MD, PhD Marc Baroncini
email: marc.baroncini@inserm.fr

MD Patrice Jissendi

PhD Eglantine Balland

PhD Pierre Besson

Jean-Pierre Pruvo

PhD; Jean-Paul Francke

Didier Dewailly

MD, PhD Serge Blond

PhD Vincent Prevot

PhD; Jean-Pierre Pruvo

MD, PhD; Didier Dewailly

Jean- Pierre Pruvo

MRI atlas of the human hypothalamus

Keywords: MRI, human hypothalamus, anatomy, coronal plane

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Functional stereotactic neurosurgery of the hypothalamus is currently used for the treatment of cluster headaches [START_REF] Leone | Stereotactic stimulation of posterior hypothalamic gray matter in a patient with intractable cluster headache[END_REF][START_REF] Fontaine | Safety and efficacy of deep brain stimulation in refractory cluster headache: a randomized placebo-controlled double-blind trial followed by a 1-year open extension[END_REF][START_REF] Franzini | Deep brain stimulation of the posteromedial hypothalamus: indications, long-term results, and neurophysiological considerations[END_REF], and has recently been shown to have beneficial effects in the treatment of paroxysmal ophthalmic pain in multiple sclerosis patients [START_REF] Cordella | Hypothalamic stimulation for trigeminal neuralgia in multiple sclerosis patients: efficacy on the paroxysmal ophthalmic pain[END_REF]. Excitingly, it has also been shown to activate neural activity in cognitive and memory circuits in patients with mild Alzheimer's disease (Laxton et al.). Additional applications of hypothalamic deep brain stimulation may be expected in the field of sleep disorders [START_REF] Vetrugno | Effect on sleep of posterior hypothalamus stimulation in cluster headache[END_REF], thermoregulation [START_REF] Jurgens | Hypothalamic deep-brain stimulation modulates thermal sensitivity and pain thresholds in cluster headache[END_REF], epilepsy [START_REF] Franzini | Deep brain stimulation of two unconventional targets in refractory non-resectable epilepsy[END_REF], disruptive behavior [START_REF] Franzini | Stimulation of the posterior hypothalamus for medically intractable impulsive and violent behavior[END_REF] and perhaps certain other diseases involving the autonomic nervous system [START_REF] Cortelli | Effect of deep brain stimulation of the posterior hypothalamic area on the cardiovascular system in chronic cluster headache patients[END_REF]. Importantly, deep brain stimulation is also currently being investigated as a weigh loss strategy in obesity [START_REF] Sani | Deep brain stimulation for treatment of obesity in rats[END_REF][START_REF] Halpern | Deep brain stimulation in the treatment of obesity[END_REF][START_REF] Hamani | Memory enhancement induced by hypothalamic/fornix deep brain stimulation[END_REF][START_REF] Mantione | Smoking cessation and weight loss after chronic deep brain stimulation of the nucleus accumbens: therapeutic and research implications: case report[END_REF]. Anatomical knowledge of the hypothalamic structures to be targeted and of the circuitry involved is thus critical to the development of these new neurosurgical treatment strategies.

The hypothalamus is the part of the diencephalon located below the thalamus; it lies along the walls of the third ventricle below the hypothalamic sulcus and continues across the floor of the ventricle [START_REF] Carpenter | Core text of Neuranatomy, 3rd Edition[END_REF]. Despite its small size (4 g), no other brain structure contains so many specialized cell groups [START_REF] Saper | The hypothalamus[END_REF]. The hypothalamus controls vital bodily processes including cardiovascular regulation, sleep, metabolism, stress, thermoregulation, water and electrolyte balance, appetite regulation, sexual behavior and endocrine and immune responses. All these functions are related to affective and emotional behavior. The wide range of tasks controlled by a very small part of the brain makes the hypothalamic region particularly prone to involvement in several disorders. Significantly, the human hypothalamus has been implicated in homeostatic and developmental disorders, including sudden infant death syndrome, Prader-Willi syndrome, disturbance of biological rhythms, infertility, and diabetes and obesity [START_REF] Swaab | The human hypothalamus in metabolic and episodic disorders[END_REF][START_REF] Gordon | Clinical practice. Functional hypothalamic amenorrhea[END_REF][START_REF] Kalsbeek | Vasopressin and the output of the hypothalamic biological clock[END_REF], as well as in episodic brain disorders such as migraine, depression, narcolepsy and cluster headaches [START_REF] Overeem | The hypothalamus in episodic brain disorders[END_REF][START_REF] Swaab | The human hypothalamus in metabolic and episodic disorders[END_REF]. Besides these, the hypothalamus is subject to many different types of lesions, including developmental abnormalities (Rathke's cleft cysts, hamartoma), primary tumors of the central nervous system (hypothalamic glioma), systemic tumors affecting the brain, inflammatory and granulomatous diseases (sarcoidosis) and lesions arising from the surrounding structures [START_REF] Loes | MR anatomy and pathology of the hypothalamus[END_REF][START_REF] Saleem | Lesions of the hypothalamus: MR imaging diagnostic features[END_REF]. Notably, modern neuroimaging techniques have revealed the presence of lesions and tumors causing structural abnormalities in the hypothalamus of patients with eating disorders [START_REF] Shinoda | Development of akinetic mutism and hyperphagia after left thalamic and right hypothalamic lesions[END_REF][START_REF] Vile | Occult intracranial tumours masquerading as early onset anorexia nervosa[END_REF], and the precocious onset of puberty [START_REF] Jung | Hypothalamic hamartoma: a paradigm/model for studying the onset of puberty[END_REF][START_REF] Trivin | Presentation and evolution of organic central precocious puberty according to the type of CNS lesion[END_REF].

