Ependymal Research Articles

TMEM106B amyloid filaments in the Biondi bodies of ependymal cells

Biondi bodies are filamentous amyloid inclusions of unknown composition in ependymal cells of the choroid plexuses, ependymal cells lining cerebral ventricles and ependymal cells of the central canal of the spinal cord. Their formation is age-dependent and they are commonly associated with a variety of neurodegenerative conditions, including Alzheimer’s disease and Lewy body disorders. Here, we show that Biondi bodies are strongly immunoreactive with TMEM239, an antibody specific for inclusions of transmembrane protein 106B (TMEM106B). Biondi bodies were labelled by both this antibody and the amyloid dye pFTAA. Many Biondi bodies were also labelled for TMEM106B and the lysosomal markers Hexosaminidase A and Cathepsin D. By transmission immuno-electron microscopy, Biondi bodies of choroid plexuses were decorated by TMEM239 and were associated with structures that resembled residual bodies or secondary lysosomes. By electron cryo-microscopy, TMEM106B filaments from Biondi bodies of choroid plexuses were similar (Biondi variant), but not identical, to the fold I that was previously identified in filaments from brain parenchyma.

YAP Signaling in Glia: Pivotal Roles in Neurological Development, Regeneration and Diseases.

Yes-associated protein (YAP), the key transcriptional co-factor and downstream effector of the Hippo pathway, has emerged as one of the primary regulators of neural as well as glial cells. It has been detected in various glial cell types, including Schwann cells and olfactory ensheathing cells in the peripheral nervous system, as well as radial glial cells, ependymal cells, Bergmann glia, retinal Müller cells, astrocytes, oligodendrocytes, and microglia in the central nervous system. With the development of neuroscience, understanding the functions of YAP in the physiological or pathological processes of glia is advancing. In this review, we aim to summarize the roles and underlying mechanisms of YAP in glia and glia-related neurological diseases in an integrated perspective.

Synergistic effects of curcumin and stem cells on spinal cord injury: a comprehensive review.

Spinal cord injury (SCI) is damage to the spinal cord that permanently or temporarily disrupts its function, causing considerable autonomic, sensory, and motor disorders, and involves between 10 and 83 cases per million yearly. Traumatic SCI happens following primary acute mechanical damage, leading to injury to the spinal cord tissue and worsening clinical outcomes. The present therapeutic strategies for this complex disease fundamentally rely on surgical approaches and conservative remedies. However, these modalities are not effective enough for neurological recovery. Therefore, it is necessary to discover more efficient methods to treat patients with SCI. Today, considerable attention has been drawn to bioactive compounds-based remedies and stem cell therapy for curing various ailments and disorders, such as neurological diseases. Some researchers have recommended that harnessing curcumin, a polyphenol obtained from turmeric, in combination with stem cells, like mesenchymal stem cells, neural stem cells, and ependymal stem cells, can remarkably improve neurological recovery-related parameters more effective than the treatment with these two methods separately in experimental models. Hereby, this literature review delves into the functionality of curcumin combined with stem cells in treating SCI with a focus on cellular and molecular mechanisms.

Comparative transcriptional analyses of the striatum in the chronic social defeat stress model in C57BL/6J male mice and the gut microbiota-dysbiosis model in Kumming mice

Depression is a complex disorder with multiple contributing factors, and chronic stress has previously been recognized as a major causative factor, while gut microbes have also been found to be involved in depression recently. However, gene expression in depression models with different etiologies is unclear. Here, we compared the transcriptomes of the striatum in chronic social defeat stress (CSDS) model of C57BL/6J male mice and fecal microbiota transplant (FMT) model of Kumming male mice. We found that the proportion of shared differentially expressed genes (DEGs) between the two models was only 24 %. The specific DEGs of FMT model were enriched in immune and inflammatory, and are associated with changes in vascular and ciliated ependymal cells. The specific DEGs of CSDS model were enriched in neuron and synapse. The results of network analysis suggested the expression patterns and biological function of depressive-like behaviors-related modules in the two models are different. Further, the alternative splicing events of CSDS are more than FMT. Our results suggested models of depression induced by different etiologies differ significantly in gene expression and biological function. Our study also suggested us to pay attention to the characteristics of models of depression of different etiologies and provided a more comprehensive understanding of the heterogeneity of depression.

