Impaired GABAergic regulation and developmental immaturity in interneurons derived from the medial ganglionic eminence in the tuberous sclerosis complex.

GABAergic interneurons play a critical role in maintaining neural circuit balance, excitation-inhibition regulation, and cognitive function modulation. In tuberous sclerosis complex (TSC), GABAergic neuron dysfunction contributes to disrupted network activity and associated neurological symptoms, assumingly in a cell type-specific manner. This GABAergic centric study focuses on identifying specific interneuron subpopulations within TSC, emphasizing the unique characteristics of medial ganglionic eminence (MGE)- and caudal ganglionic eminence (CGE)-derived interneurons. Using single-nuclei RNA sequencing in TSC patient material, we identify somatostatin-expressing (SST+) interneurons as a unique and immature subpopulation in TSC. The disrupted maturation of SST+ interneurons may undergo an incomplete switch from excitatory to inhibitory GABAergic signaling during development, resulting in reduced inhibitory properties. Notably, this study reveals markers of immaturity specifically in SST+ interneurons, including an abnormal NKCC1/KCC2 ratio, indicating an imbalance in chloride homeostasis crucial for the postsynaptic consequences of GABAergic signaling as well as the downregulation of GABAA receptor subunits, GABRA1, and upregulation of GABRA2. Further exploration of SST+ interneurons revealed altered localization patterns of SST+ interneurons in TSC brain tissue, concentrated in deeper cortical layers, possibly linked to cortical dyslamination. In the epilepsy context, our research underscores the diverse cell type-specific roles of GABAergic interneurons in shaping seizures, advocating for precise therapeutic considerations. Moreover, this study illuminates the potential contribution of SST+ interneurons to TSC pathophysiology, offering insights for targeted therapeutic interventions.

High-Fat Diet Impairs Mouse Median Eminence: A Study by Transmission and Scanning Electron Microscopy Coupled with Raman Spectroscopy.

Hypothalamic dysfunction is an initial event following diet-induced obesity, primarily involving areas regulating energy balance such as arcuate nucleus (Arc) and median eminence (ME). To gain insights into the early hypothalamic diet-induced alterations, adult CD1 mice fed a high-fat diet (HFD) for 6 weeks were studied and compared with normo-fed controls. Transmission and scanning electron microscopy and histological staining were employed for morphological studies of the ME, while Raman spectroscopy was applied for the biochemical analysis of the Arc-ME complex. In HFD mice, ME ß2-tanycytes, glial cells dedicated to blood-liquor crosstalk, exhibited remarkable ultrastructural anomalies, including altered alignment, reduced junctions, degenerating organelles, and higher content of lipid droplets, lysosomes, and autophagosomes. Degenerating tanycytes also displayed an electron transparent cytoplasm filled with numerous vesicles, and they were surrounded by dilated extracellular spaces extending up to the subependymal layer. Consistently, Raman spectroscopy analysis of the Arc-ME complex revealed higher glycogen, collagen, and lipid bands in HFD mice compared with controls, and there was also a higher band corresponding to the cyanide group in the former compared to the last. Collectively, these data show that ME ß2-tanycytes exhibit early structural and chemical alterations due to HFD and reveal for the first-time hypothalamic cyanide presence following high dietary lipids consumption, which is a novel aspect with potential implications in the field of obesity.

Dietary fats promote functional and structural changes in the median eminence blood/spinal fluid interface-the protective role for BDNF.

