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.
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.
Sturgeons are an ancient and basal group of ray-finned fishes whose morphology is critical to our understanding of the evolution of teleosts, which form most of the ray-finned fish radiation. In sturgeons, the pallium, or area dorsalis, consists of lateral, medial, and posterior divisions. The connections of these pallial divisions were determined by injections of biotinylated dextran amine and DiI. All three divisions receive olfactory input, with the posterior division receiving the bulk of the secondary olfactory projections. All three pallial divisions also have extensive intratelencephalic connections, as well as input from the thalamus and the posterior tubercle. The posterior division also receives sparse input from the midbrain and secondary gustatory nucleus of the isthmus. All three pallial divisions project heavily to the hypothalamus of the ipsilateral inferior lobe, and more sparsely to the ipsilateral optic tectum, posterior tubercle, and midbrain tegmentum. Cytological and connectional data provide no evidence that either a dorsal or central pallial division exists in sturgeons, and these pallial divisions likely arose in ancestral teleosts.
Ceramide synthases (CerS) are key enzymes in sphingolipid metabolism that regulate fundamental cellular processes, including apoptosis, cell growth, and homeostasis. Among the six known mammalian isoforms (CerS1-CerS6), CerS5 has been particularly well studied for its involvement in the synthesis of the sphingolipid C16-ceramide. However, its expression, localization, and functional significance of CerS5 in the retina remain unclear. In the present study, we investigated the presence, distribution, and functional role of CerS5 in mouse retina using CerS5 knockout (KO) mice. We performed quantitative polymerase chain reaction, X-gal staining, and immunohistochemistry to analyze the expression and localization. Electroretinography (ERG) was employed to assess the impact of CerS5 deficiency on retinal function. Our results demonstrated that CerS5 is localized to the inner nuclear layer and ganglion cell layer, co-localizing with horizontal cells and specific subsets of amacrine and ganglion cells. The retina of CerS5 KO mice showed a reduction in overall thickness, with significant thinning observed in all retinal layers except the photoreceptor, whereas the outer plexiform layer showed increased thickness. Despite these structural alterations, ERG recordings revealed no significant changes in retinal function. These findings suggest that CerS5 contributes to the maintenance of retinal structural integrity, particularly through its presence in specific retinal cell types, whereas its loss does not markedly impair retinal function in adult mice. The observed structural alterations highlight its potential role in retinal physiology and possible implications for retinal pathophysiology, warranting further investigation into compensatory mechanisms by other ceramide synthase isoforms.
The neurotransmitters vasotocin, oxytocin, dopamine, and serotonin are widely involved in vertebrate social behavior, and changes in their abundance and distribution in the brain have been linked to the evolution of complex sociality. Reptiles provide an excellent system in which to investigate the neural mechanisms of social living. Using immunohistochemistry, we compare distributions of these transmitters in two skinks differing primarily in social ecology: the family-living Liopholis whitii and the solitary Eulpamrus quoyii. We describe patterns of immunopositive signal for both cell bodies and fibers across the entire brain (excluding the olfactory bulbs). In both species, vasotocin and oxytocin were found in the preoptic area, paraventricular nucleus, supraoptic nucleus, dorsomedial hypothalamus, and supraoptic decussation, as well as surrounding the lateral forebrain bundle. Tyrosine hydroxylase (a marker for dopamine) was found in the paraventricular organ nucleus, substantia nigra, and ventral tegmental area, and serotonin was found in the raphe nuclei and superior reticular field. We found novel oxytocin cell groups in the dorsomedial hypothalamus and cerebellum of L. whitii, and novel serotonin signal in the red nucleus of E. quoyii. Immunopositive signals found only in L. whitii also include vasotocin in the ventral tegmental area, tyrosine hydroxylase in the interpeduncular nucleus, and serotonin in the suprachiasmatic nucleus. The qualitatively greater abundance of these transmitters in the family-living L. whitii suggests that these molecules may have played an important role in the evolution of social behavior in these skinks and provides a foundation for broader comparisons across the social skinks.
