage restriction and cell specification in the context of their common origin.

Lateral line system and its innervation in Tetraodontiformes with outgroup comparisons: descriptions and phylogenetic implications.

The lateral line system and its innervation in ten tetraodontiform families and five outgroup taxa were examined. Although some homology issues remained unresolved, tetraodontiforms were characterized by having two types (at least) of superficial neuromasts (defined by the presence or absence of supporting structures) and accessory lateral lines and neuromasts (except Molidae in which "accessory" elements were absent). The preopercular line in Tetraodontiformes was not homologous with that of typical teleosts, because the line was innervated by the opercular ramule that was newly derived from the mandibular ramus, the condition being identical to that in Lophiidae. Within Tetraodontiformes, the number of neuromasts varied between 70 and 277 in the main lines and between 0 and 52 in accessory elements. Variations were also recognized in the presence or absence of the supraorbital commissure, mandibular line, otic line, postotic line, ventral trunk line, and some lateral line nerve rami, most notably the dorsal branch of the opercular ramule, being absent in Aracanidae, Ostraciidae, Tetraodontidae, Diodontidae, and Molidae. Morphological characteristics derived from the lateral line system and its innervation provided some support for a sister relationship of tetraodontiforms with lophiiforms.

A G protein-coupled receptor is essential for Schwann cells to initiate myelination.

The myelin sheath allows axons to conduct action potentials rapidly in the vertebrate nervous system. Axonal signals activate expression of specific transcription factors, including Oct6 and Krox20, that initiate myelination in Schwann cells. Elevation of cyclic adenosine monophosphate (cAMP) can mimic axonal contact in vitro, but the mechanisms that regulate cAMP levels in vivo are unknown. Using mutational analysis in zebrafish, we found that the G protein-coupled receptor Gpr126 is required autonomously in Schwann cells for myelination. In gpr126 mutants, Schwann cells failed to express oct6 and krox20 and were arrested at the promyelinating stage. Elevation of cAMP in gpr126 mutants, but not krox20 mutants, could restore myelination. We propose that Gpr126 drives the differentiation of promyelinating Schwann cells by elevating cAMP levels, thereby triggering Oct6 expression and myelination.

Receptive field organization across multiple electrosensory maps. II. Computational analysis of the effects of receptive field size on prey localization.

The electric fish Apteronotus leptorhynchus emits a high-frequency electric organ discharge (EOD) sensed by specialized electroreceptors (P-units) distributed across the fish's skin. Objects such as prey increase the amplitude of the EOD over the underlying skin and thus cause an increase in P-unit discharge. The resulting localized intensity increase is called the electric image and is detected by its effect on the P-unit population; the electric image peak value and the extent to its spreads are cues utilized by these fish to estimate the location and size of its prey. P-units project topographically to three topographic maps in the electrosensory lateral line lobe (ELL): centromedial (CMS), centrolateral (CLS), and lateral (LS) segments. In a companion paper I have calculated the receptive fields (RFs) in these maps: RFs were small in CMS and very large in LS, with intermediate values in CLS. Here I use physiological data to create a simple model of the RF structure within the three ELL maps and to compute the response of these model maps to simulated prey. The Fisher information (FI) method was used to compute the optimal estimates possible for prey localization across the three maps. The FI predictions were compared with behavioral studies on prey detection. These comparisons were used to frame alternative hypotheses on the functions of the three maps and on the constraints that RF size and synaptic strength impose on weak signal detection and estimation.

Receptive field organization across multiple electrosensory maps. I. Columnar organization and estimation of receptive field size.

The electric fish Apteronotus leptorhynchus emits a high-frequency electric organ discharge (EOD) sensed by specialized electroreceptors (P-units). Amplitude modulations (AMs) of the EOD are caused by objects such as prey as well as by social interactions with conspecifics. The firing rate of P-units is modulated by the AMs due to both objects and communication signals. P-units trifurcate as they enter the medulla; they terminate topographically with three maps of the electrosensory lateral line lobe (ELL): the centromedial (CMS), centrolateral (CLS), and lateral (LS) segments. Within each map P-units terminate onto the basal dendrites of pyramidal cells. Anterograde filling of P-units and retrograde filling of the basal bushes of pyramidal cells were used to estimate their respective spreads and spacing in the three maps. These estimates were used to compute the receptive field structure of the pyramidal cells: receptive fields were small in CMS and very large in LS with intermediate values in CLS. There are several classes of pyramidal cells defined by morphological and functional criteria; these cells are organized into columns such that each column contains one member of each class and all cells within a column receive the same P-unit input.

Receptive field properties of neurons in the electrosensory lateral line lobe of the weakly electric fish, Gnathonemus petersii.

The receptive field of a sensory neuron is known as that region in sensory space where a stimulus will alter the response of the neuron. We determined the spatial dimensions and the shape of receptive fields of electrosensitive neurons in the medial zone of the electrosensory lateral line lobe of the African weakly electric fish, Gnathonemus petersii, by using single cell recordings. The medial zone receives input from sensory cells which encode the stimulus amplitude. We analysed the receptive fields of 71 neurons. The size and shape of the receptive fields were determined as a function of spike rate and first spike latency and showed differences for the two analysis methods used. Spatial diameters ranged from 2 to 36 mm (spike rate) and from 2.45 to 14.12 mm (first spike latency). Some of the receptive fields were simple consisting only of one uniform centre, whereas most receptive fields showed a complex and antagonistic centre-surround organisation. Several units had a very complex structure with multiple centres and surrounding-areas. While receptive field size did not correlate with peripheral receptor location, the complexity of the receptive fields increased from rostral to caudal along the fish's body.