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 ß2-tanycytes appear sharply defined, but other subpopulations, ß1- 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.

Cell proliferation and glial cell marker expression in the wall of the third ventricle in the tuberal region of the male mouse hypothalamus during postnatal development.

The third ventricle (3 V) wall of the tuberal hypothalamus is composed of two types of cells; specialized ependymoglial cells called tanycytes located ventrally and ependymocytes dorsally, which control the exchanges between the cerebrospinal fluid and the hypothalamic parenchyma. By regulating the dialogue between the brain and the periphery, tanycytes are now recognized as central players in the control of major hypothalamic functions such as energy metabolism and reproduction. While our knowledge of the biology of adult tanycytes is progressing rapidly, our understanding of their development remains very incomplete. To gain insight into the postnatal maturation of the 3 V ependymal lining, we conducted a comprehensive immunofluorescent study of the mouse tuberal region at four postnatal ages (postnatal day (P) 0, P4, P10, and P20). We analyzed the expression profile of a panel of tanycyte and ependymocyte markers (vimentin, S100, connexin-43 [Cx43], and glial fibrillary acidic protein [GFAP]) and characterized cell proliferation in the 3 V wall using the thymidine analog bromodeoxyuridine. Our results show that most changes in marker expression occur between P4 and P10, with a switch from a 3 V mostly lined by radial cells to the emergence of a tanycytic domain ventrally and an ependymocytic domain dorsally, a drop in cell proliferation and increased expression of S100, Cx43, and GFAP that acquire a mature profile at P20. Our study thus identifies the transition between the first and the second postnatal week as a critical time window for the postnatal maturation of the 3 V wall ependymal lining.

Analysis of a cell niche with proliferative potential at the roof of the aqueduct of Sylvius.

The aqueduct of Sylvius connects the third with the fourth ventricle and is surrounded by the Periaqueductal Grey. Here, we report a novel niche of cells in the dorsal section of the aqueduct, hereby named dorsal aqueduct niche or DAN, by applying a battery of selective markers and transgenic mouse lines. The somata of DAN cells are located toward the lumen of the ventricle forming multiple layers in close association with the cerebrospinal fluid (CSF). A single process emerges from the soma and run with the blood vessels. Cells of the DAN express radial glia/stem cell markers such as GFAP, vimentin and nestin, and the glutamate transporter GLAST or the oligodendrocyte precursor/pericyte marker NG2, thereby suggesting their potential for the generation of new cells. Morphologically, DAN cells resemble tanycytes of the third ventricle, which transfer biochemical signals from the CSF to the central nervous system and display proliferative capacity. The aqueduct ependymal lining can proliferate as observed by the integration of BrdU and expression of Ki67. Thus, the dorsal section of the aqueduct of Sylvius possesses cells that may act a niche of new glial cells in the adult mouse brain.

Developmental genes controlling neural circuit formation are expressed in the early postnatal hypothalamus and cellular lining of the third ventricle.

The arcuate nucleus of the hypothalamus is central in the regulation of body weight homeostasis through its ability to sense peripheral metabolic signals and relay them, through neural circuits, to other brain areas, ultimately affecting physiological and behavioural changes. The early postnatal development of these neural circuits is critical for normal body weight homeostasis, such that perturbations during this critical period can lead to obesity. The role for peripheral regulators of body weight homeostasis, including leptin, insulin and ghrelin, in this postnatal development is well described, yet some of the fundamental processes underpinning axonal and dendritic growth remain unclear. Here, we hypothesised that molecules known to regulate axonal and dendritic growth processes in other areas of the developing brain would be expressed in the postnatal arcuate nucleus and/or target nuclei where they would function to mediate the development of this circuitry. Using state-of-the-art RNAscope® technology, we have revealed the expression patterns of genes encoding Dcc/Netrin-1, Robo1/Slit1 and Fzd5/Wnt5a receptor/ligand pairs in the early postnatal mouse hypothalamus. We found that individual genes had unique expression patterns across developmental time in the arcuate nucleus, paraventricular nucleus of the hypothalamus, ventromedial nucleus of the hypothalamus, dorsomedial nucleus of the hypothalamus, median eminence and, somewhat unexpectedly, the third ventricle epithelium. These observations indicate a number of new molecular players in the development of neural circuits regulating body weight homeostasis, as well as novel molecular markers of tanycyte heterogeneity.