The periaxonal space as a conduit for cerebrospinal fluid flow to peripheral organs

Mechanisms controlling the movement of the cerebrospinal fluid (CSF) toward peripheral nerves are poorly characterized. We found that, in addition to the foramina Magendie and Luschka for CSF flow toward the subarachnoid space and glymphatic system, CSF outflow could also occur along periaxonal spaces (termed "PAS pathway") from the spinal cord to peripheral organs, such as the liver and pancreas. When interrogating the latter route, we found that serotonin, acting through 5-HT2B receptors expressed in ependymocytes that line the central canal, triggered Ca2+ signals to induce polymerization of F-actin, a cytoskeletal protein, to reduce the volume of ependymal cells. This paralleled an increased rate of PAS-mediated CSF redistribution toward peripheral organs. In the liver, CSF was received by hepatic stellate cells. CSF efflux toward peripheral organs through the PAS pathway represents a mechanism dynamically connecting the nervous system with the periphery. Our findings are compatible with the traditional theory of CSF efflux into the glymphatic system to clear metabolic waste from the cerebral parenchyma. Thus, we extend the knowledge of CSF flow and expand the understanding of connectivity between the CNS and peripheral organs.

Pathological Study of Demyelination with Cellular Reactions in the Cerebellum of Dogs Infected with Canine Distemper Virus

The purpose of this study was to examine the relationship between demyelination and cellular reactions in the cerebellum of Canine Distemper Virus (CDV)-infected dogs. We subdivided the disease staging by adding the degree of demyelination determined by Luxol Fast Blue staining to the previously reported disease staging from the acute stage to the chronic stage, and investigated the relationship between demyelination in the cerebellum and the number and histological changes in astroglia, microglia, and Purkinje cells in each stage. Reactions of astrocytes and microglia were observed at an early stage when demyelination was not evident. Changes progressed with demyelination. Demyelination initially began in the medulla adjoining the fourth ventricle and gradually spread to the entire cerebellum, including the lobes. CDV immune-positive granules were seen from the early stage, and inclusion bodies also appeared at the same time. CDV immune-positive reaction and inclusion bodies were observed in astrocytes, microglia, neurons, ependymal cells, and even leptomeningeal mononuclear cells. On the other hand, infiltration of CDV-immunoreactive particles from the pia mater to the gray matter and further into the white matter through the granular layer was observed from an early stage. Purkinje cells decreased from the intermediate stage, and a decrease in cells in the granular layer was also observed. There was no clear association between age and each stage, and the stages did not progress with age.

Ependymal cell lineage reprogramming as a potential therapeutic intervention for hydrocephalus.

Hydrocephalus is a common neurological condition, characterized by the excessive accumulation of cerebrospinal fluid in the cerebral ventricles. Primary treatments for hydrocephalus mainly involve neurosurgical cerebrospinal fluid diversion, which hold high morbidity and failure rates, highlighting the necessity for the discovery of novel therapeutic approaches. Although the pathophysiology of hydrocephalus is highly multifactorial, impaired function of the brain ependymal cells plays a fundamental role in hydrocephalus. Here we show that GemC1 and McIdas, key regulators of multiciliated ependymal cell fate determination, induce direct cellular reprogramming towards ependyma. Our study reveals that ectopic expression of GemC1 and McIdas reprograms cortical astrocytes and programs mouse embryonic stem cells into ependyma. McIdas is sufficient to establish functional activity in the reprogrammed astrocytes. Furthermore, we show that McIdas' expression promotes ependymal cell regeneration in two different postnatal hydrocephalus mouse models: an intracranial hemorrhage and a genetic form of hydrocephalus and ameliorates the cytoarchitecture of the neurogenic niche. Our study provides evidence on the restoration of ependyma in animal models mimicking hydrocephalus that could be exploited towards future therapeutic interventions.

Using Zebrafish to Study Multiciliated Cell Development and Disease States

Multiciliated cells (MCCs) serve many important functions, including fluid propulsion and chemo- and mechanosensing. Diseases ranging from rare conditions to the recent COVID-19 global health pandemic have been linked to MCC defects. In recent years, the zebrafish has emerged as a model to investigate the biology of MCCs. Here, we review the major events in MCC formation including centriole biogenesis and basal body docking. Then, we discuss studies on the role of MCCs in diseases of the brain, respiratory, kidney and reproductive systems, as well as recent findings about the link between MCCs and SARS-CoV-2. Next, we explore why the zebrafish is a useful model to study MCCs and provide a comprehensive overview of previous studies of genetic components essential for MCC development and motility across three major tissues in the zebrafish: the pronephros, brain ependymal cells and nasal placode. Taken together, here we provide a cohesive summary of MCC research using the zebrafish and its future potential for expanding our understanding of MCC-related disease states.

