Journal of Comparative Neurology

Adeno-associated viral (AAV) vectors are foundational tools for dissecting brain structure-function relationships, but AAV serotype tropism varies across brain regions and species, requiring empirical validation to inform experimental design. This need is especially important in non-model organisms, where molecular neuroscience tools remain underdeveloped and access to research subjects is often limited. The Arctic ground squirrel (AGS, Urocitellus parryii) is a valuable model for studying extreme physiology, including metabolic suppression during hibernation and resistance to cerebral ischemia/reperfusion, yet no studies have evaluated AAV performance in the AGS brain. Here, we investigated the ability of AAV serotypes 1, 8, 9, and DJ to transduce the AGS hypothalamus using the human synapsin (hSyn) promoter and directly compared cellular transduction rates in a region implicated in thermoregulation and hibernation. To maximize data collection from a limited experimental population, we used a within-animal, contralateral stereotaxic injection design. Recombinant AAV vectors expressing enhanced green fluorescent protein or mCherry were delivered bilaterally, and reporter expression was analyzed four weeks later. All tested serotypes produced clear and reproducible reporter expression, establishing AAV as a viable molecular tool in the AGS hypothalamus. AAV1 produced significantly greater cellular transduction rates than AAV-DJ (17.2% {+/-} 3.5% vs 8.4% {+/-} 2.9%, paired t-test, p = 0.032). AAV8 and AAV9 showed transduction rates of 22.8% {+/-} 0.6% and 20.1% {+/-} 1.5%, respectively; however, with only two biological replicates per serotype, formal statistical comparison was not performed. These findings provide the first direct characterization of AAV-mediated gene delivery in the AGS brain and establish a foundation for future molecular interrogation of hypothalamic circuits in this extreme mammalian hibernator.

The peripheral auditory system of dolphins comprises specialised bony, fatty, vascular, and neural structures adapted for underwater hearing and diving physiology. These include the external ear canal, acoustic fat bodies, sinuses, and associated neurovascular networks, which together support sound conduction, protection, and possibly sensory functions. Despite advances in gross anatomical description, the detailed integration of these tissues, particularly the innervation, neurovascular organisation, and their functional implications, remains poorly understood. Previous studies have described the presence of sensory nerve formations and vascular plexuses, but their arrangement, connectivity, and relation to each other are unresolved. Here, we combine macroscopic dissection, DICE-{micro}CT, histology, and high-resolution confocal microscopy to characterise several neurovascular and sensory components of the dolphin peripheral auditory system in several delphinid species. Macroscopic dissection and DICE-{micro}CT revealed the traditional acoustic fat body distribution with detailed morphology of the posterolateral extension that is not well-known. The cranial nerve distribution, and specifically the mandibular nerve branching patterns, are described in detail. Confocal microscopy uncovered a stratified neurovascular plexus around the external ear canal with a complex sensory system comprising lamellar corpuscles, Merkel cell-neurite complexes, and intraepithelial nerve fibres. Notably, the lamellar corpuscles formed a continuous, three-dimensional neural network with frequent merging and splitting of axonal bundles, shared perineuria, and vascular integration, features not observed in previous studies. Our findings demonstrate that the dolphin external ear canal and surrounding structures form a sophisticated, multimodal somatosensory organ, integrating structural, vascular, and neural specialisations likely adapted for proprioceptive mechanosensation in the aquatic environment. This study provides insights into the integration of the various components of the peripheral hearing apparatus. Future studies integrating anatomical, electrophysiological, and biomechanical approaches are needed to fully elucidate these adaptations.