Mosquitoes are major disease vectors that pose significant threats to human health. Recent efforts to understand the neurobiology of these insects have utilized modern research tools to advance our knowledge of the neural circuits underlying ecologically relevant behaviors in adults. However, the neurobiology of mosquito larvae remains relatively unexplored. This report focuses on the neuromuscular system of mosquito larvae, providing a morphological analysis of the abdominal neuromuscular system in Aedes aegypti. We identified 24 muscle fibers per hemi-segment and detailed their anchoring points and orientations. Using immunocytochemistry, the organization and innervation of abdominal body-wall muscles were reconstructed. Two primary nerve branches emerged from the ventral nerve cord in each segment. The branches, trajectories, and targets of the two primary nerves were named and described. At the synaptic level, motor axons arborization on muscle fibers were quantitatively analyzed. Motor axons branched extensively on muscle fibers, covering up to 90% of large fibers' length and approximately 30% of small fibers. Varicosities along axonal branches showed considerable size variation, with antibody labeled active zones present in all varicosities. Furthermore, active zone densities per varicosity on smaller muscle fibers were significantly higher than those on larger fibers. These observations provide a functional and morphological framework for understanding the physiology of mosquito larval locomotion.
Habituation is a simple form of nonassociative learning that is characterized by a decrease in response to a repetitive stimulus. As escape responses can be energetically costly and disruptive to normal behavior, it is important that prospective prey learn whether a perceived stimulus is a genuine threat or an innocuous stimulus that they can ignore. In response to a visual looming stimulus, larval zebrafish perform a characteristic escape swim that reliably habituates, and because they are small and transparent, they have been an important model for characterizing brain-wide activity patterns during habituation. In this study, we explore the spatial properties of visual adaptation to gauge whether it is mediated by local, regional, or brain-wide circuits. We present repetitive visual loom stimuli either in a fixed position in visual space or in variable positions, while also performing brain-wide calcium imaging. Across the brain, we identify both neural responses that are specific to looms at particular positions within the visual field and responses that occur regardless of where the loom is presented. By quantifying the degree of adaptation across these responses, we show that brain-wide adaptation occurs more rapidly when the position of the loom remains unchanged and that alternate looms occurring in different parts of the visual field minimally contribute to adaptation for looms at the original position. We found that the tectum, homologous to the superior colliculus, has response profiles and spatial sensitivity indicative of important contributions to this position-specific visual adaptation.
The primate anterior entorhinal cortex (EC) receives rich projections from the amygdala and from multimodal association areas, including the medial prefrontal, anterior cingulate, and orbitofrontal cortices. Axon terminations from these structures on the anterior EC facilitate processing of the emotional aspects of stimuli and events. The EC projects to hippocampus, which is associated with episodic memory. Processing in the anterior EC is modulated by inhibitory neurons, which in primates express the calcium-binding proteins (CBPs): calretinin (CR), or calbindin (CB) or parvalbumin (PV), which collectively account for most inhibitory neurons in the primate cortex. Here, stereological analysis of these neurochemical classes of inhibitory neurons in the anterior half of EC in humans revealed similar patterns as in rhesus monkeys. In both primate species, the densest neuronal subpopulation of presumed inhibitory neurons expressed CR, followed by CB, and lastly by PV. In both species CR neurons were most prevalent in layers I and II, CB neurons in layers II and III and PV neurons in the middle-deep layers. Moreover, the medial and lateral sectors of the anterior EC had different densities of neurons expressing these CBPs. Further analysis revealed that in the human anterior EC, virtually all PV neurons expressed the GABAergic marker GAD67/GAD1 (glutamate decarboxylase 67/glutamate decarboxylase 1), whereas only two-thirds of CB neurons and only one-third of CR neurons colocalized with GAD67/GAD1. In the entire neuronal population of the anterior half of human EC estimated by stereology, 10% expressed GAD67/GAD1, comparable to the collective population of CBP-positive neurons that colocalized with GAD67/GAD1. These findings reveal that the medial and lateral sectors of anterior EC have distinct inhibitory microenvironments, which likely affect the processing of input and output of hippocampus.
To navigate its environment, an animal extracts salient information from sounds using temporal and intensity cues. In birds, the nucleus laminaris (NL) detects the submillisecond differences in the arrival time of sound to the two ears, the interaural time differences (ITDs), to localize sounds. This ability is facilitated by inhibitory long-range projection neurons from the ipsilateral superior olivary nucleus (SON) that enable NL neurons to remain sensitive to ITDs across a large range of sound intensities. It is well known that the excitatory inputs to NL, from nucleus magnocellularis (NM), innervate a narrow isofrequency band along the ITD axis. However, the organization of the inhibitory input from the SON remains largely unknown. We analyzed the innervation pattern of individual axons from SON neurons within the chicken NL. SON axonal arborizations vary greatly in size and topographic organization. On average, an inhibitory SON neuron innervates one-third of both the tonotopic and ITD axes, markedly larger target regions than do the excitatory inputs from NM. Unlike the excitatory axons that are confined to one dendritic lamina (separating inputs from the two ears), most SON cells innervate both laminae to similar extents, as well as the somata of the NL neurons. In addition, we found that some NL-projecting SON neurons also send collateral axons to NM or the nucleus angularis. The pattern of synapses along SON axons suggests that the inhibitory activity of individual NL neurons is shaped by many SON neurons. A single SON neuron contributes only a small proportion of the inhibition on each NL neuron. This broad innervation pattern of SON neurons is well-suited to control the overall activity of NL, supporting accurate ITD detection in a broad range of sound environments.
