Journal of Comparative Neurology

Siphonophores are colonial hydrozoans with unprecedented differentiation and specialization, in which individual zooids are transformed into functional organs rather than autonomous polyps capable of feeding. As a result, the entire colony acts as a single, modular-individual with the highest level of coordination and integration, from development through behavior. Deciphering these integrative mechanisms requires understanding the microanatomical organization of the nervous system in all elements of the colony. Here, using two immunohistochemical markers (anti-tubulin and anti-RFamide antibodies), we systematically characterize the neural systems across the entire Nanomia colony, encompassing pneumatophore, stem and all zooid classes (nectophores, gastrozooids, palpons, male and female gonophores, and protective zooids). The use of two neuronal markers enables visualization of distinct neural subpopulations, some of which are not revealed by a single marker. We provide evidence of neuroanatomical interactions within all elements of the colony, including contributions of giant axons, stem polygonal networks, and RFamide-ir neural rings at the base of each zooid, as well as describe different subpopulations of neural networks in the body of various zooids. The presented mapping facilitates identification of novel conductive and signaling pathways for future analysis of the cellular basis of behavioral integration within decentralized, broadly distributed networks and non-neuronal elements of these unique superorganisms.

Understanding how neural circuits transform sensory and bodily signals into motivational states and adaptive behavior is a central problem in neuroscience. In teleost fish, the dorsomedial telencephalon (Dm) is a key pallial region implicated in both sensory processing and aversive behavior, yet whether these functions arise from a functionally uniform region or from interactions among specialized pallial domains has remained unknown. Here we show that the teleost dorsomedial telencephalon exhibits a previously unrecognized functional organization in which distinct but interconnected pallial domains perform complementary computations that progressively transform multimodal sensory and bodily representations into aversive motivational value and adaptive behavioral control. Wide-field voltage-sensitive dye imaging revealed that tactile, auditory, and gustatory stimuli evoke spatially organized, modality-specific activity exclusively within the caudal subdivision of Dm (Dmc), whereas the rostral subdivision (Dmr) showed little or no sensory responsiveness. In contrast, focal intracerebral microstimulation demonstrated that activation of Dmr, but not Dmc, is sufficient to generate robust, flexible, and reversible conditioned place avoidance, identifying Dmr as a pallial node causally involved in the assignment of negative motivational value. Anatomical tracing revealed a circuit in which sensory and bodily-related inputs converge onto Dmc, are relayed intrapallially to Dmr, where they are transformed into an aversive motivational signal before being conveyed to hypothalamic and brainstem centers involved in autonomic and behavioral regulation. Immunohistochemical analyses confirmed the pallial identity of both subdivisions and their distinct rostrocaudal organization, while providing no evidence that Dm corresponds to a classical pallial amygdaloid territory. This functional architecture more closely resembles the distributed organization of mammalian corticolimbic networks than either a unitary pallial amygdala or a neocortical sensory hierarchy, suggesting that the transformation of sensory and bodily representations into motivational control may represent a conserved organizational feature of the pallium that emerged early during vertebrate evolution. Short abstract / Significance statementThis study shows that the teleost dorsomedial pallium is organized into complementary functional domains that dissociate multimodal sensory representation from negative motivational processing while forming an interconnected pallial circuit associated with adaptive behavioral control. Our findings reveal a distributed pallial organization resembling mammalian corticolimbic architectures and provide a new framework for understanding the evolution of vertebrate pallial function.

Sensory systems share common circuit organization motifs across mammalian species, however, anatomical subdivisions show varying degrees of complexity depending on ecological niche and species-specific sensory requirements. While coarse neuroanatomical connections seem preserved within the visual system, it remains unknown if molecularly defined cell-types share a similar degree of conservation, regarding not only their functional properties but also connectivity. Here we analyze the organization, molecular marker expression, and connections between two prominent visual centers, the superior colliculus (SC) and the dorsal lateral geniculate nucleus of the thalamus (dLGN), in the mouse and the tree shrew, a highly visual, diurnal species closely related to primates. Previous attempts to link molecular markers to subdivisions and connectivity of the dLGN have shown lack of conservation across species, thus preventing the systematic investigation of brain-wide interactions involved in vision. Leveraging recent single-cell and single-nucleus RNA sequencing studies, our results unravel a conserved molecular marker that shows spatial restriction in the dLGN and correlates with the location of connections from the SC in both the mouse and the tree shrew. We extend our findings by confirming the presence of this molecular marker in the human dLGN. These results provide a molecular definition and genetic access point for SC to dLGN connections in the mouse and tree shrew, enabling cell-type specific studies of the parallel processing of visual information.