The evolutionary origin of nervous systems in animals remains elusive and is largely hidden from the fossil record. Ctenophores, one of the earliest-branching animals possessing neurons, are instrumental to our understanding of nervous system origin, and a few rare ctenophore fossils preserve traces of nervous tissue as carbonaceous remains. Cambrian ctenophores appear to exhibit a more diverse neuroanatomy than that of modern species, suggesting secondary loss in extant ctenophores. However, much remains unknown about the origin and ontogeny giving rise to the structural organization of modern ctenophore nervous systems. Here, by investigating the neural anatomy of the ctenophore Mnemiopsis leidyi during development, we identified a ladder-like nerve net (LNN) beneath the comb rows that converges into condensed neurites and connects to the aboral organ. Examination of carbon-rich areas of Ctenorhabdotus capulus, an extinct ctenophore from the Burgess Shale, reveals a pattern similar to that of M. leidyi, consistent with a shared neural organization. Furthermore, M. leidyi exhibits a condensed comb nerve, resembling the longitudinal nerve preserved in the Cambrian ctenophore Fasciculus vesanus and the giant axon of extant Euplokamis dunlapae. Our study reveals conserved evolutionary constraints shaping nervous system architectures linked to locomotory organs and indicates that the different modes of nervous system organization observed in Cambrian ctenophores are variably retained in modern species.

The basal ganglia (BG) form anatomically and functionally segregated yet integrative parallel circuits, but the molecular mechanisms specifying them remain unclear. We immunohistochemically mapped the expression of three {delta}2-protocadherin ({delta}2-PCDH) cell adhesion molecules--PCDH10, PCDH17, and PCDH19--in the BG of macaques. Within the striatum, each PCDH exhibited regional gradients of expression along the rostro-caudal and ventromedial-dorsolateral axes. The three PCDHs showed complementary distributions that continuously delineated molecular boundaries corresponding to functional subdivisions in a graded fashion. Such complementary distributions were also observed in the BG output nuclei. Given that neurons expressing the same {delta}2-PCDH in distinct BG structures preferentially connect with each other, the three {delta}2-PCDH expression patterns could define functional territories within parallel BG circuits. Together, the complementary expression of PCDH10, PCDH17, and PCDH19 broadly align with the distinct BG circuits, respectively, suggesting molecular codes underlying the segregated yet integrative parallel organization of the primate BG.

The brain is one of the most complex animal organs but the development of the many different neuron types remains enigmatic. A set of brain-specific transcription factors is known to be involved in brain patterning but their specific contributions remain to be elucidated in most cases, including foxQ2II. This transcription factor is known to be conserved in anterior neuroectodermal patterning of most animals while it has been lost from vertebrates. However, the contribution of foxQ2II-positive neurons to the adult brain has remained enigmatic. Here, we use an enhancer trap, immunostainings and our newly established beetle brainbow system to categorize Tc-foxQ2II-positive neurons into nine clusters with different projection patterns. All clusters contain neurons with the fast activating neurotransmitters acetylcholine and glutamate while no Tc-foxQ2II positive neuron is GABA-ergic or serotonin-positive. Interestingly, we found that many dopaminergic neurons were Tc-foxQ2II positive and we homologize them with dopaminergic neurons of the PPL2c, PPM1 and PPL1 cluster described in the Drosophila brain. Our results show that Tc-foxQ2II marks subsets of fast-acting interneurons contributing to the higher order brain centers mushroom bodies and central complex. Taken together, our work expands the known functional range of foxQ2 genes from sensory and neurosecretory cell specification to interneurons involved in the function of higher order brain centers.

Recent studies have shown that symbiotic bacteria can have drastic effects on host neurobiology, but few simple, accessible models currently exist in which to study these interactions. Hawaiian bobtail squid (Euprymna scolopes) participate in a binary symbiosis with the bacterium Vibrio fischeri, a population of which resides in a specialized hindgut-derived organ called the light organ. Upon colonization by V. fischeri, the light organ undergoes transcriptional changes that suggest neurons are impacted by the initiation of symbiosis, but the nascent light organs innervation has remained uncharacterized. Here, we show that the light organ-associated nervous system (LONS) in hatchling E. scolopes is a remarkably complex segment of the peripheral nervous system. The LONS is largely plexiform and originates from two primary nerves connected by a local commissure. The abundance of synapsin-like immunoreactivity (-lir) indicates that the lobe plexus is highly interconnected. We also highlight a small number of serotonin-lir neurites that innervate the anterior appendages whose developmental fate may be directly affected by symbiont-driven light organ morphogenesis. Finally, we present evidence that a limited but diverse population of neurons reside within the light organ and are often located near internal symbiont-interacting structures. This description of the E. scolopes LONS serves to provide a foundation from which to investigate how beneficial bacterial symbionts affect host peripheral neurobiology in a tractable model system.