Magnetic resonance imaging is the technique of choice for evaluating the anatomy of the hypothalamus in vivo. However, the very small size of this region requires the use of specific high-resolution MRI sequences. The identification of gray or white matter within the hypothalamus, where some nuclei are about one millimeter across, is difficult and must be treated with caution, with frequent reference to sequential anatomical and histological sections. An atlas of the anatomical, histological and radiological correlates of the human hypothalamus in the coronal plane would thus be very useful for MRI identification of the hypothalamic structures. However, to our knowledge, such an atlas does not exist. Braak and Braak (Braak andBraak, 1987, 1992) proposed a review of the anatomy of the chiasmatic and tuberal region, but their article was only illustrated by histological staining among different species. [START_REF] Young | A three-dimensional reconstruction of the human hypothalamus[END_REF] proposed a threedimensional reconstruction of the human hypothalamus, but this work did not contain imaging correlates. In the latest edition of their atlas of the human brain, Mai and colleagues [START_REF] Mai | Atlas of the human brain, 3rd Edition[END_REF] presented a macroscopic correlation between anatomical and MRI sections without the resolution necessary to identify hypothalamic nuclei in the latter. Finally, Miller and colleagues [START_REF] Miller | Imaging white matter tracts and nuclei of the hypothalamus: an MR-anatomic comparative study[END_REF] examined the correlation between anatomical and MRI sections of the hypothalamus, but this was only done in vitro, while Saeki and colleagues [START_REF] Saeki | Heavily T2weighted MR imaging of white matter tracts in the hypothalamus: normal and pathologic demonstrations[END_REF] focused their work exclusively on the white matter fascicles of the human hypothalamus.

Here, we provide a comprehensive atlas comparing anatomical, histological and magnetic resonance images of the human hypothalamus, and show that MRI, using specific sequences of acquisition, enables the clear-cut identification of several hypothalamic areas, nuclei and white matter fascicles.

Materials and methods

Population

The study was carried out with the approval of the local ethical committee and informed consent was obtained from each subject. Twenty healthy volunteers (10 men and 10 women) with a normal clinical and neurological examination and subjected to a 1.5 T MRI were used in the present study. There was no difference between the age of men (21.7 +/-1.9 years) and women (21.3 +/-1 years) using a Student's test for independent samples (p> 0.05). The Body Mass Index (BMI) was not different between the 2 groups (men: 22.7 +/-2.7; women: 20.9 +/-1.5 ; p> 0.05; Student's test).

Conventional MRI

MRI was performed on a 1.5T scanner (ACHIEVA R1.5.1, Philips, Best, The Netherlands) using a 8-channel SENSE head coil. Sequences were designed to cover the entire thalamus and hypothalamus. The anatomical limits used were the genu of the corpus callosum anteriorly, and the posterior aspect of the callosal splenium, posteriorly. The protocol included T1-weighted 3D Fast Field Echo (T13D) (TR/TE: 25/4.6 ms; NEX: 2; flip angle: 30; FOV: 230/183/60; matrix: 272x216 mm; 75 slices; voxel size: 0.85/0.85/0.80 mm), T1weighted inversion-recovery (T1IR) (TR/TI/TE: 7910/400/15 ms; NEX: 2; FOV: 230/195/73; matrix: 512x261 mm; 20 slices; slice thickness: 3 mm; slice gap: 0.7 mm), T2-weighted Turbo Spin Echo (T2TSE) (TR/TE: 3500/100 ms; NEX: 4; flip angle: 90; TSE factor: 25; FOV: 260/183/80; matrix: 512x262 mm; 32 contiguous slices; slice thickness: 2.5 mm), T2-weighted 2D Fast Field Echo (T2FFE) (TR/TE: 646/23 ms; NEX: 2; flip angle: 18; FOV: 250/188/73; matrix: 304x171 mm; 20 slices; slice thickness: 3 mm), all in the coronal plane.

Anatomical and histological coronal sections

For the anatomical sections, a human brain was frozen at -10 ° C for one week and then cut in the coronal plane at 5 mm intervals. The brain was taken from a patient without neurological disorders, who donated his body to science in compliance with French laws on bioethics.

