A central theme in Jon Kaas' research, celebrated in this issue, is the relationship between brain structure and function. As a tribute, we examine how certain neuroanatomical structures enable higher order (higher arity) functions whose joint effects cannot be recovered from pairwise measurements. Specifically, we argue that neurons exploit coincident input detection to realize higher order operations that cannot be derived by studying any of the pairwise combinations in isolation. Pairwise statistics can miss these irreducible higher order (more-than-pairwise) effects. We advocate for an extension of graph-based models to hypergraphs to formally capture and analyze these irreducible higher order neural interactions. Based on several recent studies, we discuss both the applicability and benefits of higher order neural data analyses.

Understanding retinal development is important for better explaining retinal function and disease and as a model for generating retinal organoids and retinal regeneration strategies. Although development of mouse retina is studied in great detail, timing of retinal cell type development in humans is still poorly understood. This study aims to compare developmental steps in the human midgestational and mouse early postnatal retinas and to establish timeline correspondents and their anatomical landmarks. Immunofluorescence analysis of retinal cryosections was performed on human retinas collected from fetuses of 15 to 27 gestational weeks (GW15-GW27) and mouse retina from pups ranging from 0 to 14 days (P0-P17). Markers for retina vascularization, cell type development, and layer lamination were determined and compared across species and developmental ages. Vascularization emerges and progresses over the investigated developmental timespan in both species. In parallel, lamination of nuclear layers progresses from two (ganglion cell layer = GCL vs. neuroblast layer = NBL) layers to the mature pattern of three layers (GCL-inner nuclear layer = INL-outer nuclear layer = ONL). Molecular markers of retinal cell types and markers of inner plexiform layer (IPL) lamination are appearing at the onset of the studied time points and progressively settle into the adult pattern of localization. A strong central-to-peripheral delay is observed in human retinas at GW18 and GW20. It therefore appears that human midgestation retina development is mirrored in the first 2 weeks of mouse postnatal development. The present work closes a gap in the literature concerning the correlation between human and mouse retinal maturation timelines. The stages identified and their anatomical landmarks may provide as a basis for future comparative studies.

Subplate neurons (SpNs) are early-born neurons in the mammalian neocortex and play a pivotal role in cortical development. It is widely believed that SpNs are predominantly lost due to postnatal cell death, with only a few remnants contributing to layer 6b (L6b) neurons in the adult neocortex, but the extent to which SpNs persist as L6b neurons remains largely unknown. To address this, we conducted a comprehensive birthdate analysis using multiple ethynyl deoxyuridine (EdU) injections over 24 h, thereby precisely tracking the survival and distribution of SpNs in the adult mouse neocortex. We further estimated the total number of SpNs and L6b neurons based on the total volumes of the subplate and L6b, quantified by employing 3D tissue clearing. Our findings reveal that a significant proportion of L6b neurons are remnants of SpNs and comprise diverse subtypes reminiscent of those in the postnatal period (SpNs: 319,588 ± 49,263, L6b neurons: 316,720 ± 11,268). This study provides direct evidence showing that most L6b neurons in the adult mouse neocortex originate as SpNs during the earliest stages of neurogenesis and persist throughout life.

Comparative morphology has revealed fascinating structural and organizational differences in the eyes of cyclostomes (jawless vertebrates) compared to gnathostomes (jawed vertebrates). This work has helped us to better understand the evolutionary origin of the vertebrate retina, but certain questions remain unresolved. Here we complement that approach by examining retinal development. We sought to characterize post-larval ("adult") retinogenesis in a representative cyclostome, the Pacific hagfish (Eptatretus stoutii). Knowledge of hagfish neurodevelopment is limited by poor access to embryos, but evidence suggests continued retinal neurogenesis occurs late into their ontogeny. A brief pulse of EdU was used to detect proliferating cells, if any, in the retina. This was followed by in situ hybridization to test whether homologs of gnathostome genes might have conserved expression patterns that direct retinogenesis in a jawless vertebrate. EdU+ cells were detected at the retinal periphery of the hagfish (a region reminiscent of the ciliary marginal zone of gnathostomes) and within the central retina. In addition, hagfish eyes were found to express several key genes required for vertebrate retinal neurogenesis. At least two of these genes, OtxA and Rx (retinal homeobox), were enriched within the proliferative retinal periphery. These findings support adult retinogenesis as an ancient and deeply conserved trait within the vertebrate lineage.