Vestibular neurons are core elements of the pathways involved in vestibulo-motor functions, such as vestibulo-spinal and vestibulo-ocular reflexes. To meet behavioral needs, electrophysiological neuronal properties are adequately adapted to the sensory-motor computation sustaining these distinct vestibular reflexes. During frog metamorphosis, there is a complete reorganization of the posturo-locomotor system while the oculomotor system remains minimally changed, probably associated to so far unknown changes in vestibular neuronal properties. We used this unique model to investigate the central developmental mechanisms underlying such a reconfiguration of vestibular-associated behaviors. Central vestibular neurons exhibit two types of electrophysiological phenotypes: tonic neurons with a continuous discharge and phasic neurons with a transitory discharge mainly due to the activation of Kv1.1 channel. Electrophysiological recordings and Kv1.1 immunolabeling of vestibulospinal (VS) and vestibulo-ocular (VO) neurons at both larval and juvenile stages revealed that the majority of VS neurons exhibited a tonic discharge in larvae but a phasic discharge in juvenile, while VO neurons remained mainly tonic throughout development. Changes in phasic and tonic neurons proportions in VS population are partly explained by neurogenesis. But we provide evidences that an electrophysiological phenotype switch is a concomitant developmental mechanism participating in the maturation of these central vestibular neurons. All together our results showed that the maturation process in central vestibular neuronal groups is highly related to the metamorphosis-induced remodeling of vestibulo-motor functions they are involved in, with the ultimate purpose of ensuring an adequate adaptation of neuronal elements properties to the developmental changes of behavioral constrains.

To avoid image blurring, the vestibulo-ocular (VOR) and the optokinetic (OKR) reflexes stabilize gaze. In all vertebrates, the VOR is mediated via direct projections from the vestibular nuclei to the motor nuclei that control the extraocular muscles. Lampreys show three vestibular nuclei that are well characterized in terms of their projections and sensory inputs, but much less is known about their inputs from other brain regions and the connectivity between them. Using tracer injections and electrophysiological recordings, we show that the lamprey vestibular nuclei are largely interconnected, while their inputs from other brain regions are scarce. The main rostral areas projecting to the vestibular nuclei are the pretectum and the ventral tier of the thalamus, which send ipsilateral inputs to the three vestibular nuclei.

Despite being essential mediators of pain processing, the molecular identity of N-methyl-D-aspartate receptor (NMDAR) subtypes in nociceptive dorsal horn circuits is poorly understood, especially between sexes and in humans. Given the importance of GluN2 subunits in shaping NMDAR function and plasticity, we investigated the expression and localization of specific GluN2 NMDAR variants in the dorsal horn of viable spinal cord tissue from male and female rodents and human organ donors. Analysis of single-cell/nuclei sequencing datasets and quantitative reverse transcriptase polymerase chain reactions (qRT-PCR) revealed that the GluN2A (GRIN2A) and GluN2B (GRIN2B) subunits are robustly expressed in dorsal horn neurons of mice, rats and humans, with moderate expression of GluN2D (GRIN2D). Immunohistochemistry (IHC) with antigen retrieval demonstrated that GluN2A, GluN2B, and GluN2D proteins are all preferentially localized to the superficial dorsal horn of both adult rats and humans, which is conserved between males and females. Surprisingly, we found that these GluN2 NMDAR subunits are enriched in the lateral superficial dorsal horn in rats but not in humans, while presynaptic and neuronal markers are symmetrically distributed across the rat mediolateral axis. A dramatic shift in localization of GluN2A to the lateral superficial dorsal horn was observed across later postnatal development (PD21-PD90) in both male and female rats, with a corresponding change in synaptic NMDAR currents. This discovery of changes in NMDAR subunit distribution during maturation and between species will shed light on the physiological roles of NMDARs and their potential as therapeutic targets for pain. SIGNIFICANCE STATEMENTWe used complementary single-cell/nuclei analysis, immunostaining, quantitative reverse transcriptase polymerase chain reactions, RNAscope in situ hybridization, and electrophysiological approaches to compare the relative expression of N-methyl-D-aspartate receptor (NMDAR) GluN2 subunits in dorsal horn spinal cord pain circuits of mouse, rat, and human spinal cord tissue. Through these comparisons, we find that the transcripts and proteins of the GluN2A, GluN2B, and GluN2D NMDAR subunits are robustly expressed in superficial dorsal horn neurons, with conserved expression across sex but important differences in expression and localization patterns across late development and between species. These discoveries shed light on the physiological roles of NMDARs and their utility as potential therapeutic targets for pain.