The lateral habenula (LHb) shapes reward and aversion learning via projections to midbrain monoaminergic centers. Recent studies have demonstrated significant genetic, anatomical, and electrophysiological diversity within the LHb. However, it remains unclear how genetic or intrinsic electrophysiological characteristics relate to in vivo neuronal activity patterns. Additionally, there are few descriptions of transgenic mouse lines labeling specific LHb neuronal subtypes. Here we describe spatial gene expression patterns, electrophysiological characteristics, and projection targets for specific subpopulations of neurons in the LHb targeted via existing transgenic mouse lines. Furthermore, we demonstrate that two genetically defined subpopulations differentially respond to value, prediction errors, and directional movement during flexible, reward-guided behavior. These findings indicate that specific, genetically targetable, neuronal subpopulations in LHb may control discrete aspects of motivated behavior through parallel circuits targeting serotonergic and dopaminergic midbrain centers.

The origins and early diversification of intercellular signaling molecules in animals remain poorly understood because comparative data across basal metazoan lineages are limited. Cnidarians form the sister group to bilaterian animals, and characterizing their transmitter systems is critical to understanding how complex adaptations within integrative systems shape evolutionary trajectories. Although glutamate is a well-established transmitter in bilaterian animals, its role in cnidarians remains unclear, and information on its neuronal function and signaling is limited. For most studied cnidarians, glutamate has been suggested to be a non-neuronal signaling molecule. Here, using glutamate immunoreactivity (Glu IR) in eight hydrozoan species with distinct ecologies (Aequorea victoria, Eutonina indicans, Clytia gregaria, Bougainvillia principis, Euphysa flammea, Polyorchis penicillatus, Aglantha digitalis, Nanomia septata), we identified and visualized distinct populations of glutamate-immunoreactive (Glu-ir) cells, including nematocytes, neurons, and muscle cells. A broad diversity of Glu-ir nematocytes was found in all studied species. Glu-ir neural cells were found only in three species (Aequorea, Nanomia, and Aglantha); their morphology and localization were species-specific. In addition, some striated and smooth myoepithelial cells were found to be either Glu-ir or GABA-ir. We propose that both glutamatergic and GABAergic systems were independently recruited more than 3 times as neurotransmitters across cnidarians, and that these recruitments are fundamentally rooted in bioenergetic demands.

Glucagon is a key metabolic hormone regulating blood glucose and appetite, yet little is known about its actions within the brain. Here, we investigated its receptor (GCGR) localization in periventricular brain barrier interfaces in young rats using immunohistochemical and immunofluorescence approaches. GCGR was enriched in the proximal region of motile ependymal cilia lining the ventricles, as well as in tanycytic primary cilia and cytoplasmic extensions within the hypothalamus. Additional immunostaining was observed in ciliated cells of the subcommissural organ and, more heterogeneously, in choroid plexus epithelium and associated primary cilia, while other circumventricular organs lacked detectable GCGR. These findings identify brain cilia and tanycytes as previously unrecognized sites of glucagon receptor localization and suggest that glucagon signaling at brain barrier interfaces may contribute to integrating peripheral metabolic cues with central homeostatic circuits.

Tree shrews (Tupaia belangeri) are increasingly used in comparative neuroscience, yet their respiratory physiology remains poorly characterized. We quantified spontaneous breathing and respiratory rhythm variability in awake adult tree shrews (n = 10; 5 males, 5 females) using whole-body plethysmography. Respiratory frequency decreased by approximately 16% with acclimatization to the recording chamber, while respiratory timing, body-mass-normalized respiratory amplitude, inspiratory flow, and minute ventilation remained relatively stable. After acclimatization, mean respiratory parameters were similar between sexes, but short-term breath-to-breath variability (SD1) was greater in males than females, whereas SD2 was comparable. These findings establish baseline respiratory characteristics in awake tree shrews and identify sex-dependent differences in short-term respiratory rhythm stability.