BACKGROUND: The consumption of large amounts of dietary fats activates an inflammatory response in the hypothalamus, damaging key neurons involved in the regulation of caloric intake and energy expenditure. It is currently unknown why the mediobasal hypothalamus is the main target of diet-induced brain inflammation. We hypothesized that dietary fats can damage the median eminence blood/spinal fluid interface. METHODS: Swiss mice were fed on a high-fat diet, and molecular and structural studies were performed employing real-time PCR, immunoblot, immunofluorescence, transmission electron microscopy, and metabolic measurements. RESULTS: The consumption of a high fat diet was sufficient to increase the expression of inflammatory cytokines and brain-derived neurotrophic factor in the median eminence, preceding changes in other circumventricular regions. In addition, it led to an early loss of the structural organization of the median eminence ß1-tanycytes. This was accompanied by an increase in the hypothalamic expression of brain-derived neurotrophic factor. The immunoneutralization of brain-derived neurotrophic factor worsened diet-induced functional damage of the median eminence blood/spinal fluid interface, increased diet-induced hypothalamic inflammation, and increased body mass gain. CONCLUSIONS: The median eminence/spinal fluid interface is affected at the functional and structural levels early after introduction of a high-fat diet. Brain-derived neurotrophic factor provides an early protection against damage, which is lost upon a persisting consumption of large amounts of dietary fats.

Neural Stem Cell or Human Induced Pluripotent Stem Cell-Derived GABA-ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy.

Grafting of neural stem cells (NSCs) or GABA-ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug-resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self-renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). On the other hand, GPCs could be generated from the medial and lateral ganglionic eminences of the embryonic brain and from hESCs and hiPSCs. To provide comprehensive methodologies involved in testing the efficacy of transplantation of NSCs and GPCs in a rat model of chronic TLE, NSCs derived from the rat medial ganglionic eminence (MGE) and MGE-like GPCs derived from hiPSCs are taken as examples in this unit. The topics comprise description of the required materials, reagents and equipment, methods for obtaining rat MGE-NSCs and hiPSC-derived MGE-like GPCs in culture, generation of chronically epileptic rats, intrahippocampal grafting procedure, post-grafting evaluation of the effects of grafts on spontaneous recurrent seizures and cognitive and mood impairments, analyses of the yield and the fate of graft-derived cells, and the effects of grafts on the host hippocampus. © 2016 by John Wiley & Sons, Inc.

Expanding the spectrum of human ganglionic eminence region anomalies on fetal magnetic resonance imaging.

INTRODUCTION: Ganglionic eminence (GE) is a transient fetal brain structure that harvests a significant amount of precursors of cortical GABA-ergic interneurons. Prenatal magnetic resonance (MR) imaging features of GE anomalies (i.e., cavitations) have already been reported associated with severe micro-lissencephaly. The purpose of this report was to illustrate the MR imaging features of GE anomalies in conditions other than severe micro-lissencephalies. METHODS: Among all the fetuses submitted to prenatal MR imaging at our center from 2005 to 2014, we collected eight cases with GE anomalies and only limited associated brain anomalies. The median gestational age at the time of MR imaging was 21 weeks ranging from 19 to 29 weeks. Two senior pediatric neuroradiologists categorized the anomalies of the GE region in two groups: group one showing cavitation in the GE region and group two showing enlarged GE region. For each fetal case, associated cranial anomalies were also reported. RESULTS: Five out of the eight cases were included in group one and three in group two. Besides the GE region abnormality, all eight cases had additional intracranial anomalies, such as mild partial callosal agenesis, vermian hypoplasia and rotation, cerebellar hypoplasia, ventriculomegaly, enlarged subarachnoid spaces, molar tooth malformation. Ultrasound generally detected most of the associated intracranial anomalies, prompting the MR investigation; on the contrary in none of the cases, GE anomalies had been detected by ultrasound. CONCLUSIONS: Our observation expands the spectrum of human GE anomalies, demonstrating that these may take place also without associated severe micro-lissencephalies.

Development of neuroendocrine components of the thyroid axis in the direct-developing frog Eleutherodactylus coqui: formation of the median eminence and onset of pituitary TSH production.