RGS22 maintains the physiological function of ependymal cells to prevent hydrocephalus.

Ependymal cells line the wall of cerebral ventricles and ensure the unidirectional cerebrospinal fluid (CSF) flow by beating their motile cilia coordinately. The ependymal denudation or ciliary dysfunction causes hydrocephalus. Here, we report that the deficiency of regulator of G-protein signaling 22 (RGS22) results in severe congenital hydrocephalus in both mice and rats. Interestingly, RGS22 is specifically expressed in ependymal cells within the brain. Using conditional knock-out mice, we further demonstrate that the deletion of Rgs22 exclusively in nervous system is sufficient to induce hydrocephalus. Mechanistically, we show that Rgs22 deficiency leads to the ependymal denudation and impaired ciliogenesis. This phenomenon can be attributed to the excessive activation of lysophosphatidic acid receptor (LPAR) signaling under Rgs22-/- condition, as the LPAR blockade effectively alleviates hydrocephalus in Rgs22-/- rats. Therefore, our findings unveil a previously unrecognized role of RGS22 in the central nervous system, and present RGS22 as a potential diagnostic and therapeutic target for hydrocephalus.

Treatment Strategies for Unknown Anatomic Pathology Ventriculus Terminalis Case Series: Single Center Experience

The ventriculus terminalis is a cavity in the conus medullaris, bounded by ependymal cells, associated with the central canal. It is an anatomical structure that is very rare in adults, with a limited number of surgical cases that have been reported in the literature. In children, it is regarded as a normal congenital variation, known to regress before five years of age, and very few symptomatic cases have been reported in both pediatric and adult populations. It is often asymptomatic in adults and is detected incidentally. Although potentially nonsignificant individually, symptoms can range from nonspecific low back pain to sphincter dysfunction and focal neurologic deficits. Our purpose is to discuss our management strategy in comparison to the existing literature. A retrospective review was conducted of all adult patients (aged 17 years and older) diagnosed with ventriculus terminalis who were referred to the hospital between 2010 and 2020. Clinical classification was made according to the classification defined by Batista. In addition, Ganau's classification was also used. Five patients were included in the study. The majority of these patients (n=4, 80%) were symptomatic at the time of diagnosis, with nonspecific back pain being the most common symptom (n=3, (60%). None of the patients required neurosurgical intervention during the follow-up period of 21.6±8.9 months, as there was no clinical deterioration observed. Ventriculus terminalis is a rare pathology that may develop de novo in adults, often remaining undiagnosed until the cyst enlarges, and can manifest with a wide spectrum of symptoms. When identified, it requires careful management, involving surgery when necessary and a conservative approach when appropriate.

A cholinergic signaling pathway underlying cortical circuit activation of quiescent neural stem cells in the lateral ventricle.

Neural stem cells (NSCs) in the subventricular zone (SVZ) located along the lateral ventricles (LVs) of the mammalian brain continue to self-renew to produce new neurons after birth and into adulthood. Quiescent LV cells, which are situated close to the ependymal cells lining the LVs, are activated by choline acetyltransferase-positive (ChAT+) neurons within the subependymal (subep) region of the SVZ when these neurons are stimulated by projections from the anterior cingulate cortex (ACC). Here, we uncovered a signaling pathway activated by the ACC-subep-ChAT+ circuit responsible for the activation and proliferation of quiescent LV NSCs specifically in the ventral area of the SVZ. This circuit activated muscarinic M3 receptors on quiescent LV NSCs, which subsequently induced signaling mediated by the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1). Downstream of IP3R1 activation, which would be expected to increase intracellular Ca2+, Ca2+-/calmodulin-dependent protein kinase II δ and the MAPK10 signaling pathway were stimulated and required for the proliferation of quiescent LV NSCs in the SVZ. These findings reveal the mechanisms that regulate quiescent LV NSCs and underscore the critical role of projections from the ACC in promoting their proliferative activity within the ventral SVZ.

Endogenous opioid signalling regulates spinal ependymal cell proliferation.

