Transcriptome characterization of three main ganglia in Patinopecten yessoensis provides novel insights into a monophyletic origin of the bilaterian central nervous system.

Sensory information is fundamental for navigation. Visual motion is used by animals to estimate their traveling distance and direction, and landmarks allow animals to tether their location and orientation to their environment. How such signals are integrated in the vertebrate brain is poorly understood. Here we investigate the representation of directional whole field visual motion and landmark position in the larval zebrafish head direction circuit. Using calcium imaging we show that these stimuli are represented in the habenula, interpeduncular nucleus and anterior hindbrain. In the dorsal interpeduncular nucleus, both stimuli are topographically arranged and align with the representation of the heading signal. Neuronal ablations show that the landmark responses, but not the whole field motion responses, require intact habenula input. Our findings suggest the interpeduncular nucleus as a site for integration of the heading signal with visual information, shedding light on how navigational signals are processed in the vertebrate brain.

Glutamate uptake activity in retina Müller cells: Circadian modulation.

Purkinje neurons are critical for the functioning of the cerebellum, which is among the oldest and most conserved regions of the vertebrate brain. In mammals and in larval zebrafish, Purkinje neurons can generate tonic firing even when isolated from the network. Here we investigated the ionic basis of tonic firing in Purkinje neurons of larval zebrafish using voltage clamp for isolation of membrane currents along with pharmacology. We discovered that these neurons express L-type and P/Q-type high voltage-gated calcium currents, T-type low voltage-gated calcium currents and SK and BK-type calcium-dependent potassium currents. Among these, L-type calcium currents and SK-type calcium-dependent potassium currents were indispensable for tonic firing, while blocking T-type, P/Q-type and BK currents had little effect in comparison. We observed that action potentials were broadened when either L-type or SK channels were blocked. Based on these results, we propose that calcium entry via L-type calcium channels activates SK potassium channels leading to faster action potential repolarization, in turn aiding the removal of inactivation of sodium channels. This allows larval zebrafish Purkinje neurons to continue to fire tonically for sustained periods. In mammals also, tonic firing in Purkinje neurons is driven by calcium channels coupling to calcium-dependent potassium channels, yet the specific types of channels involved are different. We therefore suggest that coupling of calcium channels and calcium-dependent potassium channels could be a conserved mechanism for sustaining long bouts of high frequency firing. KEY POINTS: Tonic firing is an intrinsic property of Purkinje neurons in mammals and fish. These neurons express multiple types of voltage-gated conductances including L-type, T-type and P/Q-type calcium currents and SK- and BK-type calcium-dependent potassium currents. Blocking L-type calcium channels and SK-type calcium-dependent potassium channels resulted in spike broadening and reduced tonic firing. L-type calcium currents were activated during the repolarization of the spike. Based on this we conclude that calcium entry via L-type channels activates SK channels causing faster repolarization of the spike and therefore sustained tonic firing.

Secretagogin (scgn), a novel calcium-binding protein (CBP), regulates Ca2+ homeostasis in neurons, controls exocytosis, and serves as a Ca2+-sensor. Scgn is widely expressed in the mammalian brain and has emerged as a novel neuroendocrine regulator. Although scgn is considered as a phylogenetically conserved CBP, little is known about its significance in the brain of nonmammalian vertebrates. Herein, we characterized the scgn-encoding cDNA and mapped scgn-equipped elements in the brain and spinal cord of a gecko, Hemidactylus leschenaultii (hl). In the brain, a 1303-bp hlscgn transcript containing 822-bp cds and 481-bp 3' untranslated region (UTR) was identified. The hlscgn transcript predicted to encode a hexa EF-hand 273-aa hlscgn showed 70%-90% similarity with scgn from other vertebrates. The EF2-motif in hlscgn showed highest Ca2+-binding affinity as compared to that in scgn from other vertebrates. The anti-scgn antiserum detected a ∼32-kDa band in the brain, and cell bodies/fibers in the cortex, dorsal ventricular ridge, amygdalae, striatum/septum, hypothalamic/thalamic nuclei, substantia nigra, interpeduncular, solitary tract, and raphe nuclei, and spinal cord. The scgn neurons in the paraventricular (PVN) and supraoptic nuclei co-expressed either thyrotropin-releasing hormone or vasotocin, but not mesotocin. As insulin regulates the activity of PVN neurons in mammals, and scgn expression in the mammalian/teleost brains, the superfused hypothalamic slices of H. leschenaultii were treated with insulin, and the changes in scgn-ir/hlscgn-mRNA in PVN were analyzed. Compared to control, insulin treatment significantly increased scgn expression. Thus, scgn seems to be a highly conserved protein in the vertebrate hierarchy with a variety of attributes, inclusive of neuroanatomy, neuroendocrine control, and regulation by insulin.