Direct-developing frogs lack, wholly or in part, a wide range of larval features found in metamorphosing species and form adult-specific features precociously, during embryogenesis. Most information on thyroid regulation of direct development relies on hormone manipulations; the ontogeny of many thyroid axis components has not been fully described. This analysis examines differentiation of the median eminence of the hypothalamus and production of thyroid-stimulating hormone (TSH) by the pituitary of the direct-developing frog Eleutherodactylus coqui. The median eminence is established two-thirds of the way through embryogenesis. Cells immunoreactive to human TSHß antibodies are first detected during embryogenesis and quantitative changes in TSHß-IR cells resemble those in metamorphosing amphibians. Formation of the median eminence of the hypothalamus and TSHß production by the pituitary precede or coincide with morphological changes during embryogenesis that occur during metamorphosis in biphasic anurans. Thus, while the onset of neuroendocrine regulation has changed during the evolution of direct development, it is likely that these thyroid axis components still mediate the formation of adult features.

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.

Third ventriculostomy site as a neuroreceptorial area.

PURPOSE: Endoscopic third ventriculostomy is an established method for treating hydrocephalus. The third ventriculostomy site is considered a safe area that can be disrupted during surgical endoscopic procedures. The question of the clinical consequences of an apparently unavoidable injury to the floor of the third ventricle has been sporadically addressed in the literature. The aim of this study is to describe our anatomical and operative findings during endoscopic procedures performed in fluorescent mode after intravenous fluorescein injection and address the possible role of fluorescein-enhanced visualization of the median eminence as an accessory tool in order to partially spare this functional structure when performing ventriculostomy. METHODS: We prospectively administered intravenously 500 mg of fluorescein sodium in 12 consecutive endoscopic surgery cases. A flexible scope equipped with dual observation modes for both white light and fluorescence was used. Taking into account the position of the basilar apex and the need for a conveniently sized stoma, a perforation area was chosen and dilated using a Fogarty balloon, guided by fluorescein-enhanced visualization of the median eminence. RESULTS: After a mean of 20 s in the fluorescent mode, the fluorescein enhanced the visualization of the median eminence-tuber cinereum complex. In our preliminary experience, by opening the stoma in the fluorescence mode, almost half of the visible median eminence surface can be spared from iatrogenic sacrifice. CONCLUSIONS: Tailoring fluorescence-guided ventriculostomy is a feasible way of trying to preserve the median eminence and may have implications for the site and safety of this common surgical procedure.

Rac1-dependent cell cycle exit of MGE precursors and GABAergic interneuron migration to the cortex.

Cortical γ-aminobutyric acid (GABA)ergic interneurons are characterized by extraordinary neurochemical and functional diversity. Although recent studies have uncovered some of the molecular components underlying interneuron development, including the cellular and molecular mechanisms guiding their migration to the cortex, the intracellular components involved are still unknown. Rac1, a member of the Rac subfamily of Rho-GTPases, has been implicated in various cellular processes such as cell cycle dynamics, axonogenesis, and migration. In this study, we have addressed the specific role of Rac1 in interneuron progenitors originating in the medial ganglionic eminence, via Cre/loxP technology. We show that ablation of Rac1 from Nkx2.1-positive progenitors, results in a migratory impairment. As a consequence, only half of GABAergic interneurons are found in the postnatal cortex. The rest remain aggregated in the ventral telencephalon and show morphological defects in their growing processes in vitro. Ablation of Rac1 from postmitotic progenitors does not result in similar defects, thus underlying a novel cell autonomous and stage-specific requirement for Rac1 activity, within proliferating progenitors of cortical interneurons. Rac1 is necessary for their transition from G1 to S phase, at least in part by regulating cyclin D levels and retinoblastoma protein phosphorylation.

MET signaling in GABAergic neuronal precursors of the medial ganglionic eminence restricts GDNF activity in cells that express GFRα1 and a new transmembrane receptor partner.