The mesencephalic trigeminal nucleus (MTN) contains the proprioceptive sensory neurons that innervate mechanoreceptors in the jaw closing muscles. In the chick embryo, MTN neurons are the first neurons generated in the mesencephalon. They arise bilaterally adjacent to the roof plate and then extend their axons ventrally before projecting caudally towards the rhombencephalon. MTN axons remain in a mid - dorsoventral position and pioneer the lateral longitudinal fasciculus. Notably, MTN axons never cross the roof plate, raising the question of which mechanisms underlie this restriction. Here, we investigated the effects of tissue transplants on the guidance of MTN axons. We found that both the diencephalon and the notochord exert repulsive effects on MTN axons, which could partially explain their early trajectory. We have also analysed the potential roles of the guidance cues BMP2/4, GDF7, SLIT and NETRIN in MTN axon navigation, both in vivo and in vitro. We found no evidence for a role of BMP2/4 or GDF7 in directing MTN axons. However, SLIT-ROBO signaling was found to play a significant role. SLIT proteins are repulsive guidance cues expressed by roof and floor plate. Loss or reduced expression of ROBO2 led to aberrant axon meandering within the dorsal midbrain. Most axons eventually reoriented posteriorly, and only a small fraction crossed the roof plate. Unexpectedly, in the absence of ROBO2, MTN somata migrated into the roof plate, resulting in the loss of a defined roof plate region. Taken together, these results suggest that SLIT2-ROBO2 signaling not only prevents MTN axons from crossing the roof plate but also maintains MTN cell bodies adjacent to the roof plate. With regards to MTN neuron guidance, we conclude that additional roof plate - derived factors are likely to co-operate with SLIT proteins to prevent crossing of the roof plate. Another possibility could be that SLIT might signal through additional receptors.

Homeostatic plasticity of intrinsic excitability (IE) in the visual system has been essentially shown at the cortical level but whether thalamic nuclei also express homeostatic plasticity of IE is unknown. We show here that 4 days of monocular deprivation (MD) at eye opening induces a homeostatic change in IE in dorsal lateral geniculate nucleus (dLGN) neurons. Neurons recorded in the dLGN region activated by the deprived eye are more excitable than neurons recorded in the dLGN region activated by the open eye. No significant changes were observed following 7 days of MD, however. Enhanced excitability in neurons from the deprived side after 4 days of MD was associated with a reduced Kv1-dependent LTP-IE, a smaller voltage ramp, and a reduced inter-spike interval, suggesting that Kv1 channels are down-regulated in deprived dLGN neurons. Furthermore, the ankyrin G signal of the axon initial segment was larger in deprived dLGN neurons compared with open ones, indicating that Nav1 channel number also undergoes homeostatic regulation, and Kv1.1 channel signals were lower in deprived neurons compared to open ones. In addition, electrical coupling was found to be strengthened in neurons displaying enhanced IE following either brief (4 days) or long (10 days) MD. These results suggest that homeostatic and Hebbian plasticity in the dLGN share common expression mechanisms involving the regulation of Kv1 channels, Nav1 channels and electrical coupling between relay neurons.