Neuronal circuits are a core component required for proper brain function and organization. How these circuits are built remains incompletely understood. The vast majority of studies on forebrain development have investigated mammals. Whether the described observations found in mammals apply to other vertebrates is unknown. The present report examined the development of forebrain circuits that interconnect the dorsal thalamus and the telencephalon in Alligator mississippiensis. A tracer, neurobiotin, which is transported in both an anterograde and retrograde fashion, was used. Four circuits were identified after neurobiotin placement into either the diencephalon or in the forebrain bundles themselves. Three circuits ascended from the dorsal thalamus to the telencephalon. These were: the ipsilateral lateral forebrain bundle and the ipsilateral and contralateral medial forebrain bundles. The other circuit descended from the basal nuclei to connect with the dorsal thalamus. The first circuit to develop was the basal nuclei to dorsal thalamus circuit followed by the lateral forebrain bundle and lastly by the medial forebrain bundle. Because it preceded the formation of the three other circuits, the basal nuclei to the dorsal thalamus tract is suggested to serve as a scaffold for the other three circuits to follow. The formation of synapses by lateral forebrain bundle axons in the telencephalon was determined by immunohistochemistry using a synaptic vesicle glycoprotein. Synapse formation in the telencephalon from lateral forebrain bundle axons occurred before hatching. This suggested that information carried by the lateral forebrain bundle was critical for survival upon hatching. Medial forebrain bundle axon connections may become functional only after hatching.

The objective of the current investigation was to determine the effect of neonatal castration on the expression of androgen receptor (AR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) in the neurons of the anterior pelvic ganglion (APG) in male pigs. Receptor expression was quantitatively assessed using real-time polymerase chain reaction (qPCR) and immunohistochemistry. Ganglia were sampled from intact pigs at 7 days of age (the day of castration) and from both intact and castrated animals at 3 and 6 months postsurgery. qPCR analysis of the intact boars revealed a significantly elevated gene expression level (GEL) of AR at 6 months of age compared to 3 months and 7 days of age. Conversely, the GEL of ERα was comparable across all age groups in the intact animals. Gene expression analysis of ERβ in the pelvic ganglia of both intact and castrated pigs consistently indicated very low levels. In the 3-month-old castrated cohort, the GELs of AR and ERα were significantly elevated compared to those in age-matched intact pigs. Conversely, the GEL of ERβ did not exhibit a significant difference between the castrated and intact groups. Immunofluorescence investigations complemented the qPCR data, revealing that numerous APG neurons across all control and experimental groups demonstrated immunoreactivity for AR, ERα, and ERβ, confirming the translation and presence of all three receptor types within the neuronal somata. These data collectively suggest that, in the male pig, pelvic neurons constitute a component of autonomic circuits that are highly dependent on sex steroids and are implicated in the regulation of urogenital function. Furthermore, the sensitization of these neurons to sex hormones, likely involving not only androgens but also estrogens (primarily mediated via ERα), appears to be a dynamic, maturational process that occurs during the prepubertal period.

Many decapod crustaceans occupy different habitats during their ontogeny and undergo a niche shift during the transition from the larval to the juvenile phase. Their life cycle comprises pelagic larvae and, in most species, benthic juvenile-adult stages. Already at hatching, decapod larvae possess a wealth of organ systems, necessary to autonomously survive and develop in the plankton. They also exhibit a rich behavioral repertoire to appropriately respond to environmental stimuli such as light, gravity, hydrostatic pressure, tidal currents, temperature, salinity, chemical cues, and food concentration. Considering the impressive larval behavioral performance, neuroanatomists in the past have already studied selected aspects of the functional morphology of larval nervous systems, but this knowledge is distributed across a wide taxonomic range. Our study aims to provide a concise description of the morphology of selected sensory organs and that of the central nervous system in the Zoea 1 of the Asian shore crab Hemigrapsus sanguineus using multiple neuroanatomical methods such as three-dimensional (3D) reconstruction from histological section series, scanning electron microscopy, and immunohistochemistry combined with confocal laser-scan microscopy. The larval developmental cycle of this includes five zoea stages and one megalopa stage. In the Zoea 1, our data reveal well-developed visual and olfactory organs comprising hundreds of sensory receptor neurons and also complex primary visual and olfactory neuropils related to processing this sensory input. We observed a particularly high investment of nervous tissue in the central visual pathway. Furthermore, the larval brain comprises several characteristic secondary neuropils which, as higher order neuropils, in the adult crustacean brain are thought to integrate highly processed multimodal sensory input. These are, for example, the lateral protocerebrum (which in adult crabs will house the mushroom bodies) and the median protocerebrum that includes the central body. The discussion of our findings is framed against the knowledge of the larval behavioral performance and, considering the larval investment in higher order neuropils, the possibility of complex behavioral decisions that integrate aspects of learning and memory.