Proline-rich transmembrane protein 2 (PRRT2) plays a pivotal role in the control of voluntary movements, as PRRT2 mutations cause paroxysmal kinesigenic dyskinesia (PKD) in a loss-of-function manner. Although the cerebellum is considered a region responsible for PKD, we recently reported that Prrt2 also regulates dopaminergic activity in the striatum, suggesting that Prrt2 functions not only in the cerebellum but also in the basal ganglia motor circuits. However, the relationship between neuronal cell types expressing Prrt2 and motor functions remains poorly understood. In this study, we determined the neurochemical types of Prrt2-positive neurons using immunofluorescence staining of mouse midbrain primary neurons and brain sections. Prrt2 was expressed mainly in glutamatergic and GABAergic neurons, but not in dopaminergic or cholinergic neurons. We found that Prrt2 was expressed preferentially in Vglut1-positive, rather than Vglut2-positive, cortical projection neurons and cerebellar granule cells, and in GABAergic medium spiny neurons of the basal ganglia, where Prrt2 was localized in axonal tracts and at or near presynaptic terminals. Taken together, we conclude that Prrt2 is variably expressed across excitatory and inhibitory neurons in motor-related neural circuits, where it might play more diverse roles in the regulation of neuronal excitability and voluntary movement.
The transcription factor Orthopedia (Otp) plays a key role in neuronal differentiation, regional specification, and long-term maintenance of neuronal identity across vertebrates. Although Otp expression is widely used as a landmark for hypothalamic regionalization, its distribution in bony fishes has remained poorly characterized. To assess the evolutionary conservation of Otp expression, we analyzed its immunohistochemical distribution in the central nervous system of representative species from all major clades of bony fishes: cladistians, chondrosteans, holosteans, teleosts, and lungfish. To refine anatomical localization and evaluate potential cellular coexpression, we additionally examined markers including calbindin, tyrosine hydroxylase (TH), and the transcription factors Islet-1, Pax7, and Satb1/2. Otp-immunoreactive neurons were consistently observed in the medial amygdala, preoptic area, paraventricular region, all basal hypothalamic domains except the mamillary region, as well as in a population in alar rhombomere 1, the dorsal interpeduncular nucleus, central gray, reticular formation, and the dorsal horn of the spinal cord. Notably, Otp-positive cells were detected in the posterior tubercle only in actinopterygian fishes, in contrast to lungfish. Overall, this Otp expression pattern is highly conserved in relation to that reported in cartilaginous fishes and tetrapods. This study closes a major phylogenetic gap and demonstrates that the brain-wide expression pattern of Otp is largely conserved across vertebrates, supporting its fundamental and ancient role in neuronal differentiation and cell-type specification in forebrain and hindbrain.
The vomeronasal system (VNS) in most mammals detects species-specific chemicals. Vomeronasal type 1 (V1R) and type 2 (V2R) receptors in the VNS, respectively, couple with G protein α subunits i2 (Gαi2) and o (Gαo). Most artiodactyls and carnivores have neurons that express only V1R/Gαi2 in the peripheral vomeronasal organ and project axons to the entire accessory olfactory bulb (AOB) (uniform type). In contrast, most rodents have V1R/Gαi2 and V2R/Gαo-neurons that respectively project to the rostral and caudal parts of the AOB (segregated type). However, the California ground squirrel (Otospermophilus beecheyi; family Sciuridae, subfamily Xerinae) has a V1R/Gαi2 AOB, which is the only exception to the segregated profiles of rodents to date. Here, we immunohistochemically analyzed the AOBs of four Pallas's (Callosciurus erythraeus; subfamily Callosciurinae) and three Eurasian red (Sciurus vulgaris; subfamily Sciurinae) squirrels using anti-Gαi2 and anti-Gαo antibodies. The AOBs of both arboreal species consisted of the vomeronasal nerve (VNL), glomerular (GL), external cellular (ECL), and internal cellular (ICL) layers. We found both Gαi2- and Gαo-positive regions in the VNL and GL in all squirrels examined in this study, which suggested that they can detect nonvolatile cues as pheromones or kairomones via V2R like rodents in general. However, the V1R/Gαi2 and V2R/Gαo domains were not equivalent in the AOBs of these squirrels, as a large area corresponded to the V1R/Gαi2-domain. Considering these results together with findings from ground squirrels, V2R expression in the VNS seems to be reduced in the family Sciuridae among rodents.