Despite glutamates widespread role as the dominant excitatory transmitter in vertebrate brains, the early evolution of glutamate and its recruitment into neural signaling remain largely unknown. The major limitation is the lack of information on its distribution in early-branching basal metazoans, such as ctenophores (comb jellies). Here, using glutamate immunoreactivity (IR) in two ctenophore species with distinct ecologies (Pleurobrachia bachei and Beroe abyssicola), we show that glutamate IR is present in subpopulations of neurons within the subepithelial neural network and in small groups of mesogleal neuron-like cells, and that it differentially labels some muscle fibers. Remarkably, we also observed an enriched glutamate-ir signal within the nuclei of subepithelial neurons in Beroe. However, glutamate expression levels are species-specific, suggesting a tight coupling of glutamate recruitment for neural communication with energetic demands.

ObjectiveGlucagon-like peptide-1 (GLP-1), a peptide neurotransmitter in the brain, is synthesized from proglucagon, encoded by the glucagon gene (Gcg). Besides medullary GLP-1 neurons, Gcg-expressing neuron populations were identified in the olfactory bulb and basolateral amygdala. However, several lines of evidence suggest that additional Gcg neuron populations might exist. MethodsWe conducted a brain-wide mapping of Gcg-expressing cells by fluorescent in situ hybridization in C57BL/6J and FVB/Ant mice. Proglucagon and GLP-1 expression were studied with immunofluorescence. We characterized a Gcg-Cre;tdTomato mouse line and studied the expression of proglucagon-processing enzymes in Gcg-expressing neuron populations. We used adeno-associated virus-mediated tracing in Gcg-Cre mice to map the projections of hypothalamic Gcg neurons. ResultsGcg-expressing neuron populations were identified in the olfactory bulb, claustrum, piriform cortex, basolateral amygdala, posterior hippocampus, posterior hypothalamic nucleus (PH), periaqueductal gray/dorsal raphe, and dorsal nucleus of the lateral lemniscus. These neurons express lower Gcg mRNA levels than medullary GLP-1 neurons. Proglucagon and GLP-1-immunoreactivity (C-terminus) were detected in almost all Gcg-expressing neuron populations, along with the mRNAs for prohormone convertases 1/3 and 2, enzymes generating GLP-1 or glucagon, respectively. Fasting markedly increased Gcg mRNA, proglucagon and GLP-1 synthesis in the PH. PH Gcg neurons project densely to the ventral and intermediate lateral septum, preoptic region, ventrolateral preoptic nucleus, lateral hypothalamus and zona incerta, establishing close contacts with both GLP-1 receptor-positive and -negative neurons. ConclusionsProglucagon is expressed in 9 distinct neuron populations. Feeding status regulates GLP-1 synthesis in PH neurons that likely control feeding- or energy balance-related functions.

Unipolar brush cells (UBCs) are excitatory interneurons that have a characteristic dendritic brush that amplifies and extends incoming signals in the cerebellum. UBCs transform synaptic input through their ionotropic and metabotropic glutamate receptors. Differential regulation of receptor subunits is a critical developmental process, but how the expression of glutamatergic receptors changes in UBCs as they develop is unclear. NMDA-type glutamate receptors (NMDARs) are particularly important for development and plasticity. We examined the expression of NMDAR subunits during development and tested whether signaling through these receptors is necessary for the development of the elaborate dendritic structure and unusual synaptic function of UBCs. Whole-cell patch clamp recordings from UBCs in acute brain slices revealed tonic and synaptic NMDAR-mediated currents in early postnatal UBCs that decrease during development. RNAscope in situ hybridization revealed differential developmental regulation of GluN2C/D subunits. Cell-type specific constitutive NMDAR knockout had no apparent effect on dendritic brush development, but increased UBC number in adulthood, suggesting a role in programmed cell death. Both pharmacological blockade or genetic deletion of NMDARs produced a paradoxical increase in excitability, which was calcium dependent and was occluded by inhibition of calcium activated potassium channels. Thus, NMDA receptors are dispensable for migration and dendritic development but may be involved in cell death pathways. Their functional roles include synaptic signaling as well as providing a tonic calcium flux that dampens excitability in developing UBCs and may influence transformations of vestibular signals essential for smooth movements and balance.