An organism's vision is dependent on the photoreceptor types in the eye and how these cells are organized across the retina. The turquoise killifish (Nothobranchius furzeri) is an annual killifish species that lives in the turbid waters of ephemeral pools. Although turquoise killifish are gaining popularity as a model, we know little about their photoreceptors. Here, the turquoise killifish retina was investigated for photoreceptor subtype identity and mosaic organization at the adult and larval stages to assess specialization for their light environment. Turquoise killifish were determined to have cone-dominant retinas, with expression of UV, blue, green, and red cone opsins, as well as rhodopsin. Adult fish were found to have an ordered square mosaic, wherein double cones formed squares with single cones in the center and corners of the squares. Double cones could be comprised of a red and green or two red cone units, while the single cones could be UV, blue, red, or - very rarely - green in identity. Although ordered, the mosaic did not have regularity of cone subtype orientation across the retina. Larvae were found to have retinal regionalization, with a dorsonasal region of increased short-wavelength cone density. In both larvae and adult retinas, red cones were the predominant cone subtype. This study presents the first description of the photoreceptor types and mosaic in an annual killifish, and provides a foundation for future work investigating killifish photoreceptor biology and visual function.

The lateral septum is a key subcortical structure and has been implicated in social memory. One aspect of social memory, the ability to recognize relatives, is conserved across vertebrate species and reflected in stable, lifelong memories. Synapses are considered to be the smallest unit of memory storage. Excitatory synapses are typically found on dendritic spines, whereas inhibitory synapses are mostly located on the dendritic shaft. Here, we investigate the synaptic architecture of unusual somatic spines, an apparent synaptic specialization of septal GABAergic neurons. We uncover the formation and molecular organization of septal somatic spines found in the lateral septum in in vivo and in vitro model systems using various microscopy approaches. We use classical label-free methods such as transmission electron microscopy and Golgi stainings and established new culturing methods for dissociated and organotypic septal slices that were kept in culture over multiple weeks. We describe the presence, morphology, ultrastructure, and molecular composition of excitatory somatic spines across multiple developmental stages in various model systems. We propose that the ability to develop such spines is an intrinsic feature of somatospiny neurons that does not depend on extra-septal connectivity. While smaller than dendritic spines, somatic spines exhibited distinct features, frequently containing secretory organelles such as autophagosomes, multivesicular bodies, and endosomes, but often lacking a spine apparatus and ribosomes. Our findings offer insights into the molecular architecture of septal somatic spines and establish a basis for further investigations into the somatic spines of the lateral septum.

Olfaction in locusts offers a useful model for understanding how the brain encodes environmental information. While previous studies of olfaction in locusts have focused mainly on the antennal pathway, most insects, including locusts, possess olfactory receptors on their palps. Although they are traditionally considered to be gustatory or mechanosensory structures, accumulating molecular, electrophysiological, and behavioral evidence shows locust palps also mediate olfactory processing. The palps therefore initiate a second olfactory pathway, whose neural architecture and sensory functions remain largely unexplored. Here, we used anatomical approaches to characterize the chemosensory system of the palps in Schistocerca americana, mapping pathways from the peripheral sensilla through the brain to third-order neurons. We found that sensory input from the palps projects to two distinct regions: the gnathal ganglion, which integrates gustatory input from multiple head appendages, and the glomerular lobe of the cerebral ganglion, which, traditionally thought of as a gustatory center, appears instead to receive only olfactory input. The glomerular lobe, in turn, provides olfactory input to the accessory calyx of the mushroom body, a region traditionally considered gustatory. Notably, the palp and antennal olfactory pathways remain anatomically segregated through at least the third-order neurons. Our systematic characterization of the palp olfactory system anatomy challenges existing views of olfactory and gustatory integration in hemimetabolous insects and establishes a framework for comparative studies of antennal and palp-based olfactory coding.

The spinal cord plays a central role in sensorimotor integration and exhibits substantial diversity across vertebrates in relation to ecological and behavioral demands. In teleost fish, however, detailed morphological and cellular analyses of the adult spinal cord remain scarce. Here, we provide the first comprehensive characterization of the adult spinal cord of the annual fish Garcialebias charrua, a species displaying pronounced environmental adaptation and sexual dimorphism. Using adult males and females, the spinal cord was systematically partitioned into five equally sized rostrocaudal regions (SI-SV) to evaluate regional variation in morphology, neurochemical organization, and cell proliferation. We analyzed gross morphology and cross-sectional features, the distribution and morphology of NADPH-diaphorase-positive (NADP-d+) neurons as indicators of nitric oxide-related signaling, and proliferative activity using 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Our results reveal marked rostrocaudal heterogeneity in spinal cord morphometry, with dimorphic variation in the segment SIII associated with the dorsal fin, region-specific patterns of NADPH-d+ neuronal populations, and sustained cell proliferation throughout the entire spinal cord in both gray and white matter. Focused analysis of segment SIII, which exhibits distinctive anatomical features, demonstrated higher proliferative activity in the central canal and dorsal regions compared to ventral areas in both sexes, with males showing significantly increased proliferation across all analyzed regions. Finally, the combination of BrdU labeling with a neuronal lineage marker provides the first evidence of adult spinal cord neurogenesis in G. charrua. These findings highlight the spinal cord as a dynamic and sexually dimorphic substrate underlying neuroplasticity in annual fishes.