For the histological sections, the brains of five women were obtained from autopsies 6-48 hrs postmortem. After whole brain removal, blocks of 20 mm per side encompassing the hypothalamus were harvested with the optic chiasm as the anterior limit and the mamillary bodies as the posterior limit. Hypothalami were immersion-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 week, cryoprotected in 20% buffered sucrose for 48 h, embedded in Tissue Tek (Miles, Elkhart, IN, USA) and frozen in isopentane. Coronal cryostat sections (14 m) were mounted on chrome-alum-gelatin coated slides, air dried, and immersed in a solution of 0.3 % Sudan Black B (Sigma, St Quentin Fallavier, France) in 70 % ethanol for ten minutes or in Nissl stain for 4 minutes. Because of the likelihood of tissue distortion during the histological sectioning process, no attempt was made to fuse histological and MRI data. Histological analyses provided the anatomical landmarks that served as standards for the reliable identification of hypothalamic areas, nuclei and white matter fascicles in MRI coronal sections and volumes.

Data analysis

An anatomist and an experienced neuroradiologist reviewed all MRI sets in order to score the visibility of gray and white structures within the hypothalamus. Structures were identified visually based on our knowledge of specific landmarks obtained from anatomical-histological cross sections and with reference to previously published atlases [START_REF] Swaab | Functional neuroanatomy and neuropathology of the human hypothalamus[END_REF][START_REF] Koutcherov | Organization of the human paraventricular hypothalamic nucleus[END_REF][START_REF] Koutcherov | Organisation of the human dorsomedial hypothalamic nucleus[END_REF][START_REF] Koutcherov | Organization of the human medial preoptic nucleus[END_REF][START_REF] Mai | Atlas of the human brain, 3rd Edition[END_REF]; the landmarks used, such as the optic tract, the floor of the diencephalon, the third ventricle, and the fornix, were readily and reliably identifiable in MR scans. Each structure was given a score as follows: 1 (non-identifiable), 2 (identifiable but poorly delineated), and 3 (identifiable and welldelineated). The agreement between the two scorers for 20 brains, as assessed by Cohen's kappa coefficient was very good ( = 0.96, p < 0.0001).

All MRIs were reviewed on a Mac OS computer using the software OsiriX v. 3.2.1 (www.osirix-viewer.com). The gray and white structures that were targeted are summarized in Table 1. Other abbreviations, not expanded in Table 1, are summarized in Table 2.

For each subject, all T1 3D images acquired underwent automated linear registration onto standard Montreal Neurological Institute (MNI) coordinates system by maximizing the crosscorrelation between the individual image and the MNI template [START_REF] Collins | Automatic 3D Intersubject Registration of MR Volumetric Data in Standardized Talairach Space[END_REF]. Since the high-resolution images acquired did not encompass the entire head, which resulted in the loss of some landmarks usually used for automated linear registration such as skull, eyes and top of the neck, quality of each registration was carefully assessed visually using the MNI-Register tool. Registrations were found to be accurate for all subjects. Individual 3Dreconstructed MRI volumes were visually inspected and the coordinates of the center of each manually identified gray and white hypothalamic structure calculated using SPM8.

Statistical analysis

Differences between MRI sequences in terms of the detection of the hypothalamic nuclei were assessed using the Friedman test (a non-parametric equivalent of a one-sample repeated measures design). A p-value of less than 0.05 was considered significant. A Wilcoxon signed ranks test was then done to determine the best sequence for the identification of these structures.

Results

Anatomical sections were annotated using data from the literature, especially the atlas of Mai et al. [START_REF] Mai | Atlas of the human brain, 3rd Edition[END_REF] and review articles from Braak andBraak (Braak andBraak, 1987, 1992), [START_REF] Swaab | Functional neuroanatomy and neuropathology of the human hypothalamus[END_REF] and [START_REF] Koutcherov | Organization of the human paraventricular hypothalamic nucleus[END_REF][START_REF] Lustig | Hypothalamic obesity: the sixth cranial endocrinopathy[END_REF][START_REF] Koutcherov | Organization of the human medial preoptic nucleus[END_REF].

All data are summarized in Tables 3 (for white matter and preoptic area) and 4 (for anterior hypothalamic area, tuberal and mammillary regions). Figures 1, 3 and 5 illustrate a series of anatomical and histological sections in the coronal plane. In Figures 2,4 and 6 are illustrated a series of MRI sections in the coronal plane of the corresponding hypothalamic regions. All MRI images used in these figures were acquired from the same subject. Figure 7 shows the display of additional hypothalamic nuclei in different volunteers. Table 5 provides the average coordinates (n = 20 volunteers) of the identified hypothalamic areas, nuclei and white matter fascicles in anatomical MNI space.

White matter

The diagonal band (DB) was well identifiable both in T1 3D (high signal intensity) and in T1IR sequences (low signal intensity). It forms the posterior border of the anterior perforated substance (Fig. 7A).

The optic tract (opt) was remarkably easily identifiable in all sequences, except, in T1IR where it was still poorly defined in some cases. Each optic tract partially encircled the hypothalamus and the rostral portions of the crus cerebri (Fig 2,4).