Background: Brain banks usually provide small tissue blocks fixed by immersion in neutral-buffered formalin (NBF). While still underexploited for research, gross anatomy laboratories could provide full brains fixed by perfusion with solutions better suited for gross anatomy dissection. However, the chemicals in these solutions might have a different impact on histology protocols for cell quantification than in NBF-fixed brains. The main goal of this study is to compare the effects on the number and size of labeled neurons of the primary motor cortex (PMC) of mouse brains fixed with three different solutions: (1) NBF, typical of brain banks, (2) a saturated salt solution (SSS), and (3) an alcohol-formaldehyde solution (AFS), both used in human anatomy laboratories. Methods: 27 C57BL/6J mouse brains were perfused with the NBF (N=9), SSS (N=9) or AFS (N=9), then cut in 40-m slices and processed with immunohistochemistry to target neurons. Various quantitative variables were assessed manually and automatically on photomicrographs of 3 regions of interest (ROIs) of the PMC per specimen, namely the total and individual neuronal profile areas, number and diameters. The effects of the three fixatives on these variables were compared using ANOVA or Kruskal-Wallis, depending on the distribution. For measures on individual cells, a generalized linear mixed model was applied. Dice coefficients and correlations were applied to evaluate the agreement of the manual and automatic methods. Results: There was no significant difference between the brains fixed by the three fixatives for the total and individual cell areas, the total cell count and the cell diameters. The values obtained from manual and automatic measures had an overall good agreement (Dice coefficients > 0.79). Conclusion: It was found that the SSS and AFS had similar impacts on the quantitative variables in the tissue as the NBF. These results are promising for neuroscientists interested in using brains from anatomy laboratories for quantitative research on neurons from the PMC.

In mammals, sex differences in the brain arise from genetic and hormonal factors, including organizational effects of perinatal testosterone. Epigenetic mechanisms including DNA methylation and demethylation have emerged as critical mediators of brain masculinization; specifically, their regulatory enzymes are upregulated in neonatal mice during the critical period of sexual differentiation, with their inhibition abolishing sex-specific cellular phenotypes. Here, we assessed sex differences in gene expression of the DNA demethylation machinery (Tet1, Tet2, Tet3, Gadd45a, Gadd45b and Tdg) during and after the critical period, and examined how these differences relate to the oxytocinergic system. mRNA expression was measured in the prefrontal cortex (PFC), preoptic area (POA) and paraventricular nucleus of the hypothalamus (PVN) at postnatal day (P) 7 and P18. At P7, males showed higher expression of all six genes than females in PFC, with no differences in POA or PVN; by P18, no regional differences remained. Oxytocin (OXT) immunoreactivity was surveyed across periventricular nucleus (Pe), anteroventral periventricular nucleus (AVPe), POA, PVN and supraoptic nucleus (SON). OXT was undetectable in the POA, AVPe and Pe at P7, and no sex differences were found in PVN or SON at either age, or in AVPe at P18. At P18, females showed higher OXT-immunoreactivity in the Pe and POA, than males. For Oxtr, qPCR revealed higher mRNA expression in the PFC of males at P7, with no other regional differences and none remaining at P18. Together, these findings suggest that sex differences in oxytocinergic regions arise from sex-specific epigenetic regulation during the critical period, and that perinatal testosterone may program DNA methylation dynamics underlying sex-specific gene expression in the developing brain. Our results support a model in which testosterone-dependent epigenetic mechanisms contribute to the sexual differentiation of neuroendocrine circuits, linking hormonal signals to long-term brain organization.

The extent to which conserved neural circuit architectures depend on shared molecular specification programs remains unclear. Here, we address this question by examining the somatosensory system of the little skate, Leucoraja erinacea, an early-diverging vertebrate that retains ancestral features of both finned and limb-based body plans. We show that core features of somatosensory circuit organization, including laminar organization of the spinal cord and dorsally restricted targeting of sensory afferents, are deeply conserved. Unexpectedly, the molecular programs specifying dorsal root ganglion (DRG) sensory subtypes diverge extensively from those of mammals. Although DRG neuron subtype specification and spinal connectivity rely on target-derived cues, skates employ distinct neurotrophin receptor and transcription factor identity codes. These findings support a model in which conserved spinal circuit architectures provide a stable scaffold that leverages flexible sensory neuron specification programs, enabling the evolutionary diversification of vertebrate somatosensory systems. HighlightsO_LIIntegrated analysis of spinal cord and DRG neuronal diversity in Leucoraja erinacea C_LIO_LILaminar organization of the dorsal spinal cord is an ancestral vertebrate feature C_LIO_LIDivergent neurotrophin receptor and transcription factor codes in sensory neurons C_LIO_LIConserved target-dependent regulation of sensory identity and connectivity C_LI