The Lactate Receptor HCA1 Is Present in the Choroid Plexus, the Tela Choroidea, and the Neuroepithelial Lining of the Dorsal Part of the Third Ventricle.

The volume, composition, and movement of the cerebrospinal fluid (CSF) are important for brain physiology, pathology, and diagnostics. Nevertheless, few studies have focused on the main structure that produces CSF, the choroid plexus (CP). Due to the presence of monocarboxylate transporters (MCTs) in the CP, changes in blood and brain lactate levels are reflected in the CSF. A lactate receptor, the hydroxycarboxylic acid receptor 1 (HCA1), is present in the brain, but whether it is located in the CP or in other periventricular structures has not been studied. Here, we investigated the distribution of HCA1 in the cerebral ventricular system using monomeric red fluorescent protein (mRFP)-HCA1 reporter mice. The reporter signal was only detected in the dorsal part of the third ventricle, where strong mRFP-HCA1 labeling was present in cells of the CP, the tela choroidea, and the neuroepithelial ventricular lining. Co-labeling experiments identified these cells as fibroblasts (in the CP, the tela choroidea, and the ventricle lining) and ependymal cells (in the tela choroidea and the ventricle lining). Our data suggest that the HCA1-containing fibroblasts and ependymal cells have the ability to respond to alterations in CSF lactate in body-brain signaling, but also as a sign of neuropathology (e.g., stroke and Alzheimer's disease biomarker).

Third Ventricular Injection of CCL2 in Rat Embryo Stimulates CCL2/CCR2 Neuroimmune System in Neuroepithelial Radial Glia Progenitor Cells: Relation to Sexually Dimorphic, Stimulatory Effects on Peptide Neurons in Lateral Hypothalamus.

Clinical and animal studies show maternal alcohol consumption during pregnancy causes in offspring persistent alterations in neuroimmune and neurochemical systems known to increase alcohol drinking and related behaviors. Studies in lateral hypothalamus (LH) demonstrate in adolescent offspring that maternal oral administration of ethanol stimulates the neuropeptide, melanin-concentrating hormone (MCH), together with the inflammatory chemokine C-C motif ligand 2 (CCL2) and its receptor CCR2 which are increased in most MCH neurons. These effects, consistently stronger in females than males, are detected in embryos, not only in LH but hypothalamic neuroepithelium (NEP) along the third ventricle where neurons are born and CCL2 is stimulated within radial glia progenitor cells and their laterally projecting processes that facilitate MCH neuronal migration toward LH. With ethanol's effects similarly produced by maternal peripheral CCL2 administration and blocked by CCR2 antagonist, we tested here using in utero intracerebroventricular (ICV) injections whether CCL2 acts locally within the embryonic NEP. After ICV injection of CCL2 (0.1 µg/µl) on embryonic day 14 (E14) when neurogenesis peaks, we observed in embryos just before birth (E19) a significant increase in endogenous CCL2 within radial glia cells and their processes in NEP. These auto-regulatory effects, evident only in female embryos, were accompanied by increased density of CCL2 and MCH neurons in LH, more strongly in females than males. These results support involvement of embryonic CCL2/CCR2 neuroimmune system in radial glia progenitor cells in mediating sexually dimorphic effects of maternal challenges such as ethanol on LH MCH neurons that colocalize CCL2 and CCR2.

Isolation and characterization of cluster of differentiation 9-positive ependymal cells as potential adult neural stem/progenitor cells in the third ventricle of adult rats.