Electrical transmission is mediated by intercellular channels that cluster into structures known as gap junctions (GJ). In vertebrates, GJ channels are encoded by the gene family of connexin (Cx) proteins that assemble as hexamers, termed hemichannels, in the pre- and postsynaptic membranes, and that subsequently dock to form GJ channels. Auditory contacts on the fish Mauthner cells serve as model to study the properties and organization of vertebrate electrical synapses. Electrical transmission at these synapses is mediated by multiple co-existing GJs at which the presence of intercellular channels is regulated by a molecular scaffold. Zebrafish contain four homologs of the neuronal Cx36: Cx35.5 and Cx35.1 (gjd2a and b, respectively), and Cx34.1 and Cx34.7 (gjd1a and b). Cx mutations suggested that GJs are formed by heterotypic channels made of presynaptic Cx35.5 and postsynaptic Cx34.1. Using transgenic fish in which Cxs were tagged, we found that a second Cx, Cx34.7, is present together with Cx34.1 on the postsynaptic side at some but not all GJs at these terminals. When exogenously expressed, both Cx34.1 and Cx34.7 formed heterotypic functional channels with Cx35.5, each with substantially different voltage-dependent properties, indicating they can serve differential functions. However, we previously demonstrated that electrical transmission is lost in Cx34.1 but not Cx34.7 null mutants, suggesting that Cx34.7 cannot compensate for the loss of Cx34, despite the intrinsic ability of Cx34.1 and Cx34.7 to create functional channels. The findings reveal an unanticipated functional organization in the electrical synapse, where Cx34.1 is obligatory and Cx34.7 accessory, roles that appear to be defined by the postsynaptic molecular scaffold, with two postsynaptic Cxs possibly assembling under specific functional contexts. Thus, our results indicate that electrical synapses share an organizational motif with chemical synapses, akin to how they combine postsynaptic receptor types to modify synaptic function.

The addictive properties of opioids are due in part to these drugs ability to alter ventral tegmental area (VTA) activity via activation of mu opioid receptors (MORs) on local and distal inputs. Prior studies have identified numerous opioid-modulated afferents to the VTA, some of which show differing levels of functional modulation by opioids, but the degree to which this parallels differences in receptor expression is not known. Hence, we used retrograde labeling combined with RNAscope to examine oprm1 mRNA expression in VTA-projecting afferents arising from a variety of distal brain regions. Because opioids are thought to be particularly influential on GABAergic afferents to the VTA, we also examined colocalization of oprm1 with GABAergic markers in VTA-projecting neurons. Interestingly, we found that oprm1 mRNA is present in both GABAergic and non-GABAergic VTA-projecting neurons. However, many (though not all) GABAergic afferents expressed higher levels of oprm1 compared to most non-GABAergic afferents (especially those arising from the cortex). These results complement previous anatomical studies that had examined oprm1 expression in these regions but in a non-quantitative way and without regard to their efferent targets. Our findings encourage future work to examine the functional implications of MOR sensitivity within these afferent pathways.

The cerebellar nuclei form the main output structures of the cerebellum and are composed of a deeply conserved set of cell types. Two excitatory cell classes, Class-A and -B, are present in each cerebellar nucleus and mediate all excitatory output of the cerebellum. To provide genetic access to these cell types, here we identified Acan as a marker gene for Class-B cells and generated a knock-in Acan-P2A-Cre mouse line. We demonstrate that this Acan-Cre line selectively labels Class-B neurons in the cerebellar nuclei and validate its use in viral projection tracing. This new mouse line provides a valuable genetic tool to study cerebellar nuclei organization and function.

Elite athletes exhibit sport-specific neural adaptations, yet it remains unclear whether such changes reflect general effects of training or the unique demands of individual sports. Skiing requires postural control and whole-body coordination under dynamically unstable environments, placing high demands on somatosensory processing and sensorimotor integration. The present study aimed to identify structural brain characteristics specific to elite skiers by comparing them with athletes from other sports disciplines and non-athletes. T1-weighted MRI data were analyzed using voxel-based morphometry in 13 skiers, 23 non-ski control athletes and 25 non-athletes. Whole-brain analysis comparing skiers with non-ski athletes revealed a significant cluster showing greater gray matter volume in skiers compared with non-ski athletes in the left postcentral gyrus, extending into the superior parietal lobule. The identified cluster primarily encompassed cytoarchitectonic Areas 2 and 5L. These regions are involved in higher-order somatosensory processing and multisensory integration. Importantly, region-of-interest analysis demonstrated that gray matter volume within this cluster was greater in skiers compared with non-ski athletes and non-athletes, with no difference between non-ski athletes and non-athletes. These findings highlight the relative prominence of structural adaptations within somatosensory-parietal networks, reflecting the unique integration of proprioceptive and other sensory information required for elite skiing. Overall, these findings provide evidence for sport-specific structural brain differences in elite athletes and highlight the importance of somatosensory and parietal regions in sensorimotor integration relevant to skiing. These findings may have implications for understanding neural markers of expertise and may inform future approaches to training and performance evaluation in skiing.