Activated leukocyte cell adhesion molecule A (Alcama) plays a role in axonal guidance, cell differentiation, and retinal lamination in a developing retina and was identified as a marker for activated Muller glial cells in adult zebrafish. However, its spatiotemporal localization and its involvement in retina regeneration remains unclear. Here we induced focal photoreceptor damage in zebrafish using laser photocoagulation and examined the expression and localization of Alcama at different time points post lesion. Immunohistochemistry in wild type fish and Tg(kdrl-EGFP) fish showed Alcama localized to the blood retina barrier with increased expression in Muller glial end feet and radial processes in a regenerating retina. To confirm its role in retina regeneration, alcama expression was transiently knocked down using morpholinos in adult fish. Scanning laser ophthalmoscopy, Zpr1 immunostaining and EdU staining showed delayed retina regeneration in alcama knockdown fish, indicating a possible role for Alcama in zebrafish retina regeneration.

Neural circuits produce reliable activity even after environmental disturbances. This occurs because neurons respond to perturbations in a compensatory manner, a process termed homeostatic plasticity. Bullfrogs undergo prolonged periods underwater during winter, when lung ventilation and its neural control system ceases activity, but air-breathing resumes unscathed when environmental temperatures increase weeks to months later. Compensatory neural mechanisms that contribute involve upregulation of excitatory synaptic transmission on motoneurons driven by inactivity, but whether inactivity or acclimation to low temperatures drive other forms of compensation is not known. The GABAA receptor contribution to respiratory rhythm generation is downregulated following overwintering, which promotes network excitability. Therefore, we disentangled the contributions of cold temperature acclimation and inactivity experienced during overwintering on reduced GABAergic signaling. Here, we show that cold temperature, and not inactivity, reduces GABAA signaling in the respiratory rhythm generating network, without influencing GABAA transmission onto motoneurons. Therefore, cold temperature acclimation drives reduced GABAergic signaling selectively in inter-neuronal rhythm generating circuits, while excitatory motoneurons synapses are strengthened by inactivity in the overwintering environment. Most work interprets compensatory plasticity as activity-dependent during activity perturbations, but we reveal that different aspects of a disruptive environment elicit distinct forms of plasticity across a motor network.

Retinal ganglion cells (RGCs) are the sole output neurons of the retina, responsible for transmitting visual information to the brain. Recent transcriptomic studies have revealed extensive molecular diversity among RGCs that parallels their known morphological and functional heterogeneity. Although large-scale efforts have unified the transcriptomic, morphological, and physiological features for a limited number of RGC subtypes, the majority of molecularly defined types remain poorly characterized. Developing approaches that can be used to identify RGC subtypes in a reliable and reproducible manner is critical for understanding their roles in visual processing. We used a MafbmCherry-2A-Cre mouse line to genetically label four uncharacterized RGC subtypes, along with two well-established -RGC subtypes, and we systematically characterized their molecular markers, central brain targets, dendritic morphologies, and light response properties. Within the population of previously uncharacterized MAFB+ RGCs, we identified two OFF-responsive subtypes which we termed "MAFB-midi-OFF" and "MAFB-asymmetric-OFF", and two ON-OFF-responsive subtypes termed "MAFB-equal bistratified" and "MAFB-unequal bistratified". Notably, no single molecular, morphological, or functional characteristic was sufficient to distinguish all MAFB+ subtypes. Instead, accurate subtype classification emerged only through the integration of multiple complementary features, highlighting the importance of multimodal approaches for defining RGC subtype identity and resolving neuronal diversity within the retina.