Ependymal cells located above the ventricular zone of the lateral, third, and fourth ventricles and the spinal cord are thought to form part of the adult neurogenic niche. Many studies have focused on ependymal cells as potential adult neural stem/progenitor cells. To investigate the functions of ependymal cells, a simple method to isolate subtypes is needed. Accordingly, in this study, we evaluated the expression of cluster of differentiation (CD) 9 in ependymal cells by in situ hybridization and immunohistochemistry. Our results showed that CD9-positive ependymal cells were also immunopositive for SRY-box 2, a stem/progenitor cell marker. We then isolated CD9-positive ependymal cells from the third ventricle using the pluriBead-cascade cell isolation system based on antibody-mediated binding of cells to beads of different sizes and their isolation with sieves of different mesh sizes. As a result, we succeeded in isolating CD9-positive populations with 86% purity of ependymal cells from the third ventricle. We next assayed whether isolated CD9-positive ependymal cells had neurospherogenic potential. Neurospheres were generated from CD9-positive ependymal cells of adult rats and were immunopositve for neuron, astrocyte, and oligodendrocyte markers after cultivation. Thus, based on these findings, we suggest that the isolated CD9-positive ependymal cells from the third ventricle included tanycytes, which are special ependymal cells in the ventricular zone of the third ventricle that form part of the adult neurogenic and gliogenic niche. These current findings improve our understanding of tanycytes in the adult third ventricle in vitro.

Three-plane description of astroglial populations of OVLT subdivisions in rat: Tanycyte connections to distant parts of third ventricle.

This study demonstrates glial and gliovascular markers of organon vasculosum laminae terminalis (OVLT) in three planes. The distribution of glial markers displayed similarities to the subfornical organ. There was an inner part with vimentin- and nestin-immunopositive glia whereas GFAP and the water-channel aquaporin 4 were found at the periphery. This separation indicates different functions of the two regions. The presence of nestin may indicate stem cell-capabilities whereas aquaporin 4 has been reported to promote the osmoreceptor function. Glutamine synthetase immunoreactivity was sparse like in the area postrema and subfornical organ. The laminin and ß-dystroglycan immunolabelings altered along the vessels such as in the subfornical organ indicating altering gliovascular relations. The different subdivisions of OVLT received glial processes of different origins. The posterior periventricular zone contained short vimentin-immunopositive processes from the ependyma of the adjacent surface of the third ventricle. The lateral periventricular zone received forceps-like process systems from the anterolateral part of the third ventricle. Most interestingly, the "dorsal cap" received a mixed group of long GFAP- and vimentin-immunopositive processes from a distant part of the third ventricle. The processes may have two functions: a guidance for newly produced cells like radial glia in immature brain and/or a connection between distant parts of the third ventricle and OVLT.

Low-dose infusions of leptin into the nucleus of the solitary tract increase sensitivity to third ventricle leptin.

Previous studies suggest that weight loss occurs when leptin receptors in both the forebrain and hindbrain are activated. Experiments described here tested whether this integration is mediated through a neural connection or by leptin diffusion through the subarachanoid space. If the hypothalamus and hindbrain communicated through a neural pathway, then a very low dose of leptin infused directly into the nucleus of the solitary tract (NTS) would enhance the response to third ventricle (3V) leptin but would have no effect if infused into the fourth ventricle (4V). A 12-day infusion of 10 ng/24 h into the 4V or the NTS reduced body fat. Leptin at 5 ng/24 h into the 4V or NTS had no effect on food intake or body composition, but infusion of 5 ng of leptin/24 h into the NTS combined with a 3V injection of 0.1 µg of leptin inhibited food intake between 6 and 12 h after injection. Cumulative intake was inhibited for up to 36 h. 3V leptin had no effect on food intake of rats receiving the 4V leptin infusion. Similar results were found using infusions of 5 ng leptin/24 h and a 3V injection of 0.025 µg leptin. These data suggest that activation of leptin receptors in the NTS lowers the threshold for response to leptin in the forebrain through a neural network.

One-week exposure to a free-choice high-fat high-sugar diet does not disrupt blood-brain barrier permeability in fed or overnight fasted rats.