T-cells infiltrate somatosensory ganglia in response to nerve damage, autoimmune disease, and infection, contributing to sensory abnormalities and pain. In naive states, T-cells are rare in the rodent dorsal root ganglion (DRG) but have been reported in human and non-human primates without known relevant exposures. It remains unclear whether there are inherent evolutionary or species differences in DRG T-cell residence. Using a comparative biology approach, we investigated the frequency and distribution of T-cells in the mammalian DRG across humans, non-human primates, pigs, and rodents, and in humans investigated the contributions of sex and age. Spatial transcriptomics and immunofluorescence independently verified the robust presence of DRG T-cells at similar levels in humans, non-human primates, and pigs, but were fewer in rats and largely absent in mice. In humans, premenopausal females were more likely to have elevated DRG endoneurial T-cells than post-menopausal females or adult males. T-cells were detected in human dorsal root ganglion at as early as two months of age but were less abundant within the perineuronal niche. Most human DRG T-cells expressed distinct markers consistent with a resident memory (Trm) phenotype. We discuss the importance of studying the functional roles of DRG-resident T-cells and raise broader considerations for modelling peripheral nervous system disease.

BackgroundThe optic nerve serves as a vital conduit for visual signaling, and its degeneration in optic neuropathy results in irreversible vision loss. It is also a widely used model for studying central nervous system (CNS) injury and repair. Although adeno-associated virus (AAV) and lentivirus are extensively applied in CNS research, their transduction efficiency and cell-type specificity within the optic nerve remain poorly characterized. This study aimed to identify the most effective viral vector, serotype, and promoter for direct gene delivery to the adult rat optic nerve. MethodsSprague-Dawley rats (7-10 weeks) received intra-optic nerve injections of lentiviral or AAV vectors encoding GFP under different promoters (CAG, CMV, or GFAP). Two to three weeks post-injection, optic nerves were collected for immunohistochemistry with markers of oligodendrocytes (Olig2), astrocytes (GFAP, Sox9), and microglia (IBA1). Transduction efficiency and cell-type specificity were assessed using confocal microscopy. ResultsAAV2, AAV5, and lentivirus showed minimal transduction, with only sparse GFP-positive cells observed near injection sites. In contrast, AAV-PHP.eB carrying the CAG promoter yielded robust and widespread GFP expression near the injection site. Quantitative analysis revealed that approximately 90% of transduced cells were Olig2-positive oligodendrocytes, indicating strong tropism for this glial population. ConclusionAAV-PHP.eB driven by the CAG promoter enables efficient gene delivery to the optic nerve, with a predominant tropism for oligodendrocytes. This targeted intra-optic nerve injection approach offers a reliable platform for manipulating oligodendrocytes and investigating mechanisms of CNS development, injury, and repair relevant to both optic neuropathies and other CNS diseases.

Among mammals, spiny mice (Acomys spp.) exhibit the unique capacity to regenerate parts of their nervous system. Studying this phenomenon has the potential to reveal new targets that can slow or halt human neurodegenerative disorders. Unfortunately, research tools (e.g., transgenic lines, gene delivery vehicles) are lacking compared to those available for other rodent models. Here, we tested systemic adeno-associated viral vectors (AAVs) in Acomys dimidiatus and identified three promising candidates: X1.1, CAP-Mac, and MaCPNS1. Characterizing their tropism following intravenous delivery, we found that in the brain, MaCPNS1 and X1.1 primarily transduced astrocytes. In the peripheral nervous system, MaCPNS1 efficiently transduced dorsal root ganglia, axon bundles of the ear pinnae, and enteric neurons throughout the gastrointestinal tract. As a proof-of-concept, we used MaCPNS1 to chemogenetically modulate the activity of enteric neurons, successfully decreasing gastric motility in vivo and increasing colonic motility ex vivo. We expect these findings to enable functional studies of the uniquely regenerative nervous system of Acomys, which may in turn help advance neuroregenerative therapeutics for humans. Summary StatementIdentification of an AAV tool to efficiently deliver transgenes to the central and peripheral nervous systems of spiny mice enables functional studies of the nervous system in a mammalian model of regeneration.