The mammillothalamic tract (mt) is a small structure that was sometimes identified in T13D (high signal intensity) or T2TSE sequences (without a significant difference between these two sequences). It represents the dorsal component of the principal mammillary fasciculus (Fig. 567). It contains fibers that project from the medial mammillary nucleus to the anterior thalamic nuclei [START_REF] Carpenter | Core text of Neuranatomy, 3rd Edition[END_REF].

The principal mammillary fasciculus (pm), was most easily identifiable in T1 3D sequences (p=0,001), followed by T2 TSE and FFE. It was clearly identified in most cases under the shape of a small high-intensity structure, concave underneath and on the outside, surrounding the mammillary nucleus (Fig . 67).

The fornix (fx) is composed of arch-shaped white matter tracts connecting the hippocampus to the mammillary body (Fig 1 -7). The main parts of the fornix are the two crura, the commissure, the body and the two columns [START_REF] Carpenter | Core text of Neuranatomy, 3rd Edition[END_REF]. Each column further divides into pre-and postcommissural fornices (25 % and 75 % respectively) at the level of the anterior commissure. The precommissural fibers extend to the septal, lateral preoptic and anterior hypothalamic nuclei. The postcommissural fornix projects to the mammillary body and terminates mainly in the medial nucleus. The fornix was easily identifiable in all sequences except T1IR.

The anterior commissure (ac) crosses the median plane as a compact fiber bundle immediately in front of the anterior columns of the fornix (Fig. 1). It was easily identifiable in The medial forebrain bundle (mfb) is a complex group of fibers arising from the basal olfactory regions, the septal nuclei and the periamygdaloid region, which passes to and through the lateral preoptic and lateral hypothalamic regions [START_REF] Carpenter | Core text of Neuranatomy, 3rd Edition[END_REF]. This tract is well developed in lower vertebrates, but is small in humans (Fig. 1), and it was not possible to identify it in the different MRI sequences.

Preoptic area

This region constitutes the periventricular gray of the most rostral part of the third ventricle.

The medial preoptic nucleus (MPO) (Fig. 1) is a structure composed of predominantly small cells, indented laterally by the lateral hypothalamic area (LHA) and bulging medially toward the paraventricular nucleus (Pa) [START_REF] Koutcherov | Organization of the human medial preoptic nucleus[END_REF]. This nucleus was only identifiable in T13D sequence as a hyposignal (Fig. 2).

The uncinate nucleus (Un) has been described as being a tight group of medium-sized neurons invading the lateral boundary of the anterodorsal Pa and bordered laterally by the MPO (Fig. 1C) [START_REF] Braak | Anatomy of the human hypothalamus (chiasmatic and tuberal region)[END_REF][START_REF] Koutcherov | Organization of the human medial preoptic nucleus[END_REF]. However, none of the approaches used in the present study could identify this nucleus.

The intermediate nucleus (InM) (or sexually dimorphic nucleus/INAH-1) was revealed with Nissl staining as a compact nuclear gray formed of a homogeneous population of nerve cells (Fig 1 .C-D). The nucleus shows rounded contours and is located at the level of the optic chiasm between the supraoptic and paraventricular nucleus. The intermediate nucleus is known to be twice as large (0.20 mm 3 ) and contains twice as many cells in men as in women [START_REF] Swaab | Functional neuroanatomy and neuropathology of the human hypothalamus[END_REF]. Because of its small size, this nucleus was not seen using MRI either in male or in female volunteers.

Anterior hypothalamic area

This region, also called the chiasmatic region, contains two of the most striking and sharply defined hypothalamic nuclei, the paraventricular nucleus and the supraoptic nucleus (fig. 1).

The paraventricular nucleus of the hypothalamus (Pa) forms a vertical plate of densely packed cells immediately beneath the ependyma of the third ventricle (Fig. 1, 3) [START_REF] Carpenter | Core text of Neuranatomy, 3rd Edition[END_REF][START_REF] Koutcherov | Organization of the human paraventricular hypothalamic nucleus[END_REF]. Ventral to the fornix, the main body of the Pa rises vertically along the wall of the third ventricle throughout the preoptic and tuberal regions of the hypothalamus [START_REF] Koutcherov | Organization of the human paraventricular hypothalamic nucleus[END_REF]. The paraventricular nucleus was seen very clearly in T1IR and in T13D sequences (Fig. 2). It was also visible in T2 sequences, but not as sharply.

The supraoptic nucleus (SO) caps the optic chiasm and straddles the optic tract laterally [START_REF] Mai | Evidence for subdivisions in the human suprachiasmatic nucleus[END_REF]. This nucleus is composed mainly of uniformly large cells (Fig 1). The supraoptic nucleus was usually correctly identifiable using T13D; it was not visible in other sequences (Fig 2).