After injury, mammalian spinal cords develop scars to confine the lesion and prevent further damage. However, excessive scarring can hinder neural regeneration and functional recovery1,2. These competing actions underscore the importance of developing therapeutic strategies to dynamically modulate scar progression. Previous research on scarring has primarily focused on astrocytes, but recent evidence has suggested that ependymal cells also participate. Ependymal cells normally form the epithelial layer encasing the central canal, but they undergo massive proliferation and differentiation into astroglia following certain injuries, becoming a core scar component3-7. However, the mechanisms regulating ependymal proliferation in vivo remain unclear. Here we uncover an endogenous κ-opioid signalling pathway that controls ependymal proliferation. Specifically, we detect expression of the κ-opioid receptor, OPRK1, in a functionally under-characterized cell type known as cerebrospinal fluid-contacting neuron (CSF-cN). We also discover a neighbouring cell population that expresses the cognate ligand prodynorphin (PDYN). Whereas κ-opioids are typically considered inhibitory, they excite CSF-cNs to inhibit ependymal proliferation. Systemic administration of a κ-antagonist enhances ependymal proliferation in uninjured spinal cords in a CSF-cN-dependent manner. Moreover, a κ-agonist impairs ependymal proliferation, scar formation and motor function following injury. Together, our data suggest a paracrine signalling pathway in which PDYN+ cells tonically release κ-opioids to stimulate CSF-cNs and suppress ependymal proliferation, revealing an endogenous mechanism and potential pharmacological strategy for modulating scarring after spinal cord injury.

Multiciliated ependymal cells: an update on biology and pathology in the adult brain.

Mature multiciliated ependymal cells line the cerebral ventricles where they form a partial barrier between the cerebrospinal fluid (CSF) and brain parenchyma and regulate local CSF microcirculation through coordinated ciliary beating. Although the ependyma is a highly specialized brain interface with barrier, trophic, and perhaps even regenerative capacity, it remains a misfit in the canon of glial neurobiology. We provide an update to seminal reviews in the field by conducting a scoping review of the post-2010 mature multiciliated ependymal cell literature. We delineate how recent findings have either called into question or substantiated classical views of the ependymal cell. Beyond this synthesis, we document the basic methodologies and study characteristics used to describe multiciliated ependymal cells since 1980. Our review serves as a comprehensive resource for future investigations of mature multiciliated ependymal cells.

Vitamin D Deficiency Does Not Affect Cognition and Neurogenesis in Adult C57Bl/6 Mice.

Vitamin D deficiency is a global problem. Vitamin D, the vitamin D receptor, and its enzymes are found throughout neuronal, ependymal, and glial cells in the brain and are implicated in certain processes and mechanisms in the brain. To investigate the processes affected by vitamin D deficiency in adults, we studied vitamin D deficient, control, and supplemented diets over 6 weeks in male and female C57Bl/6 mice. The effect of the vitamin D diets on proliferation in the neurogenic niches, changes in glial cells, as well as on memory, locomotion, and anxiety-like behavior, was investigated. Six weeks on a deficient diet was adequate time to reach deficiency. However, vitamin D deficiency and supplementation did not affect proliferation, neurogenesis, or astrocyte changes, and this was reflected on behavioral measures. Supplementation only affected microglia in the dentate gyrus of female mice. Indicating that vitamin D deficiency and supplementation do not affect these processes over a 6-week period.

Abstract MP05: A Preliminary 10X Genomics Xenium Profiler Spatial Transcriptomics Map of Renin-Angiotensin System Genes in the Mouse Subfornical Organ

The subfornical organ (SFO) is a brain region rich in angiotensin-II (ANG) AT1 receptors (AT1R) which regulates water and salt intake and communicates with other hypothalamic nuclei to regulate blood pressure (BP). Activation of the classical G protein-mediated AT1R signaling in the brain induces pressor and natri-dipsogenic responses. TRV027, a synthetic AT1R β-arrestin-biased agonist, decreases BP and increases saline avoidance. We examined the spatial transcriptomic makeup of the SFO to balanced or biased AT1R activation using 10X Genomics Xenium. We treated C57BL/6J mice with either aCSF, ANG (0.64 µg/h), or TRV027 (0.56 µg/h) through continuous ICV infusion for 11-days. Water intake was monitored on days 6-11. ANG caused an increase in water intake compared to aCSF or TRV027 groups (7.0±2.0, 3.0±0.2, and 1.9±0.6 mL/day, respectively, n=4, p<0.05). On day 11, brains were perfused, collected, and fixed. Subsequently, 5 µm paraffin-embedded coronal sections were mounted on Xenium slides and tested with the 10X mouse brain set of 247 genes plus 100 custom add-on genes including all RAS genes. A third of the cells expressed AT1R, and the pattern of AT1R-expressing cells Xenium-detected was qualitatively similar to NZ44 mice, which express GFP under the control of the AT1R locus. Expression of the prorenin receptor ( Atp6ap2 ) was abundant throughout the SFO. A few cells in the SFO and more prominently in the adjacent blood vessels were angiotensinogen positive. Single cells were identified that expressed the ANG type 2 receptors (AT2R). Expression of Mas receptor was undetectable. Most of the AT1R-expressing cells were excitatory glutamatergic neurons (expressing Slc17a6 , VGLUT2) but a few were inhibitory GABAergic neurons co-expressing Slc32a1 (VGAT). Each section had cells classified as astrocytes, neurons, ependymal cells, and immune cells (e.g., microglia or macrophages). Increased numbers of immune cells were detected in the SFO of ANG-treated mice. Interestingly, although there was no evidence for expression of renin in the SFO, renin-expressing cells were identified in the choroid plexus which is consistent with highly sensitive in situ RNA hybridization data. Additional qualitative and quantitative analyses are ongoing to compare gene expression patterns in AT1R-positive cell types. Future studies will identify gene expression patterns in SFO neurons with connectivity to other brain regions controlling water/salt intake and BP.