IntroductionVagus nerve stimulation modulates laryngeal, cardiac, pulmonary, and gastrointestinal functions. Knowledge of where along the vagal trunk organ-specific branches emerge may support alternative surgical placements of stimulation devices to engage targeted functions while avoiding off-target effects. However, no quantified map of how vagal branches emerge and how they relate to surgically relevant anatomical landmarks exists in humans. MethodsFifty-eight vagus nerves (29 left, 29 right) from 29 embalmed donor bodies (15 females) were dissected from the jugular foramen through the thoracic cavity. Branches were traced to end organs and allocated to seven groups -- sympathetic, muscular, vascular, cardiac, pulmonary, esophageal, and multiple targets -- and several sub-groups. Distances between branch emergence and the jugular foramen (JF) were normalized to three anatomical landmarks: carotid bifurcation, laryngeal prominence, and superior border of clavicle. ResultsBranch emergence follows a proximal-to-distal order: sympathetic (5.28 cm from JF), muscular (9.59 cm), vascular (10.70 cm), cardiac (19.65 cm), pulmonary (25.36 cm), and esophageal (26.57 cm). Vagal branches emerge into two embryological domains separated near the clavicle: pharyngeal arch-targeting branches cluster proximally (9.34 cm) and primitive mediastinum-targeting branches cluster distally (23.74 cm), with sympathetic, muscular, and vascular sub-groups occupying distinct zones within the proximal domain. The largest branch-free intervals occur above the left clavicle (2.33 {+/-} 2.80 cm) and below the left carotid bifurcation (2.58 {+/-} 3.17 cm). Alternate placement regions separating targeted organs from off-targets: sympathetic vs. cervical visceral at 6/8 cm (L/R), cardiac vs. carotid sinus/bifurcation at 14/10 cm, and recurrent laryngeal vs. other cervical visceral at 18/13 cm from JF. Overall, no differences were found between male and female donors. ConclusionsThis study provides a quantified, landmark-registered map of cervical and thoracic vagal branch emergence, offering a standardized anatomical template that may inform strategies for more function-selective vagal neuromodulation.

Phagocytic and immune-like cells have been observed in the satellite envelope of neuronal somata in peripheral sensory ganglia of many species for several decades. These cells likely play an important role in normal function of sensory neurons and they may also play an important role in neuronal dysfunction and neurodegeneration seen with neuropathy. Recent findings have described a satellite macrophage population transcriptomically similar to microglia in peripheral ganglia of some mammalian species. The function of these cells, and the mechanisms by which they may influence neurons in neuropathy are unclear. We sought to understand the phenotype and localization of these cells in the human dorsal root ganglion (hDRG) using large-scale single nucleus and spatial transcriptomic datasets from individuals with and without a history of peripheral diabetic neuropathy. We observed a large population of macrophages that express classical microglia makers such as TMEM119 and P2RY12 in the hDRG, as previously described. Our findings confirm that these microglia-like cells (MLCs) localize to the satellite envelope around neuronal somata, yet are transcriptomically distinct from all glial cell types characterized in the hDRG. These MLCs exhibit changes in abundance and localization with diabetic painful neuropathy (DPN) in both the hDRG and sural nerves suggesting that they are not exclusively localized to the DRG. We conclude that microglia-like cells are likely the resident tissue macrophage (RTM) of the hDRG, and perhaps the peripheral nervous system (PNS) given their localization to the sural nerve and other ganglia, where they are predicted to regulate homeostatic neuronal functions and response to injury. HighlightsO_LIMLCs are likely the RTM of hDRGs C_LIO_LIMLCs localize to the satellite envelope and recede with Nageotte nodule formation C_LIO_LIMLC activation state and signaling shift with diabetic neuropathy C_LIO_LIMLCs are also present in other ganglia and sural nerve C_LI