The suprachiasmatic nucleus (SCh) is small and situated above the optic chiasm, close to the ependymal lining of the third ventricle (Fig 1). Anterolaterally, it blends into the chiasmatic gray, and posteriorly it gradually merges with the retrochiasmatic nucleus. It receives projections from the retina and is involved in the circadian rhythm [START_REF] Swaab | Functional neuroanatomy and neuropathology of the human hypothalamus[END_REF]. It is sexually dimorphic -it is more elongated in women and more spherical in men [START_REF] Swaab | Functional neuroanatomy and neuropathology of the human hypothalamus[END_REF]. It was occasionally possible to pinpoint the suprachiasmatic nucleus in T13D sequences, even though its boundaries remained poorly delineated.

The retrochiasmatic nucleus (RC), also called the anterior hypothalamic nucleus, is localized above, between and below the fibers of the supraoptic commissures (Fig. 7B).

Laterally and inferiorly it borders upon the infundibular (arcuate) nucleus of the tuberal region. It corresponds to the less well-differentiated gray of the anterior hypothalamic region.

In the coronal plane, it was in isosignal but not easily identifiable, and was never seen in T1IR.

The lateral hypothalamic area (LHA) is bounded medially by the mammillothalamic tract and the anterior column of the fornix (Fig 1, 3, 5); its lateral boundary is the medial margin of the internal capsule and the subthalamic region. LHA could be identified using T13D MRI sequences (Fig 2 ,7).

Tuberal region

In this region, the hypothalamus reaches its widest extent. Medially, it is generally possible to distinguish a ventromedial and a posteromedial nucleus. The ventromedial nucleus of the hypothalamus (VMH) is the largest cell group in the tuberal region. It has a round or oval shape and is surrounded by a cell-poor zone that can helps to delineate its boundaries. In the coronal plane it creates a small bulge between the median eminence and the optic tract (Fig 3C), which made identification relatively easy by MRI, particularly in T13D (Fig. 4).

The dorsomedial hypothalamic nucleus (DMH) is a less distinct aggregation of cells that borders the third ventricle. Indeed, the DMH is composed of a poorly differentiated group of loosely dispersed neurons whose morphology resembles the morphology of neurons of the surrounding hypothalamic gray [START_REF] Braak | Anatomy of the human hypothalamus (chiasmatic and tuberal region)[END_REF][START_REF] Koutcherov | Organisation of the human dorsomedial hypothalamic nucleus[END_REF]. The DMH covers the anterior and superior poles of the ventromedial nucleus of the hypothalamus. Its identification by MRI was difficult and, again, the T13D sequence was the most useful in highlighting it (Fig. 4,7).

The horseshoe-shaped infundibular nucleus (inf) (or arcuate nucleus of the hypothalamus) is located in the most ventral part of the third ventricle near the entrance to the infundibular recess and extends into the median eminence (ME) [START_REF] Koutcherov | Organization of human hypothalamus in fetal development[END_REF] (Fig. 3). The small cells of this nucleus are in close contact with the ependyma lining the ventricle. This nucleus was clearly visible in T13D (hypersignal) or T1IR (hyposignal) sequences (Fig. 4). T2 sequences were less useful in highlighting it.

Finally, the perifornical nucleus (PeF) was not identified in MRI, although it was easily identifiable by Nissl staining in the basal and lateral part of the column of the fornix (Fig 3).

Mammillary region

This region consists of the mammillary bodies and the dorsally located posterior hypothalamic nucleus (fig. 4). Each mammillary body is composed almost entirely of the large spherical medial mammillary nucleus (MM), consisting of relatively small cells invested by a capsule of myelinated fibers. The medial part of this nucleus was clearly visible in MRI, especially in T2TSE, T1IR and T13D sequences (Fig 5), without a significant difference between these sequences (Table 4). The lateral part of the mammillary body, the lateral mammillary nucleus (LM), a well-defined group of large cells (Fig 5C ), were not distinctly visible in MRI.

The lateral tuberal nucleus (LTu) could never be identified in MRI. Between the LTu and the MM, and surrounding them, are found neurons of the tuberomammillary nucleus (TM), part of which accompanies the fornix (forming the perifornical nucleus). This nucleus was sometimes visible as a hyposignal in T13D sequences.

The posterior hypothalamic area (PH) lies dorsal to the mammillary body (Fig. 5) and caudal portions of the ventromedial nucleus of the hypothalamus. The large cells of the posterior hypothalamus are especially numerous in humans and extend caudally into the periaqueductal gray. The posterior hypothalamic area, limited on the outside by the mammillothalamic tract (mt), was fairly visible as hyposignal in T13D thanks to the identification of the mammillothalamic tract. Similarly, the posterior part of the lateral hypothalamic area was identifiable by MRI because it was limited on the inside by the mammillary body and the mammillothalamic tract (fig. 3).