GDNF (glial cell line-derived neurotrophic factor) promotes the differentiation and migration of GABAergic neuronal precursors of the medial ganglionic eminence (MGE). These functions are dependent on the GPI-anchored receptor GFRα1, but independent of its two known transmembrane receptor partners RET and NCAM. Here we show that soluble GFRα1 is also able to promote differentiation and migration of GABAergic MGE neurons. These activities require endogenous production of GDNF. Although GDNF responsiveness is abolished in Gfra1(-/-) neurons, it can be restored upon addition of soluble GFRα1, a result that is only compatible with the existence of a previously unknown transmembrane signaling partner for the GDNF-GFRα1 complex in GABAergic neurons. The roles of two candidate transmembrane receptors previously implicated in GABAergic interneuron development--MET, a receptor for hepatocyte growth factor (HGF), and ErbB4, the neuregulin receptor--were examined. GDNF did not induce the activation of either receptor, nor did inhibition of MET or ErbB4 impair GDNF activity in GABAergic MGE neurons. Unexpectedly, however, inhibition of MET or HGF per se promoted neuronal differentiation and migration and enhanced the activity of GDNF on MGE neurons. These effects were dependent on endogenous GDNF and GFRα1, suggesting that MET signaling negatively regulates GDNF activity in the MGE. In agreement with this, Met mutant MGE neurons showed enhanced responses to GDNF and inhibition of MET or HGF increased Gfra1 mRNA expression in MGE cells. In vivo, expression of MET and GFRα1 overlapped in the MGE, and a loss-of-function mutation in Met increased Gfra1 expression in this region. Together, these observations demonstrate the existence of a novel transmembrane receptor partner for the GDNF-GFRα1 complex and uncover an unexpected interplay between GDNF-GFRα1 and HGF-MET signaling in the early diversification of cortical GABAergic interneuron subtypes.

Vacuolar pathology in the median eminence of the hypothalamus after hyponatremia.

The median eminence of the hypothalamus is an important conduit by which neurosecretory hormones from hypothalamic nuclei are delivered to the pars nervosa (neural lobe) of the pituitary en route to the bloodstream. Dilutional hyponatremia was produced in adult rats to determine the effect on the morphology of the median eminence of the hypothalamus. Hyponatremia was caused by reducing electrolyte and organic osmolyte reserves to block the excretion of water through delivery of the nephrotoxin mercuric chloride (HgCl2). Histological examination of the brain 1 day after a hyponatremic insult revealed vacuolation within the median eminence of the hypothalamus. No other lesions were found in other parts of the brain after hyponatremia. The hyponatremic lesion consisted of a band of closely packed vacuoles that crossed the floor of the third ventricle. Vacuoles associated with hyponatremia were predominantly in the subependymal, fiber, reticular, and palisade layers of the median eminence. Vacuolation was not observed in the tanycyte layer of the median eminence. This study indicates that the median eminence is a potentially vulnerable site in human hyponatremic conditions that should be evaluated further in relevant animal models.

Glycogen stores are impaired in hypothalamic nuclei of rats malnourished during early life.

Perinatal nutrition has persistent influences on neural development and cognition. In humans and other animals, protein malnutrition during the perinatal period causes permanent changes, inducing to adulthood metabolic syndrome. Feeding is mainly modulated by neural and hormonal inputs to the hypothalamus. Hypothalamic glycogen stores are a source of glucose in high energetic demands, as during development of neural circuits. As some hypothalamic circuits are formed during lactation, we studied the effects of malnutrition, during the first 10 days of lactation, on glycogen stores in hypothalamic nuclei involved in the control of energy metabolism. Female pregnant rats were fed ad libitum with a normal protein diet (22% protein). After delivery, each dam was kept with 6 male pups. During the first 10 days of lactation, dams from the experimental group received a protein-free diet and the control group a normoprotein diet. By post-natal day 10 (P10), glycogen stores were very high in the arcuate nucleus and median eminence of control group. Glycogen stores decreased during development. In P20 control animals, glycogen stores were lower when compared to P10 control animals. Animals submitted to malnutrition presented a staining even lower than control ones. After P45, it was difficult to determine differences between control and diet groups because glycogen stores were reduced. We also showed that tanycytes were the cells presenting glycogen stores. Our data reinforce the concept that maternal nutritional state during lactation may be critical for neurodevelopment since it resulted in a low hypothalamic glycogen store, which may be critical for establishment of neuronal circuitry.