BackgroundBrain regions integrate neural information arriving from several convergent projection sources. At the mesoscale level, neural projections can potentially span both hemispheres and extend along the entire rostrocaudal axis, which complicates efforts to map their full extent. To address this issue, we describe a novel method for mapping such mesoscale connectivity in vivo and ex vivo. Our neurotomographic approach utilizes micro-computed tomography (micro-CT) to image the spatial distribution of neural tracers bound to high Z-elements, e.g, gold. MethodsIn this study, we conjugated colloidal gold to a retrograde tracer wheat-germ agglutinin apo-horseradish peroxidase (WGA-HRP) and then stereotactically injected the gold-bound tracer (WAHG) into the mouse forebrain. Micro-CT was then used to image the brain in vivo and ex vivo, followed by three-dimensional reconstruction of tracer distribution. We then validated our approach by histologically processing the brains using silver enhancement to label gold particles; this enabled a direct comparison of histological labeling with the neurotomographic images. ResultsWe found that micro-CT imaging could reveal the major spatial distributions of the gold-bound tracer, which was consistent across in vivo and ex vivo imaging conditions. Moreover, the neurotomographically determined patterns corresponded with the labeling observed in histologically processed tissue, with the major sites of labeling reliably detected in reconstructed neurotomographic images. ConclusionsOverall, our findings demonstrate a potential novel method for non-destructive, three-dimensional mapping of neural tracers in vivo. This novel approach can potentially guide targeted multi-site recordings, enable validation of injection site placement, and facilitate rapid longitudinal connectomic analyses in vivo.

An animal's musculature must support its specific biomechanical needs, so muscle morphology and volume allocation may adapt when locomotor strategies diversify. We examined muscle size and morphology in two sister families of bombycoid moths, wild silkmoths (Saturniidae) and hawkmoths (Sphingidae), that have diverged in wingbeat frequency, wing morphology, and behavior. Although both families rely on the same muscles to power and steer flight, they may distribute muscle volume differently to prioritize distinct functions. We hypothesized that flight power muscle proportions are larger in hawkmoths and increase with wingbeat frequency, helping meet inertial power demands of high-frequency maneuverable flight. We also hypothesized that some individual muscles diverge in proportional volume and area to support distinct wing control strategies. To test our hypotheses, we took CT scans of twenty bombycoid species and quantified volumes and geometries of six flight muscle pairs. As expected, flight power muscle proportions positively correlate with wingbeat frequency and are generally greater in hawkmoths. Two of three steering muscles diverge substantially in relative volume and area between families. Most muscles exhibit greater length in silkmoths and greater cross-sectional area in hawkmoths. Finally, the dorsal oblique(DO) muscle diverges exceptionally in size and morphology, being highly developed in hawkmoths and smaller or absent in silkmoths. This unexpected difference supports the DO having an underappreciated role in flight control, possibly via shaping indirect strain propagation in the elastic thorax. We show that muscle volume distribution parallels bombycoids' divergent flight strategies, demonstrating how muscle allocation can adapt for specialized functional goals.

The cerebellum has long been considered a late-maturing structure subordinate to neocortical development, therefore its potential role as an early driver of cortical organization remains largely unexplored. Using two large longitudinal neuroimaging cohorts of developing children together with lesion experiments in mice, we show that early cerebellar morphology may drive neocortical maturation in a regionally specific manner. These cross-species findings implicate the cerebellum as a possible regulator of neocortical organization.

Across species, sleep amount and timing are tightly linked to the nutritional environment. While early life sleep and sleep in mature organisms are both dramatically influenced by reductions in the dietary environment, the mechanisms linking nutritional cues to conserved sleep-regulatory circuitry are not well understood. Using both early 3rd instar (L3) Drosophila larvae and adults, we examined the plasticity of sleep responses under shifting nutrient environments across the lifespan. We find that L3 larvae and adults exhibit changes in sleep duration in low sugar environments with L3 showing a loss of sleep-wake rhythms that can be rescued with additional nutrients. We show that larval and adult sleep plasticity is regulated by CCHamide-1 signaling between DN1a and Dh44 neurons and glucose metabolic genes in Dh44 neurons. Additionally, our data indicate that sleep plasticity is not dependent on anatomical and functional connectivity between clock-arousal circuitry, suggesting that peptidergic signaling alone is sufficient for diet-dependent sleep regulation. Finally, we demonstrate that Dh44 neurons in both L3 larvae and adults adjust mRNA levels of CCHamide-1 receptor (CCHa1-R) in response to changes in dietary sugar. Together, our findings suggest that organisms utilize conserved molecular signaling pathways across the lifespan to dynamically regulate their sleep in a changing environment.