A tiny vertebrate reveals brain-scale network functions.

Fractional Brownian motion is a Gaussian stochastic process with long-range correlations in time; it has been shown to be a useful model of anomalous diffusion. Here, we investigate the effects of mutual interactions in an ensemble of particles undergoing fractional Brownian motion. Specifically, we introduce a mean-density interaction in which each particle in the ensemble is coupled to the gradient of the total, time-integrated density produced by the entire ensemble. We report the results of extensive computer simulations for the mean-squared displacements and the probability densities of particles undergoing one-dimensional fractional Brownian motion with such a mean-density interaction. We find two qualitatively different regimes, depending on the anomalous diffusion exponent α characterizing the fractional Gaussian noise. The motion is governed by the interactions for α<4/3, whereas it is dominated by the fractional Gaussian noise for α>4/3. We develop a scaling theory explaining our findings. We also discuss generalizations to higher space dimensions and nonlinear interactions, the relation of our process to the "true" or myopic self-avoiding walk, as well as applications to the growth of strongly stochastic axons (e.g., serotonergic fibers) in vertebrate brains.

Protocol for synthesizing, implanting, and using polydimethylsiloxane as skull replacement in mice for imaging, electrophysiology, and optogenetics.

In the vertebrate brain, neural stem cell (NSC) quiescence is necessary for stemness maintenance. Using single-cell RNA sequencing (scRNAseq) in the zebrafish adult telencephalon, we identified different molecular clusters of quiescent NSCs, interpreted to sign different quiescence depths. Here, we show that these clusters, when challenged in vivo with an inhibitor of Notch signaling, a major quiescence promoting pathway, unfold different behaviors. Notably, deeply quiescent NSCs with astrocytic features display a unique activation phenotype that combines the maintenance of astrocytic markers with the rapid up-regulation of activation and neuronal commitment genes, reminiscent to murine periventricular astrocytes activating upon lesion. In contrast, an NSC cluster predicted to be in the deepest quiescence state resists Notch blockade, and we demonstrate that the transcription factor Nr2f1b mediates this resistance to activation in vivo. These results together link the molecular heterogeneity of quiescent NSCs with bona fide biological properties and their molecular regulators.

The hypothalamus is an ancient brain region that regulates diverse aspects of physiology and behavior, including sleep and wakefulness, appetite, energy homeostasis, anxiety, depression, and social interaction. Specific neuronal populations in the hypothalamus exert their effects via the release of neurotransmitters and neuropeptides. Whole-cell patch-clamp recording is an indispensable approach for studying the roles of these factors in synaptic transmission and brain function. However, it is challenging to access hypothalamic neurons for electrophysiological recordings in intact mammals due to their location deep within the brain. As a result, our understanding of the intrinsic properties and physiological functions of hypothalamic neurons is limited. The larval zebrafish is a useful alternative model to study hypothalamic neurons due to its transparent and small, but well-conserved, vertebrate brain. Here, we present a protocol for in vivo whole-cell patch clamp recordings of hypothalamic neurons in intact larval zebrafish. Using this technique, we can record from peptidergic neurons in the hypothalamus, examine the responses of these neurons to sensory stimuli, and explore their effects on downstream neurons. This experimental technique thus provides a useful approach to study the physiological functions of hypothalamic neuropeptidergic neurons in intact animals.