Discussion

This study was designed to provide an anatomical basis for the development of new treatment strategies involving functional neurosurgery by mapping gray and white structures in the living human hypothalamus. Indeed, the hypothalamus serves as a crucial centre for the integration and coordination of various brain functions, and as such, it is prone to involvement in several major brain disorders [START_REF] Saper | The hypothalamus[END_REF][START_REF] Overeem | The hypothalamus in episodic brain disorders[END_REF][START_REF] Sisk | The neural basis of puberty and adolescence[END_REF][START_REF] Swaab | The human hypothalamus in metabolic and episodic disorders[END_REF][START_REF] Aziz | Hypothalamic dysfunction and neuroendocrine and metabolic alterations in Huntington's disease: clinical consequences and therapeutic implications[END_REF][START_REF] Bao | The stress system in depression and neurodegeneration: focus on the human hypothalamus[END_REF][START_REF] Willis | Parkinson's disease as a neuroendocrine disorder of circadian function: dopamine-melatonin imbalance and the visual system in the genesis and progression of the degenerative process[END_REF][START_REF] Gordon | Clinical practice. Functional hypothalamic amenorrhea[END_REF][START_REF] Kalsbeek | Vasopressin and the output of the hypothalamic biological clock[END_REF]. Precise MRI identification of the gray and white structures of the hypothalamus would also be of great help in developing new research approaches aimed at investigating hypothalamic connectivity [START_REF] Lemaire | White matter connectivity of human hypothalamus[END_REF] and the functional and/or metabolic changes that occur naturally within the hypothalamus with fluctuating physiological conditions [START_REF] Baroncini | Sex steroid hormones-related structural plasticity in the human hypothalamus[END_REF] and/or upon changes to the external environment [START_REF] Goldstein | Sex differences in stress response circuitry activation dependent on female hormonal cycle[END_REF], as well as in routine clinical examination using 1.5T MRI. Besides, detailed knowledge of hypothalamic topography in vivo is a prerequisite for the future development of automatic algorithms [START_REF] Wang | A learning-based wrapper method to correct systematic errors in automatic image segmentation: consistently improved performance in hippocampus, cortex and brain segmentation[END_REF] that enable the accurate segmentation of the hypothalamus and its subregions [START_REF] Goldstein | Hypothalamic abnormalities in schizophrenia: sex effects and genetic vulnerability[END_REF], and thus the performance of morphometric MRI studies on an automated scale.

Our results are based on anatomically and histologically processed sections of the human diencephalon and hypothalamus, respectively, and their comparison with magnetic resonance images in living volunteers. The Sudan Black B served as the initial basis for the identification of fiber tracts. MRI was then used to identify them with high spatial resolution.

The reproducibility of our results in many cases and comparison with existing data in humans [START_REF] Carpenter | Core text of Neuranatomy, 3rd Edition[END_REF][START_REF] Mai | Atlas of the human brain, 3rd Edition[END_REF] allowed us to identify the bulk of the fiber tracts with reasonable confidence. Furthermore the use of Nissl staining provided supplemental criteria for the confirmation of anatomical limits and discrimination between the different tracts in addition to enabling the identification of gray structures, some of which were reliably identified by MRI, in conjunction with previously published neuroanatomical studies [START_REF] Swaab | Functional neuroanatomy and neuropathology of the human hypothalamus[END_REF][START_REF] Koutcherov | Organization of the human paraventricular hypothalamic nucleus[END_REF][START_REF] Koutcherov | Organisation of the human dorsomedial hypothalamic nucleus[END_REF][START_REF] Koutcherov | Organization of the human medial preoptic nucleus[END_REF][START_REF] Mai | Atlas of the human brain, 3rd Edition[END_REF].

Our qualitative analyses convincingly show that specific hypothalamic nuclei, areas and white matter tracts can be consistently identified using specific 1.5T MRI. To enable the identification of intra-hypothalamic structures, a compromise between the thickness of the sections and the signal-to-noise ratio had to be found. High-resolution images were thus acquired from very thin sections using weighted T13D, T1IR, T2TSE and T2FFE sequences that did not encompass the entire head, in order to keep acquisition time within reasonable limits for subjects. However, the MRI volumes acquired still retained key anatomical landmarks necessary for reliable automated linear registration onto the standard MNI coordinates system [START_REF] Collins | Automatic 3D Intersubject Registration of MR Volumetric Data in Standardized Talairach Space[END_REF] enabling a probabilistic distribution of hypothalamic subregions. Interestingly, MNI coordinates can easily be transposed to Talairach space [START_REF] Talairach | Co-planar stereotaxic atlas of the human brain Stuttgart[END_REF]) by using the appropriate transformations [START_REF] Laird | Comparison of the disparity between Talairach and MNI coordinates in functional neuroimaging data: validation of the Lancaster transform[END_REF].