The mesodiencephalic junction (MDJ) is located in a caudorostrally elongated, column-shaped region encompassing the medial accessory oculomotor nucleus and perirubral area. It relays cerebral projections to the rostral subnuclei of the inferior olive (IO), which, in turn, send climbing fibers to the cerebellar hemisphere and receive nucleo-olivary projections from the cerebellar nuclei (CN), forming a parallel, loop-shaped, modular circuitry. However, the topographical organization of the MDJ-IO projection, which may contribute to functional localization within the cerebellar hemisphere, has not been fully elucidated. Here, we analyzed the distribution of labeled neurons in the MDJ in relation to those in the CN by injecting a Lumafluor retrograde tracer into various sites within the rostral IO subnuclei of mice. Injections mainly targeting the lateral or medial parts of the rostral IO subnuclei labeled neurons predominantly in the ventral or dorsal CN, respectively (referred to as v- and d-CN-predominant injection cases). Correspondingly, v-CN-predominant injection cases labeled neurons in the rostromedial, centroventral, and caudolateral regions of the MDJ, collectively termed the "sheathing subarea," whereas d-CN-predominant injection cases labeled neurons primarily in the central "shaft subarea" of the MDJ. Considering the previously reported lobule-related topography among the IO, cerebellar cortex, and CN, the sheathing and shaft subareas of the MDJ are likely to project preferentially to crus I and to other neighboring cerebellar hemispheric lobules, respectively. We speculate that the sheathing subarea of the MDJ is more strongly involved in non-somatosensorimotor cognitive functions than the shaft subarea.
Spinal cord injury (SCI) in mammals leads to inflammation, glial scarring, and extracellular matrix (ECM) remodeling that hinder regeneration. In contrast, certain teleosts, such as zebrafish and goldfish (Carassius auratus), exhibit remarkable spinal cord regenerative capabilities and substantial functional recovery following injury. To uncover the molecular basis of this regenerative ability, we performed a proteomic analysis of goldfish spinal cord ECM-enriched post-injury and compared it to literature on mammalian models. We identified a distinct regenerative signature in goldfish, marked by the downregulation of vimentin A2, histone H1-like isoform X1, connexin 43, and biglycan-like proteins, factors typically associated with scarring and inflammation in mammals. Notably, nucleosome assembly protein 1-like 4 (NAP1L4), a chromatin remodeling protein, was uniquely upregulated in goldfish, suggesting a more permissive epigenetic environment for repair. These findings provide new insights into the molecular determinants of successful spinal cord regeneration and may inform strategies for developing regenerative therapies in humans.Teaser: Goldfish spinal cords reveal a unique regenerative proteomic signature absent in non-regenerative mammals after injury.
The mushroom bodies (MBs) in the insect brain serve sensory integration and memory formation. In the honeybee, they house two classes of intrinsic neurons: class I (spiny) and II (clawed) Kenyon cells (KCs). Both classes form postsynaptic elements in synaptic complexes (microglomeruli) comprising large axonal boutons from olfactory and visual projection neurons. To adapt their neuronal information processing systems, MB microglomeruli undergo age-, memory-, and environment-related structural plasticity. To analyze KC dendritic specializations and their connections with presynaptic boutons, we combined tracer injections in small groups of KCs from different age cohorts (freshly emerged bees to foragers) with presynaptic anti-synapsin immunolabeling. Using high-resolution confocal 3D reconstructions, we analyzed shape and contacts of class I and II KC dendrites in the olfactory (lip) and visual (collar) input sites of the MB calyx. In both KC classes, dendrites are always restricted to either the lip or the collar. We classified two types of class II KCs regarding the spatial distribution of dendritic branches: large-clustered and small-distributed. Individual claws of class II KCs largely vary regarding surface areas covered on individual axonal boutons (∼5%-70%). In class I KCs, we found four distinct morphological spine categories: stubby, thin, mushroom-shaped, and branched. Interestingly, the overall frequency of putative spine-bouton contacts in class I KCs remains largely constant throughout age cohorts. We discuss the results in the light of structural dynamics in MB microglomerular circuits and their role in multisensory information processing.
