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The sense of smell is generated by electrical currents that are influenced by the concentration of ions in olfactory sensory neurons and mucus. In contrast to the extensive morphological and molecular characterization of sensory neurons, there has been little description of the cells that control ion concentrations in the zebrafish olfactory system. Here, we report the molecular and ultrastructural characterization of zebrafish olfactory ionocytes. Transcriptome analysis suggests that the zebrafish olfactory epithelium contains at least three different ionocyte types, which resemble Na⁺/K⁺-ATPase-rich (NaR), H⁺-ATPase-rich (HR), and Na⁺/Cl^- cotransporter (NCC) cells, responsible for calcium, pH, and chloride regulation, respectively, in the zebrafish skin. In the olfactory epithelium, NaR-like and HR-like ionocytes are usually adjacent to one another, whereas NCC-like cells are usually solitary. The distinct subtypes are differentially distributed: NaR-like/HR-like cell pairs are found broadly within the olfactory epithelium, whereas NCC-like cells reside within the peripheral non-sensory multiciliated cell zone. Comparison of gene expression and serial-section electron microscopy analysis indicates that the NaR-like cells wrap around the HR-like cells and are connected to them by shallow tight junctions. The development of olfactory ionocyte subtypes is also differentially regulated, as pharmacological Notch inhibition leads to a loss of NaR-like and HR-like cells, but does not affect NCC-like ionocyte number. These results provide a molecular and anatomical characterization of olfactory ionocytes in a stenohaline freshwater teleost. The paired ionocytes suggest that both transcellular and paracellular transport regulate ion concentrations in the olfactory epithelium, while the solitary ionocytes may enable independent regulation of ciliary beating.

BACKGROUND: The habenula is a major regulator of serotonergic neurons in the dorsal raphe, and thus of brain state. The functional connectivity between these regions is incompletely characterized. Here, we use the ability of changes in irradiance to trigger reproducible changes in activity in the habenula and dorsal raphe of zebrafish larvae, combined with two-photon laser ablation of specific neurons, to establish causal relationships.

RESULTS: Neurons in the habenula can show an excitatory response to the onset or offset of light, while neurons in the anterior dorsal raphe display an inhibitory response to light, as assessed by calcium imaging. The raphe response changed in a complex way following ablations in the dorsal habenula (dHb) and ventral habenula (vHb). After ablation of the ON cells in the vHb (V-ON), the raphe displayed no response to light. After ablation of the OFF cells in the vHb (V-OFF), the raphe displayed an excitatory response to darkness. After ablation of the ON cells in the dHb (D-ON), the raphe displayed an excitatory response to light. We sought to develop in silico models that could recapitulate the response of raphe neurons as a function of the ON and OFF cells of the habenula. Early attempts at mechanistic modeling using ordinary differential equation (ODE) failed to capture observed raphe responses accurately. However, a simple two-layer fully connected neural network (NN) model was successful at recapitulating the diversity of observed phenotypes with root-mean-squared error values ranging from 0.012 to 0.043. The NN model also estimated the raphe response to ablation of D-off cells, which can be verified via future experiments.

CONCLUSION: Lesioning specific cells in different regions of habenula led to qualitatively different responses to light in the dorsal raphe. A simple neural network is capable of mimicking experimental observations. This work illustrates the ability of computational modeling to integrate complex observations into a simple compact formalism for generating testable hypotheses, and for guiding the design of biological experiments.

The analysis of neural circuits has been revolutionized by optogenetic methods. Light-gated chloride-conducting anion channelrhodopsins (ACRs)-recently emerged as powerful neuron inhibitors. For cells or sub-neuronal compartments with high intracellular chloride concentrations, however, a chloride conductance can have instead an activating effect. The recently discovered light-gated, potassium-conducting, kalium channelrhodopsins (KCRs) might serve as an alternative in these situations, with potentially broad application. As yet, KCRs have not been shown to confer potent inhibitory effects in small genetically tractable animals. Here, we evaluated the utility of KCRs to suppress behavior and inhibit neural activity in Drosophila, Caenorhabditis elegans, and zebrafish. In direct comparisons with ACR1, a KCR1 variant with enhanced plasma-membrane trafficking displayed comparable potency, but with improved properties that include reduced toxicity and superior efficacy in putative high-chloride cells. This comparative analysis of behavioral inhibition between chloride- and potassium-selective silencing tools establishes KCRs as next-generation optogenetic inhibitors for in vivo circuit analysis in behaving animals.

Photoreceptors in the vertebrate eye are dependent on the retinal pigmented epithelium for a variety of functions including retinal re-isomerization and waste disposal. The light-sensitive pineal gland of fish, birds, and amphibians is evolutionarily related to the eye but lacks a pigmented epithelium. Thus, it is unclear how these functions are performed. Here, we ask whether a subpopulation of zebrafish pineal cells, which express glial markers and visual cycle genes, is involved in maintaining photoreceptors. Selective ablation of these cells leads to a loss of pineal photoreceptors. Moreover, these cells internalize exorhodopsin that is secreted by pineal rod-like photoreceptors, and in turn release CD63-positive extracellular vesicles (EVs) that are taken up by pdgfrb-positive phagocytic cells in the forebrain meninges. These results identify a subpopulation of glial cells that is critical for pineal photoreceptor survival and indicate the existence of cells in the forebrain meninges that receive EVs released by these pineal cells and potentially function in waste disposal.

The habenula is an evolutionarily conserved structure of the vertebrate brain that is essential for behavioural flexibility and mood control. It is spontaneously active and is able to access diverse states when the animal is exposed to sensory stimuli. Here we investigate the dynamics of habenula spontaneous activity, to gain insight into how sensitivity is optimized. Two-photon calcium imaging was performed in resting zebrafish larvae at single cell resolution. An analysis of avalanches of inferred spikes suggests that the habenula is subcritical. Activity had low covariance and a small mean, arguing against dynamic criticality. A multiple regression estimator of autocorrelation time suggests that the habenula is neither fully asynchronous nor perfectly critical, but is reverberating. This pattern of dynamics may enable integration of information and high flexibility in the tuning of network properties, thus providing a potential mechanism for the optimal responses to a changing environment.

Significance Statement: Spontaneous activity in neurons shapes the response to stimuli. One structure with a high level of spontaneous neuronal activity is the habenula, a regulator of broadly acting neuromodulators involved in mood and learning. How does this activity influence habenula function? We show here that the habenula of a resting animal is near criticality, in a state termed reverberation. This pattern of dynamics is consistent with high sensitivity and flexibility, and may enable the habenula to respond optimally to a wide range of stimuli.

We report the presence of a rare cell type, the olfactory rod cell, in the developing zebrafish olfactory epithelium. These cells each bear a single actin-rich rod-like apical projection extending 5-10 μm from the epithelial surface. Live imaging with a ubiquitous Lifeact-RFP label indicates that the olfactory rods can oscillate. Olfactory rods arise within a few hours of the olfactory pit opening, increase in numbers and size during larval stages, and can develop in the absence of olfactory cilia. Olfactory rod cells differ in morphology from the known classes of olfactory sensory neuron, but express reporters driven by neuronal promoters. A sub-population of olfactory rod cells expresses a Lifeact-mRFPruby transgene driven by the sox10 promoter. Mosaic expression of this transgene reveals that olfactory rod cells have rounded cell bodies located apically in the olfactory epithelium and have no detectable axon. We offer speculation on the possible function of these cells in the Discussion.