The present study shows that the use of specific 1.5T MRI sequences with an acquisition time compatible with routine examination approaches the resolution obtained from anatomical/histological sections. The use of such high-resolution MRI sequences noticeably facilitates the accurate identification of white matter bundles within the hypothalamus, which are subject to structural alteration in some disorders, and which are also key landmarks used in the planning of surgical approaches and electrode placement in patients. The fornix, which is composed of arch-shaped white matter fibers connecting the hippocampus to the mammillary body [START_REF] Carpenter | Core text of Neuranatomy, 3rd Edition[END_REF][START_REF] Mark | The fornix[END_REF], has been suggested to play an important role in memory function [START_REF] Tsivilis | A disproportionate role for the fornix and mammillary bodies in recall versus recognition memory[END_REF][START_REF] Laxton | A phase I trial of deep brain stimulation of memory circuits in Alzheimer's disease[END_REF] and has been shown to be morphologically altered in patients with temporal epilepsy [START_REF] Supprian | The fornix of the human brain: evidence of left/right asymmetry on axial MRI scans[END_REF][START_REF] Freeman | MR imaging and spectroscopic study of epileptogenic hypothalamic hamartomas: analysis of 72 cases[END_REF][START_REF] Concha | In vivo diffusion tensor imaging and histopathology of the fimbria-fornix in temporal lobe epilepsy[END_REF]. Its detailed examination is required for the surgical planning of transcallosal or transventricular tumor resection [START_REF] Winkler | Transcallosal approach to the third ventricle: normative morphometric data based on magnetic resonance imaging scans, with special reference to the fornix and forniceal insertion[END_REF]. Within the mammillary region, the fornix that runs in between the medial hypothalamus, where the posterior hypothalamic area resides, and the lateral hypothalamic area, a candidate target area for deep brain stimulation to trigger weight loss, constitutes, together with the mammillo-thalamic tract a useful anatomical landmark for the placement of deep brain stimulation electrodes for chronic cluster headaches and in putative treatments for obesity, respectively. The anterior commissure, along with the posterior commissure, is a classic marker used to determine the bi-commissural line, an essential landmark for the calculation of stereotaxic coordinates. The diagonal band consists of various fiber bundles in association with scattered large cells, which provide direct cholinergic projections to the amygdala, hippocampus and cortical mantle [START_REF] Hedreen | Topography of the magnocellular basal forebrain system in human brain[END_REF][START_REF] Zaborszky | Stereotaxic probabilistic maps of the magnocellular cell groups in human basal forebrain[END_REF].

Intriguingly, postmortem analysis has demonstrated anatomical abnormalities in the vertical limb of the diagonal band of Broca in autistic patients [START_REF] Kemper | Neuropathology of infantile autism[END_REF]. The size and the number of magnocellular neurons have been shown to vary during the course of the disease. Recent MRI studies have shown that changes in the volume of the diagonal band of Broca are associated with a global decline in cognitive status in patients at high risk of developing Alzheimer's disease [START_REF] Grothe | Reduction of basal forebrain cholinergic system parallels cognitive impairment in patients at high risk of developing Alzheimer's disease[END_REF].