Kaposi sarcoma related to an ectopic hypothalamic adrenocorticotropic hormone-secreting adenoma: case report.

OBJECTIVE: We aim to report a case of Kaposi sarcoma (KS) with Cushing's syndrome caused by endogenic glucocorticoid-induced immunosuppression. CLINICAL PRESENTATION: A 43-year-old woman presented with delirium, hirsutism, fatigue, and hypertension. At the time of presentation, physical findings showed a Cushingoid appearance, with moon-like facies, hirsutism, and hyperpigmentation. Laboratory findings showed the following: adrenocorticotropic hormone, 86.7 pg/mL (normal range, 0-46 pg/mL); baseline cortisol level, 50 microg/dL (normal range, 6.2-19 microg/dL); potassium, 2.2 mEq/L (normal range, 3.5-5 mEq/L); and midnight cortisol level, 33 microg/dL. Serum cortisol levels failed to suppress after low and high doses of dexamethasone; these findings confirmed the diagnosis of ectopic adrenocorticotropic hormone production. Magnetic resonance imaging revealed a 12 x 15-mm, round, hypothalamic mass lesion in the center of the median eminence. INTERVENTION: Endoscopic biopsy from the floor of the third ventricle was performed, and pathological examination of the lesion showed a diffuse adrenocorticotropic hormone-secreting adenoma. The patient developed diffuse skin lesions that were proven to be a KS by skin biopsy while she was prepared for transcranial surgery. After surgical removal of the adenoma, she became hypocortisolemic and required cortisol replacement. Within 1 month after surgery, all KS lesions disappeared spontaneously. CONCLUSION: Excessive cortisol may induce immunosuppression. KS is one of the most common malignant tumors of patients with immunosuppression. To the best of our knowledge, this is the first case of Cushing's syndrome with KS caused by endogenous glucocorticoid-induced immunosuppression.

Three-dimensional properties of GnRH neuroterminals in the median eminence of young and old rats.

The decapeptide gonadotropin-releasing hormone (GnRH), which regulates reproduction in all vertebrates, is stored in, and secreted from, large dense-core secretory vesicles in nerve terminals in the median eminence. GnRH is released from these terminals with biological rhythms that are critical for the maintenance of normal reproduction. During reproductive aging in female rats, there is a loss of GnRH pulses and a diminution of the GnRH surge. However, information about the specific role of GnRH nerve terminals is lacking, particularly in the context of aging. We sought to gain novel ultrastructural information about GnRH neuroterminals by performing three-dimensional (3D) reconstructions of GnRH neuroterminals and their surrounding microenvironment in the median eminence of young (4-5 months) and old (22-24 months) ovariectomized Sprague-Dawley female rats. Median eminence tissues were freeze-plunge embedded and serial ultrathin sections were collected on slot grids for immunogold labeling of GnRH immunoreactivity. Sequential images were used to create 3D models of GnRH terminals. These reconstructions provided novel perspectives into the morphological properties of GnRH terminals and their neural and glial environment. We also noted that the cytoarchitectural features of the median eminence became disorganized with aging. Quantitative measures showed a significant decrease in the apposition between GnRH terminal membranes and glial cells. Our data suggest reproductive aging in rats is characterized by structural organizational changes to the GnRH terminal microenvironment in the median eminence.

Gonadotropin-releasing hormone neuron requirements for puberty, ovulation, and fertility.