Objectives: The hypothalamus lies adjacent to the third ventricle and is in close proximity with the median eminence (ME), a circumventricular organ with an incomplete blood-brain barrier (BBB) which controls direct entry of nutrients into the brain. The blood-CSF barrier of the hypothalamus shows dynamic changes upon neuroendocrine events and adjusts permeability with the tight junction (TJ) complex. It has been shown that chronic exposure to a high-fat diet (HFD) affects BBB permeability. HFD also induces leptin resistance and alters neuropeptide expression in the arcuate nucleus (Arc) of the hypothalamus starting early during overnutrition. We hypothesized altered integrity of the BBB to occur after exposing rats to a free-choice high-fat high-sugar (fcHFHS) diet for 1 week. Methods: We measured diffusion of Evans blue dye over the ME and assessed expression of the TJ proteins ZO-1, claudin-5, and occludin in the tanycytic wall of the third ventricle. Furthermore, we assessed protein expression of glucose transporter 1 (GLUT-1), which is highly expressed in the Arc-ME complex and facilitates glucose transport over the BBB. Results: fcHFHS-fed rats increased caloric intake compared to control, however, there was no effect of the fcHFHS diet on permeability of the BBB, nor changes in protein expression of tight TJ proteins or GLUT-1. Fasting acutely affects the BBB and we hypothesized that exposure to the fcHFHS diet affects the BBB differently compared to chow after fasting. We did not, however, find any differences in Evans blue diffusion nor protein expression between chow- and fcHFHS-fed rats when fasted overnight. Conclusions: We conclude that short-term consumption of a fcHFHS diet does not change permeability or diffusion in the hypothalamus barrier in ad libitum fed or fasted rats.

Nestin expression and in vivo proliferative potential of tanycytes and ependymal cells lining the walls of the third ventricle in the adult rat brain.

There is a disagreement in the literature concerning the degree of proliferation of cells in the walls of the third ventricle (3rdV) under normal conditions in the adult mammalian brain. To address this issue, we mapped the cells expressing the neural stem/progenitor cell marker nestin along the entire rostrocaudal extent of the 3rdV in adult male rats and observed a complex distribution. Abundant nestin was present in tanycyte cell bodies and processes and also was observed in patches of ependymal cells as well as in isolated ependymal cells throughout the walls of the 3rdV. However, we observed very limited ependymal cell or tanycyte proliferation in normal adult rats as determined by bromodeoxyuridine (BrdU) incorporation or the expression of Ki-67. Moreover, fewer than 13% of the cells that were BrdU-positive (BrdU+) or Ki-67-positive (Ki-67+) expressed nestin. These observations stand in contrast to those made in the subventricular zone of the lateral ventricle (SVZ) and subgranular zone of the hippocampal formation (SGZ), where cell proliferation measured by BrdU incorporation or Ki-67 expression is observed frequently in cells that also express nestin. Thus, while ependymal cell or tanycyte cell proliferation can be promoted by the addition of mitogens, dietary modifications or other in vivo manipulations, the proliferation of ependymal cells and tanycytes in the walls of the 3rdV is very limited in the normal adult male rat brain.

Low-dose leptin infusion in the fourth ventricle of rats enhances the response to third-ventricle leptin injection.

We previously reported that low-dose leptin infusions into the third or fourth ventricle that do not affect energy balance when given independently cause rapid weight loss when given simultaneously. Therefore, we tested whether hindbrain leptin enhances the response to forebrain leptin or whether forebrain leptin enhances the response to hindbrain leptin. Rats received fourth-ventricle infusions of saline or 0.01, 0.1, 0.3, or 0.6 µg leptin/day for 13 days. On days 9 and 13, 0.1 µg leptin was injected into the third ventricle. The injection inhibited food intake for 36 h in saline-infused rats but for 60 h in those infused with 0.6 µg leptin/day. Leptin injection increased intrascapular brown fat temperature in leptin-infused, but not saline-infused, rats. In a separate experiment, rats received third-ventricle infusions of saline or 0.005, 0.01, 0.05, or 0.1 µg leptin/day and fourth-ventricle injections of 1.0 µg leptin on days 9 and 13 Leptin injection inhibited food intake, respiratory exchange ratio, and 14-h food intake in rats infused with saline or the two lowest doses of leptin. There was no effect with higher-dose leptin infusions because food intake, body fat, and lean mass were already inhibited. These data suggest that activation of leptin receptors in the hindbrain enhances the response to third-ventricle leptin, whereas activation of forebrain leptin receptors does not enhance the response to fourth-ventricle leptin, consistent with our previous finding that weight loss in rats treated with fourth-ventricle leptin is associated with indirect activation of hypothalamic STAT3.