Sex specific differences in size and distribution of cell types have been observed in mammalian brains. How sex-specific differences in the brain are established and to what extent sexual dimorphism contributes to sex-biased neurodevelopment and neurological disorders is not well understood. Microglia are the resident immune cells of the nervous system and have been implicated in masculinizing the mammalian brain and refining neural connections to promote remodeling of neural circuitry, yet their contributions to developmental brain patterning and plasticity in zebrafish remains unclear. Here, we report anatomical and cellular differences between juvenile brains and adult female and male brains. Leveraging the plasticity of the zebrafish female brain and genetic models lacking microglia and tumor suppressor factors, we provide insight into the mechanisms that establish sex-specific brain dimorphism in zebrafish. Specifically, we identified sexually dimorphic features in the adult zebrafish brain that depend on microglia and Chek2, which may have broader implications and represent therapeutic targets for sex-biased neurological disorders.

GluA2 is a key subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) ion channels that is abundantly expressed in the vertebrate brain. Posttranscriptional Q/R editing of GluA2 renders AMPARs nearly impermeable to calcium ions, which is crucial for their normal function. Although previous studies have characterized conductivity and selectivity differences between edited and unedited GluA2 variants and heteromeric receptors incorporating GluA2, the consequences of pore editing have not been studied in all-atom simulations, which leave the atomistic mechanisms unclear. In this study, we investigate ion permeation in the context of multiple Ca2+ binding sites along the pore predicted from molecular dynamics (MD) simulations, considering both mutations and co-permeating monovalent ions. Patch clamp electrophysiology recordings confirmed a binding site at the intracellular mouth of the selectivity filter that confers selectivity for calcium over monovalent ions. A patient mutation at the same site has been previously shown to cause neurodevelopmental abnormalities. Furthermore, MD simulations of GluA2 with different arginine copy number at the Q/R site show that Ca2+ conduction is blocked in the presence of two arginines, whereas K+ is only blocked by four arginines, in explaining the results from decades of electrophysiological work. Finally, MD simulations revealed that Ca2+ reduces K+ conduction by preferentially occupying the intracellular selectivity filter binding site, whereas Na+ does not. This result is consistent with electrophysiological results from the D590 mutants and suggests that divalent binding in the selectivity filter is a major determinant of AMPAR conductance.

SUMMARYAnimals continuously extract and evaluate diverse sensory information from the environment to guide behavior. Yet, how neural circuits integrate multiple, potentially conflicting, inputs during decision-making remains poorly understood. Here, we use larval zebrafish to address this question, leveraging their robust optomotor response to coherent random dot motion and phototaxis towards light. We demonstrate that animals employ an additive behavioral algorithm of three visual features: motion coherence, luminance level, and changes in luminance. Using brain-wide two-photon imaging, we identify the loci of these computations, with the anterior hindbrain emerging as a multifeature sensory integration hub. Through single-cell neurotransmitter and morphological analyses of functionally identified neurons, we characterize potential connections within and across computational nodes. These experiments reveal three parallel and converging pathways, matching our behavioral results. Our study provides a mechanistic brain-wide account of how a vertebrate brain integrates multiple features to drive sensorimotor decisions, bridging the algorithmic bases of behavior and its neural implementation.

The myelin basic protein (MBP) is the most abundant intracellular protein of the myelin, which forms the electrically insulating sheath of axons of many actively functioning neurons. This protein binds the opposite membranes of the flattened processes of oligodendrocytes and plays a crucial role in myelin compaction. Here we show that MBP is present in amyloid form in the oligodendrocytes in the brain of vertebrates. It forms SDS-resistant insoluble aggregates and clearly colocalizes with Congo Red and Thioflavin S in vivo, ex vivo, and in vitro. The fibrils of MBP extracted from the brain are detected by electron microscopy and exhibit apple-green birefringence after Congo Red staining. We showed that the central region of MBP, spanning amino acid residues 60–119, is responsible for the formation of amyloid fibrils. Based on these data, we present a model in which MBP not only connects the opposite membranes of oligodendrocyte processes but also provides longitudinal amyloid stitching of myelin sheaths. Amyloid fibrils appear to be an ideal natural material for myelin compaction and axon insulation.