A high-fat diet influences neural stem and progenitor cell environment in the medulla of adult mice

It has been widely established that neural stem cells (NSCs) exist in the adult mammalian brain. The area postrema (AP) and the ependymal cell layer of the central canal (CC) in the medulla were recently identified as NSC niches. There are two types of NSCs: astrocyte-like cells in the AP and tanycyte-like cells in the CC. However, limited information is currently available on the characteristics and functional significance of these NSCs and their progeny in the AP and CC. The AP is a part of the dorsal vagal complex (DVC), together with the nucleus of the solitary tract (Sol) and the dorsal motor nucleus of the vagus (10 N). DVC is the primary site for the integration of visceral neuronal and hormonal cues that act to inhibit food intake. Therefore, we examined the effects of high-fat diet (HFD) on NSCs and progenitor cells in the AP and CC. Eight-week-old male mice were fed HFD for short (1 week) and long periods (4 weeks). To detect proliferating cells, mice consecutively received intraperitoneal injections of BrdU for 7 days. Brain sections were processed with immunohistochemistry using various cell markers and BrdU antibodies. Our data demonstrated that adult NSCs and neural progenitor cells (NPCs) in the medulla responded more strongly to short-term HFD than to long-term HFD. HFD increased astrocyte density in the Sol and 10 N, and increased microglial/macrophage density in the AP and Sol. Furthermore, long-term HFD induced mild inflammation in the medulla, suggesting that it affected the proliferation of NSCs and NPCs.

Volumetric Changes in the Choroid Plexus Associated with Spontaneous Intracranial Hypotension in Patients with Spinal CSF Leak.

The choroid plexus contains specialized ependymal cells responsible for CSF production. Recent studies have demonstrated volumetric and perfusion changes in the choroid plexus with age and neurodegenerative disorders, however, volumetric changes in the choroid plexus in low pressure states is not known. The purpose of this study is to evaluate volumetric differences in choroid plexus size in patients with spontaneous intracranial hypotension (SIH) resultant from spinal CSF leaks compared with healthy controls. This was a retrospective, institutional review board-approved study. Patients with MRI evidence of SIH and a spinal CSF leak diagnosed on myelography and subsequently confirmed at surgery were included in this study. All patients included in this study including age-matched healthy controls had a brain MRI performed on a either a 1.5 or 3T scanner with acquisition of 3D T1 postcontrast (eg, BRAVO, MPRAGE, etc). In all patients, the trigonum ventriculi volume, in the atria of the lateral ventricles, was contoured by using Visage-7 segmentation tools on the volumetric postcontrast T1 sequence. A basic 2-tailed t test was used to compare choroid plexus volumes between the 2 groups. Thirty-four patients were included with 17 patients with SIH with spinal CSF leak and 17 healthy control patients who were age- and sex-matched. The mean age of patients was 45 years, standard deviation 14 years. The mean volume of the choroid plexus for patients with SIH with spinal CSF leak was 1.2 cm3 (standard deviation = 0.26) compared with 0.63 cm3 (standard deviation = 0.31) in the control group (P < .0001). Results of this study demonstrate a higher choroid plexus volume in patients with SIH with spinal CSF leak compared with age- and sex-matched healthy controls. This likely reflects compensatory mechanisms to counteract intracranial hypotension by increasing CSF production as well as increased vascularity of the choroid plexus through expansion of the intracranial blood pool.