The radiological findings of chordoid glioma: report of two cases, one case with MR spectroscopy.

Chordoid glioma is a rare tumor that often originate from the third ventricle. We reported two cases of choroid glioma. Although calcification is a rare findings, dot-like calcification was present on one case. On contrast images, one case demonstrated strong, homogeneous enhancement. On MR spectroscopy (MRS) images, the tumor showed high choline (Cho), low N-acetyl aspartate (NAA) and Cho/Cr=2.38, NAA/Cr=1.19, NAA/Cr=1.19. This might mimic a low-grade tumour. MRS offers additional information and facilitate differential diagnosis for chordoid glioma.

SrGAP3 knockout mice display enlarged lateral ventricles and specific cilia disturbances of ependymal cells in the third ventricle.

In several mouse models of mental retardation, ventricular enlargements have been observed. Mutation in the SrGAP3 gene residing on chromosome 3p25 has previously been associated with intellectual disability in humans. In addition, SrGAP3 is related to Rho-GAPs signaling pathways, which play essential roles in the development and plasticity of the nervous system. About 10 % of postnatal homozygous SrGAP3-deficient mice die due to hydrocephalus, whereas the remaining mice survive into adulthood but display enlarged ventricles. We analyze the ventricular enlargement of these mice by performing a post-mortem MRI approach. We found a more than 15-fold enlargement of the lateral ventricles of homozygous SrGAP3-deficient mice. Moreover, we demonstrate that this phenotype was not accompanied by a stenosis of the aqueduct. Instead, SrGAP3 knockout mice displayed reduced densities of cilia of ependymal cells in These third ventricle compared to age-matched controls. This results indicate that the ventricular enlargement may be due to ciliopathy.

Chordoid gliomas of the third ventricle share TTF-1 expression with organum vasculosum of the lamina terminalis.

Chordoid glioma of the third ventricle (CG3V) is a rare tumor developing in a stereotyped localization. It has been related to the circumventricular organ of the lamina terminalis, in the anterior part of the third ventricle, but its oncogenesis is poorly understood. TTF-1 transcription factor is involved in the development and adult physiology of the ventral forebrain. We studied the histopathologic and immunohistochemical features of a multicentric series of 17 cases of CG3V. We described additional histologic patterns (solid, fibrosing, and fusiform) to the typical chordoid pattern. TTF-1 was constantly expressed in CG3V, as in developing and adult lamina terminalis. The anti-TTF-1 SPT24 clone was more sensitive than the 8G7G3/1 clone. No mutation of IDH1 R132, IDH2 R172, or BRAF V600 codons was found. We showed TTF-1 as a useful marker for the diagnosis of CG3V and the understanding of its oncogenesis.

Is pineal melatonin released in the third ventricle in humans? A study in movement disorders.

In order to determine sources and metabolism of melatonin in human cerebrospinal fluid (CSF), melatonin and 6-sulfatoxymelatonin (aMT6S) concentrations were measured in CSF sampled during neurosurgery in both lateral and third ventricles in patients displaying movement disorder (Parkinson's disease, essential tremor, dystonia or dyskinesia) and compared with their plasma levels. Previous determinations in nocturnal urine had showed that the patients displayed melatonin excretion in the normal range, compared with healthy controls matched according to age. A significant difference in melatonin concentration was observed between lateral and third ventricles, with the highest levels in the third ventricle (8.75±2.75pg/mL vs. 3.20±0.33pg/mL, P=0.01). CSF aMT6s levels were similar in both ventricles and of low magnitude, less than 5pg/mL. They were not correlated with melatonin levels or influenced by the area of sampling. Melatonin levels were significantly higher in third ventricle than in the plasma, whereas there was no difference between plasma and lateral ventricle levels. These findings show that melatonin may enter directly the CSF through the pineal recess in humans. The physiological meaning of these data remains to be elucidated.