Serotonergic axons (fibers) are a universal feature of all vertebrate brains. They form meshworks, typically quantified with regional density measurements, and appear to support neuroplasticity. The self-organization of this system remains poorly understood, partly because of the strong stochasticity of individual fiber trajectories. In an extension to our previous analyses of the mouse brain, serotonergic fibers were investigated in the brain of the Pacific angelshark (Squatina californica), a representative of a unique (ray-like) lineage of the squalomorph sharks. First, the fundamental cytoarchitecture of the angelshark brain was examined, including the expression of ionized calcium-binding adapter molecule 1 (Iba1, AIF-1) and the mesencephalic trigeminal nucleus. Second, serotonergic fibers were visualized with immunohistochemistry, which showed that fibers in the forebrain have the tendency to move toward the dorsal pallium and also accumulate at higher densities at pial borders. Third, a population of serotonergic fibers was modeled inside a digital model of the angelshark brain by using a supercomputing simulation. The simulated fibers were defined as sample paths of reflected fractional Brownian motion (FBM), a continuous-time stochastic process. The regional densities generated by these simulated fibers reproduced key features of the biological serotonergic fiber densities in the telencephalon, a brain division with a considerable physical uniformity and no major “obstacles” (dense axon tracts). These results demonstrate that the paths of serotonergic fibers may be inherently stochastic, and that a large population of such paths can give rise to a consistent, non-uniform, and biologically-realistic fiber density distribution. Local densities may be induced by the constraints of the three-dimensional geometry of the brain, with no axon guidance cues. However, they can be further refined by anisotropies that constrain fiber movement (e.g., major axon tracts, active self-avoidance, chemical gradients). In the angelshark forebrain, such constraints may be reduced to an attractive effect of the dorsal pallium, suggesting that anatomically complex distributions of fiber densities can emerge from the interplay of a small set of stochastic and deterministic processes.

Absence of a glutamatergic channel arrest mechanisms in hypoxic naked mole-rat cortex.

Zebrafish (Danio rerio) combine accessible behavioral phenotypes with conserved neurochemical pathways and molecular features of vertebrate brain function, positioning them as a powerful model for investigating early neurodegenerative processes and screening neuroprotective strategies. In this context, integrated behavioral and proteomic analyses provide valuable insights into the initial pathophysiological events shared by conditions such as Parkinson's disease and related disorders-including mitochondrial dysfunction, oxidative stress, and synaptic impairment-which emerge before overt neuronal loss and offer a crucial window to understand disease progression and evaluate therapeutic candidates prior to irreversible damage. To investigate this early window of dysfunction, zebrafish larvae were exposed to 500 μM 1-methyl-4-phenylpyridinium (MPP+) from 1 to 5 days post-fertilization and evaluated through integrated behavioral and label-free proteomic analyses. MPP+-treated larvae exhibited hypokinesia, characterized by significantly reduced total distance traveled, fewer movement bursts, prolonged immobility, and a near-complete absence of light-evoked responses-mirroring features of early Parkinsonian-like motor dysfunction. Label-free proteomic profiling revealed 40 differentially expressed proteins related to mitochondrial metabolism, redox regulation, proteasomal activity, and synaptic organization. Enrichment analysis indicated broad molecular alterations, including pathways such as mitochondrial translation and vesicle-mediated transport. A focused subset of Parkinsonism-related proteins-such as DJ-1 (PARK7), succinate dehydrogenase (SDHA), and multiple 26S proteasome subunits-exhibited coordinated dysregulation, as visualized through protein-protein interaction mapping. The upregulation of proteasome components and antioxidant proteins suggests an early-stage stress response, while the downregulation of mitochondrial enzymes and synaptic regulators reflects canonical PD-related neurodegeneration. Together, these findings provide a comprehensive functional and molecular characterization of MPP+-induced neurotoxicity in zebrafish larvae, supporting its use as a relevant in vivo system to investigate early-stage Parkinson's disease mechanisms and shared neurodegenerative pathways, as well as for screening candidate therapeutics in a developmentally responsive context.