Fasting induces metabolic switches and spatial redistributions of lipid processing and neuronal interactions in tanycytes

The ependyma lining the third ventricle (3V) in the mediobasal hypothalamus plays a crucial role in energy balance and glucose homeostasis. It is characterized by a high functional heterogeneity and plasticity, but the underlying molecular mechanisms governing its features are not fully understood. Here, 5481 hypothalamic ependymocytes were cataloged using FACS-assisted scRNAseq from fed, 12h-fasted, and 24h-fasted adult male mice. With standard clustering analysis, typical ependymal cells and β2-tanycytes appear sharply defined, but other subpopulations, β1- and α-tanycytes, display fuzzy boundaries with few or no specific markers. Pseudospatial approaches, based on the 3V neuroanatomical distribution, enable the identification of specific versus shared tanycyte markers and subgroup-specific versus general tanycyte functions. We show that fasting dynamically shifts gene expression patterns along the 3V, leading to a spatial redistribution of cell type-specific responses. Altogether, we show that changes in energy status induce metabolic and functional switches in tanycyte subpopulations, providing insights into molecular and functional diversity and plasticity within the tanycyte population.

Absence of Aquaporin-4 (AQP4) Prolongs the Presence of a CD11c+ Microglial Population during Postnatal Corpus Callosum Development.

Aquaporin-4 (AQP4) expression is associated with the development of congenital hydrocephalus due to its structural role in the ependymal membrane. Gene expression analysis of periaqueductal tissue in AQP4-knockout (KO) mice at 11 days of age (P11) showed a modification in ependymal cell adhesion and ciliary protein expression that could alter cerebrospinal fluid homeostasis. A microglial subpopulation of CD11c+ cells was overexpressed in the periaqueductal tissue of mice that did not develop hydrocephalus, suggesting a possible protective effect. Here, we verified the location of this CD11c+ expression in the corpus callosum (CC) and cerebellum of AQP4-KO mice and analysed its time course. Immunofluorescence labelling of the CD11c protein in the CC and cerebellum of WT and KO animals at P3, P5, P7 and P11 confirmed an expanded presence of these cells in both tissues of the KO animal; CD11c+ cells appeared at P3 and reached a peak at P11, whereas in the WT animal, they appeared at P5, reached their peak at P7 and were undetectable by P11. The gene expression analysis in the CC samples at P11 confirmed the presence of CD11c+ microglial cells in this tissue. Among the more than 4000 overexpressed genes, Spp1 stood out with the highest differential gene expression (≅600), with other genes, such as Gpnmb, Itgax, Cd68 and Atp6v0d2, also identified as overexpressed. Therefore, CD11c+ cells appear to be necessary for normal corpus callosum development during postnatal life, and the absence of AQP4 prolonged its expression in this tissue.

The central oxytocinergic system of the prairie vole

Oxytocin (OXT) is a peptide hormone and a neuropeptide that regulates various peripheral physiological processes and modulates behavioral responses in the central nervous system. While the humoral release occurs from the axons arriving at the median eminence, the neuropeptide is also released from oxytocinergic cell axons in various brain structures that contain its receptor, and from their dendrites in hypothalamic nuclei and potentially into the cerebrospinal fluid (CSF). Understanding oxytocin’s complex functions requires the knowledge on patterns of oxytocinergic projections in relationship to its receptor (OXTR). This study provides the first comprehensive examination of the oxytocinergic system in the prairie vole (Microtus ochrogaster), an animal exhibiting social behaviors that mirror human social behaviors linked to oxytocinergic functioning. Using light and electron microscopy, we characterized the neuroanatomy of the oxytocinergic system in this species. OXT+ cell bodies were found primarily in the hypothalamus, and axons were densest in subcortical regions. Examination of the OXT+ fibers and their relationship to oxytocin receptor transcripts (Oxtr) revealed that except for some subcortical structures, the presence of axons was not correlated with the amount of Oxtr across the brain. Of particular interest, the cerebral cortex that had high expression of Oxtr transcripts contained little to no fibers. Electron microscopy is used to quantify dense cored vesicles (DCV) in OXT+ axons and to identify potential axonal release sites. The ependymal cells that line the ventricles were frequently permissive of DCV-containing OXT+ dendrites reaching the third ventricle. Our results highlight a mechanism in which oxytocin is released directly into the ventricles and circulates throughout the ventricular system, may serve as the primary source for oxytocin that binds to OXTR in the cerebral cortex.