The absolute requirement for reproduction implies that the hypothalamo-pituitary-gonadal axis, controlling fertility, is an evolutionary robust mechanism. The GnRH neurons of the hypothalamus represent the key cell type within the body dictating fertility. However, the level of functional redundancy within the GnRH neuron population is unknown. As a result of a fortuitous transgene insertion event, GNR23 mice exhibit a marked allele-dependent reduction in GnRH neuron number within their brain. Wild-type mice have approximately 600 GnRH neurons, compared with approximately 200 (34%) and approximately 70 (12%) in GNR23(+/-) and GNR23(-/-) mice, respectively. Using these mice, we examined the minimal GnRH neuron requirements for fertility. Male GNR23(-/-) mice exhibited normal fertility. In contrast, female GNR23(-/-) mice were markedly subfertile, failing to produce normal litters, have estrous cycles, or ovulate. The failure of ovulation resulted from an inability of the few existing GnRH neurons to generate the LH surge. This was not the case, however, for the first cycle at puberty that appeared normal. Together, these observations demonstrate that 12% of the GnRH neuron population is sufficient for pulsatile gonadotropin secretion and puberty onset, whereas between 12 and 34% are required for cyclical control in adult female mice. This indicates that substantial redundancy exists within the GnRH neuronal population and suggests that the great majority of GnRH neurons must be dysfunctional before fertility is affected.

Hormonal, pituitary magnetic resonance, LHX4 and HESX1 evaluation in patients with hypopituitarism and ectopic posterior pituitary lobe.

OBJECTIVE: LHX4 and HESX1 are important in early stages of pituitary development and their mutations can be associated with an ectopic posterior lobe (EPL) in the pituitary of patients with hypopituitarism. The EPL can be located at the median eminence or at the path of the pituitary stalk. The aim of this study was to analyse LHX4 and HESX1 and characterize the hormonal deficiency profiles, establishing relationships with magnetic resonance imaging (MRI) findings in these patients. PATIENTS AND DESIGN: Sixty-two patients with hypopituitarism associated with EPL were submitted to evaluation of pituitary function, analysis of MRI with EPL location and molecular analysis of LHX4 and HESX1 using polymerase chain reaction (PCR), digestion with restriction enzyme and automatic sequencing. RESULTS: Forty-two patients had a nonvisualized pituitary stalk (NPS), and 20 a visualized pituitary stalk (VPS). Most patients (95%) with NPS had combined pituitary hormone deficiency (CPHD), with ACTH deficiency in 85%. In patients with VPS, CPHD was found in 50% and ACTH deficiency occurred in only 20%. The frequency of the location of EPL was similar in patients with VPS and NPS: 35% at median eminence and 65% at the path of the stalk. No mutations in LHX4 and HESX1 were identified. Three new polymorphisms in LHX4 were found. CONCLUSIONS: ACTH deficiency is frequent in patients with hypopituitarism and NPS (85%), the location of EPL at the median eminence was not predictive of the hormonal profile [isolated GH deficiency (IGHD) or CPHD], and LHX4 and HESX1 genes mutations remain rare causes of hypopituitarism associated with EPL.

Branching and nucleokinesis defects in migrating interneurons derived from doublecortin knockout mice.

Type I lissencephaly results from mutations in the doublecortin (DCX) and LIS1 genes. We generated Dcx knockout mice to further understand the pathophysiological mechanisms associated with this cortical malformation. Dcx is expressed in migrating interneurons in developing human and mouse brains. Video microscopy analyses of such tangentially migrating neuron populations derived from the medial ganglionic eminence show defects in migratory dynamics. Specifically, the formation and division of growth cones, leading to the production of new branches, are more frequent in knockout cells, although branches are less stable. Dcx-deficient cells thus migrate in a disorganized manner, extending and retracting short branches and making less long-distant movements of the nucleus. Despite these differences, migratory speeds and distances remain similar to wild-type cells. These novel data thus highlight a role for Dcx, a microtubule-associated protein enriched at the leading edge in the branching and nucleokinesis of migrating interneurons.