Prolactin regulation of kisspeptin neurones in the mouse brain and its role in the lactation-induced suppression of kisspeptin expression.

Hyperprolactinaemia is a major cause of infertility in both males and females, although the mechanism by which prolactin inhibits the reproductive axis is not clear. The aim of the present study was to test the hypothesis that elevated prolactin causes suppression of kisspeptin expression in the hypothalamus, resulting in reduced release of gonadotrophin-releasing hormone (GnRH) and consequent infertility. In oestrogen-treated ovariectomised mice, chronic prolactin-treatment prevented the rise in luteinising hormone (LH) seen in vehicle-treated mice. Kiss1 mRNA was significantly suppressed in both the rostral periventricular region of the third ventricle (RP3V) and arcuate nucleus after prolactin treatment. Exogenous prolactin treatment induced phosphorylated signal transducer and activator of transcription 5 (pSTAT5) in kisspeptin neurones, and suppression of endogenous prolactin using bromocriptine reduced levels of pSTAT5 in kisspeptin neurones, suggesting that prolactin acts directly on kisspeptin neurones. By contrast, fewer than 1% of GnRH neurones expressed pSTAT5 in either dioestrous or lactating mice. As reported previously, there was significant suppression of kisspeptin mRNA and protein in the RP3V on day 7 of lactation, although not in the arcuate nucleus. Bromocriptine treatment significantly increased Kiss1 mRNA expression in the RP3V, although not to dioestrous levels. Unilateral thelectomy, aiming to eliminate sensory inputs from nipples on one side of the body, failed to alter the reduction in the number of kisspeptin neurones observed in the RP3V. These data demonstrate that chronic prolactin administration suppressed serum LH, and reduced Kiss1 mRNA levels in both the RP3V and arcuate nucleus, consistent with the hypothesis that prolactin-induced suppression of kisspeptin secretion might mediate the inhibitory effects of prolactin on GnRH secretion. During lactation, however, the suppression of Kiss1 mRNA in the RP3V was only partially reversed by the administration of bromocriptine to block elevated levels of prolactin, suggesting that, although elevated prolactin contributes to lactational anovulation, additional non-neural factors must also contribute to the lactation-induced suppression of kisspeptin neurones.

Sustained alterations of hypothalamic tanycytes during posttraumatic hypopituitarism in male mice.

Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hypothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome.

Blockade of the cerebral aqueduct in rats provides evidence of antagonistic leptin responses in the forebrain and hindbrain.

Previously, we reported that low-dose leptin infusions into the fourth ventricle produced a small but significant increase in body fat. These data contrast with reports that injections of higher doses of leptin into the fourth ventricle inhibit food intake and weight gain. In this study, we tested whether exogenous leptin in the fourth ventricle opposed or contributed to weight loss caused by third ventricle leptin infusion by blocking diffusion of CSF from the third to the fourth ventricle. Male Sprague-Dawley rats received third ventricle infusions of PBS or 0.3 µg leptin/24 h from miniosmotic pumps. After 4 days, rats received a 3-µl cerebral aqueduct injection of saline or of thermogelling nanoparticles (hydrogel) that solidified at body temperature. Third ventricle leptin infusion inhibited food intake and caused weight loss. Blocking the aqueduct exaggerated the effect of leptin on food intake and weight loss but had no effect on the weight of PBS-infused rats. Leptin reduced both body fat and lean body mass but did not change energy expenditure. Blocking the aqueduct decreased expenditure of rats infused with PBS or leptin. Infusion of leptin into the third ventricle increased phosphorylated STAT3 in the VMHDM of the hypothalamus and the medial NTS in the hindbrain. Blocking the aqueduct did not change hypothalamic p-STAT3 but decreased p-STAT3 in the medial NTS. These results support previous observations that low-level activation of hindbrain leptin receptors has the potential to blunt the catabolic effects of leptin in the third ventricle.