Neuronal function relies on the precise spatial organization of intracellular membrane-bounded organelles involved in anabolism and Ca2+ sequestration, such as the Golgi apparatus, mitochondria and the endoplasmic reticulum (ER), along with structures involved in catabolism, such as lysosomes. Despite their known roles in energy supply, calcium homeostasis, and proteostasis, our understanding of how the anabolism-linked organelles are structurally arranged within neurons remains incomplete. Due to the tremendous complexity in the morphologies and fine structural features and interwoven nature of these intracellular organelles, particularly the ER, our understanding of their structural organization is limited, particularly, with regard to quantitative assessments of their sites of interaction and accurate measures of their volumetric proportions inside of a single large neuron. To approach this challenge, we used serial block-face scanning electron microscopy (SBEM) to generate large-scale 3D EM volumes and electron tomography on high-pressure frozen tissue of the rodent cerebellum, including the largest cells in the vertebrate brain, the cerebellar Purkinje neuron as well as the most abundant cell type in the vertebrate brain, the much smaller cerebellar granule neuron. We reconstructed the neuronal ultrastructure of these different cell types, focusing on the ER, mitochondria and membrane contact sites, to then characterize intracellular motifs and organization principles in detail, providing a first full map to quantitatively describe a neuronal endoarchitectome. At the gross level organization, we found that the intracellular composite of organelles are cell type specific features, with specific differences between Purkinje neurons and Granule cells. At the level of fine structure, we mapped ultrastructural domains within Purkinje neurons where ER and mitochondria associate directly. In addition to cell type specific differences, we observed significant subcellular regional variation, particularly within the axon initial segment (AIS) of Purkinje neurons, where we identified ultrastructural domains with sharply contrasting distributions of ER and mitochondria. These findings suggest a finely tuned spatial organization of organelles that may underpin the distinct functional demands along the axon. We expect that our subcellular map, along with the methods developed to obtain these maps, will facilitate future studies in health, aging and disease to characterize defined features, by developing a framework for quantitative analysis of the neuronal ultrastructure.

Understanding brain evolution requires detailed comparative analyses of brain structures across species. However, high-resolution anatomical and connective data remain limited for most vertebrates beyond a few well-studied model organisms. To address this gap, we collected postmortem brain samples from a range of vertebrates, primarily small amniotes, and performed magnetic resonance imaging and histological staining. Here, we present the “Animal Brain Collection (ABC),” a freely accessible database that enables researchers to examine and compare cellular and tissue-level brain architectures across species. This resource provides a foundation for cross-species investigations of brain structure and development, offering new opportunities for research into the diversity and evolution of vertebrate brains.

Network science has revealed universal brain connectivity principles across species. However, several macroscopic network features established in human neuroimaging studies remain underexplored at cellular scales in small animal models. Here, we use whole-brain calcium imaging in larval zebrafish to investigate the structural and genetic basis of functional brain networks. Mesoscopic functional connectivity (FC) robustly captures the individuality of larvae and reflects structural connectivity (SC) derived from single-neuron reconstructions. Several connectome properties, including diffusion mechanisms and indirect pathways, predict interregional correlations. SC and FC share a hierarchical modular architecture, with structural modules shaping spontaneous and stimulus-driven activity patterns. Visual stimuli and tail monitoring reveal a functional gradient that coincides with sensorimotor functions. Last, regional expression levels of specific genes predict interregional FC. Our findings reproduce key mammalian brain network features, demonstrating larval zebrafish as a powerful model for studying large-scale network phenomena in a small and optically accessible vertebrate brain.