The Cairo spiny mouse (Acomys cahirinus), a promising animal model in neuroscience, lacks a detailed neuroanatomical map of its cerebral cortex. This study aims to delineate the location and internal subdivision of the sensorimotor cortex, specifically the region controlling the hindlimb. For this purpose, retrograde tracing from the lumbar spinal cord using Fast Blue was combined with immunohistochemical characterization using neuronal markers (NeuN, SMI-32, and calbindin 28 kDa) in adult animals. Fast Blue-labeled neurons were identified in layer V within the cortical region defined as the sensorimotor cortex responsible for hindlimb control. Within this region, the general cytoarchitecture was defined using NeuN, which allowed visualization of all cortical layers. The border between the lateral and medial sub-areas was clearly identified by SMI-32 immunostaining, which showed denser labeling in the lateral zone (presumably the primary somatosensory cortex) compared to the medial zone (presumably the secondary motor cortex). Within the defined layers and sub-areas, a comprehensive analysis of the calbindin-expressing interneuron population was performed, revealing significant interareal differences in soma size and cellular density of calbindin-positive neurons exclusively within layers III-IV. These findings provide the first detailed map of the hindlimb sensorimotor cortex in spiny mice, establishing a crucial neuroanatomical foundation for future studies using this novel model in sensorimotor research.

Synapse formation and function are coordinated spatially and temporally by a host of synaptic proteins that regulate neuronal signaling, synapse specificity, and plasticity, many of which are implicated in neuropsychiatric disorders. Many members of the C1q/TNF superfamily function as synaptic organizers, shaping synapse assembly and maintenance. Among them, C1QL3 plays a putative role in trans-synaptic adhesion and modulation of synaptic strength, but the lack of a reliable antibody to detect it has severely limited the ability to map its endogenous localization and study its biochemical properties. Here, we present a novel epitope-tagged knock-in mouse line (C1ql3[2HA]), in which two hemagglutinin (HA) epitopes were inserted near the N-terminus of the endogenous C1QL3 protein. This model enables purification, detection, and subcellular localization of native C1QL3 protein (C1QL3-2HA) with high specificity, eliminating the need for overexpression or custom antibodies. We validated that C1ql3[2HA] mice maintain normal mRNA expression, biochemical properties, and behavior. Using native PAGE, we determined the endogenous oligomeric state of C1QL3-2HA. Brain-wide light-sheet microscopy uncovered an expanded neuroanatomical map of C1QL3-2HA expression, including newly identified populations in cortical and subcortical regions as well as the retina. Dual immunohistochemistry confirmed cell-type-specific expression patterns, and super-resolution STED microscopy localized C1QL3-2HA to hippocampal mossy fiber synapses, positioned between pre- and postsynaptic markers, supporting its hypothesized role in trans-synaptic complexes. This knock-in mouse line is a valuable tool for studying the anatomical, molecular, and synaptic biology of C1QL3 in all cellular/tissue contexts, enabling future studies into its potential roles in the nervous system and beyond.

Understanding how the brain processes bodily signals requires mapping the circuits that transform interoceptive information into coordinated responses. Visceral signals converge in the nucleus of the solitary tract (NTS), which coordinates appetite, breathing, cardiovascular reflexes, and digestion. The NTS contains many intermingled subpopulations of neurons, and deciphering their functions requires understanding their connections. Here, we used cell-type-specific tracing to test whether molecularly distinct NTS neurons exhibit unique connectivity patterns. First, we found that Lmx1b-expressing excitatory neurons provide output to a broad array of NTS target regions in both the brainstem and forebrain, while inhibitory neurons in this region project predominantly within the brainstem. Next, we found that several genetically defined excitatory subpopulations-catecholaminergic (Th), neuropeptidergic (Cck, Npff, or Pdyn), and aldosterone-sensitive (Hsd11b2)-exhibit unique output patterns across multiple targets. As examples, the ventrolateral medulla receives moderate Th, Cck, and Pdyn, light Npff, and no Hsd11b2 input. The outer rim of the external lateral parabrachial subnucleus receives concentrated Th, Cck, and Npff input, contrasting a more uniform Pdyn input and a lack of Hsd11b2 input. The subcommissural bed nucleus of the stria terminalis receives broad Th, light Cck, sparse Pdyn, and virtually no Npff input, contrasting the focal Hsd11b2 input to its fusiform subnucleus. These divergent patterns demonstrate that molecular identity predicts connectivity and define the organizational logic by which interoceptive signals are transformed into coordinated autonomic and behavioral responses.