The current work also shows that some hypothalamic gray structures can be identified using 1.5T MRI with specific high-resolution sequences. The ventromedial nucleus of the hypothalamus and the infundibular (arcuate) nucleus are readily and consistently identifiable using T13D sequences, whereas T1IR and T2TSE are optimal sequences in which to spot the paraventricular nucleus of the hypothalamus and the mammillary body, respectively. The mammillary bodies have repeatedly been implicated in amnesia although their importance has been questioned (see for review [START_REF] Vann | The mammillary bodies: two memory systems in one?[END_REF]). Damage to mammillary body efferents is characteristic of thalamic strokes that cause amnesia, and mammillary body atrophy is always found in Korsakoff's amnesic syndrome, even though this atrophy may not always be the best predictor of amnesia. Although more restricted, mammillary body damage, for example, by tumors, has also been associated with amnesia [START_REF] Kapur | Anterograde but not retrograde memory loss following combined mammillary body and medial thalamic lesions[END_REF][START_REF] Tsivilis | A disproportionate role for the fornix and mammillary bodies in recall versus recognition memory[END_REF], the deficits can be mild and transient [START_REF] Hildebrandt | Are some memory deficits unique to lesions of the mammillary bodies?[END_REF]. MRIbased anatomical studies have shown that the volume of the region containing the mammillary bodies is increased in schizophrenic patients and non-psychotic first-degree relatives, and that this enlargement is associated with anxiety [START_REF] Goldstein | Hypothalamic abnormalities in schizophrenia: sex effects and genetic vulnerability[END_REF]. Deep brain stimulation of the mammillary bodies and the mammillothalamic tract are currently being investigated as potential treatments for seizure control [START_REF] Van Rijckevorsel | Deep EEG recordings of the mammillary body in epilepsy patients[END_REF][START_REF] Khan | High frequency stimulation of the mamillothalamic tract for the treatment of resistant seizures associated with hypothalamic hamartoma[END_REF]. The paraventricular nucleus of the hypothalamus is a complex motor relay, integrating essential endocrine and autonomic responses that sustain homeostasis [START_REF] Thompson | Structural characterization of a hypothalamic visceromotor pattern generator network[END_REF][START_REF] Watts | Neural network interactions and ingestive behavior control during anorexia[END_REF]. Neuroanatomical studies have reported alterations in the cytoarchitecture of this nucleus in patients with homeostatic control deficits, such as Prader-Willi syndrome, diabetes insipidus, depression and AIDS [START_REF] Swaab | Functional neuroanatomy and neuropathology of the human hypothalamus[END_REF][START_REF] Swaab | Development of the human hypothalamus[END_REF][START_REF] Overeem | The hypothalamus in episodic brain disorders[END_REF][START_REF] Swaab | The human hypothalamus in metabolic and episodic disorders[END_REF]. Lesions in the region of the paraventricular nucleus are associated with hyperactivity of the autonomic nervous system and with diabetes insipidus [START_REF] Carmel | Surgical syndromes of the hypothalamus[END_REF][START_REF] Ropper | Acute autonomic emergencies and autonomic storms[END_REF]. As for the mammillary bodies, MRI studies show that the volume of the paraventricular nucleus of the hypothalamus is abnormally enlarged in schizophrenia, which is associated with high rates of endocrine disorders [START_REF] Goldstein | Hypothalamic abnormalities in schizophrenia: sex effects and genetic vulnerability[END_REF]. The ventromedial nucleus of the hypothalamus is consistently associated with the regulation of sexual behavior, appetite and metabolism, and had been implicated in the etiology of obesity [START_REF] Mcclellan | Development of the ventromedial nucleus of the hypothalamus[END_REF][START_REF] Blaustein | Neuroendocrine regulation of feminine sexual behavior: lessons from rodent models and thoughts about humans[END_REF][START_REF] Mietus-Snyder | Childhood obesity: adrift in the "limbic triangle[END_REF]. Like its near neighbor, the infundibular nucleus (also termed the arcuate nucleus of the hypothalamus), damage to the ventromedial nucleus of the hypothalamus has been linked to obesity syndromes in humans [START_REF] Bray | Manifestations of hypothalamic obesity in man: a comprehensive investigation of eight patients and a reveiw of the literature[END_REF][START_REF] Carmel | Surgical syndromes of the hypothalamus[END_REF][START_REF] Lustig | Hypothalamic obesity: the sixth cranial endocrinopathy[END_REF][START_REF] Swaab | The human hypothalamus in metabolic and episodic disorders[END_REF]. The infundibular nucleus plays a key role in relaying peripheral information to the metabolic and the reproductive brain [START_REF] Dudas | Three-dimensional representation of the neurotransmitter systems of the human hypothalamus: inputs of the gonadotrophin hormone-releasing hormone neuronal system[END_REF][START_REF] Morton | Central nervous system control of food intake and body weight[END_REF][START_REF] Hill | Hypothalamic pathways linking energy balance and reproduction[END_REF][START_REF] Simpson | Hypothalamic regulation of food intake and clinical therapeutic applications[END_REF]. The ventromedial hypothalamus, which comprises both the ventromedial nucleus of the hypothalamus and the infundibular nucleus, might be an interesting site to target for body weight control in morbidly obese patients [START_REF] Halpern | Deep brain stimulation in the treatment of obesity[END_REF]. Another interesting candidate for deep brain stimulation in the treatment of obesity is the lateral hypothalamic area, which is a diffuse structure that can be seen using high-resolution T13D MRI sequences and that has long been implicated in the control of feeding behavior and energy expenditure [START_REF] Sawchenko | Toward a new neurobiology of energy balance, appetite, and obesity: the anatomists weigh in[END_REF]. Interestingly, the stereotactic electrocoagulation of this hypothalamic area in obese patients has been shown to cause significant, although transient, appetite suppression and a slight reduction in weight [START_REF] Quaade | Stereotaxic stimulation and electrocoagulation of the lateral hypothalamus in obese humans[END_REF]. In contrast, bilateral lesions have been associated with anorexia and emaciation [START_REF] Carmel | Surgical syndromes of the hypothalamus[END_REF]. Finally, the dorsomedial hypothalamic nucleus, which may constitute an important neural relay in the central regulation of homeostasis in humans [START_REF] Dai | Postmortem anterograde tracing of intrahypothalamic projections of the human dorsomedial nucleus of the hypothalamus[END_REF][START_REF] Elias | Characterization of CART neurons in the rat and human hypothalamus[END_REF][START_REF] Koutcherov | Organisation of the human dorsomedial hypothalamic nucleus[END_REF], as it is known to be in rodents [START_REF] Bellinger | The dorsomedial hypothalamic nucleus and its role in ingestive behavior and body weight regulation: lessons learned from lesioning studies[END_REF][START_REF] Thompson | Structural characterization of a hypothalamic visceromotor pattern generator network[END_REF], can also be spotted using the T13D MRI sequences. The target for deep brain stimulation in the treatment of chronic cluster headaches, i.e., the posterior hypothalamic area [START_REF] Leone | Stereotactic stimulation of posterior hypothalamic gray matter in a patient with intractable cluster headache[END_REF][START_REF] Fontaine | Safety and efficacy of deep brain stimulation in refractory cluster headache: a randomized placebo-controlled double-blind trial followed by a 1-year open extension[END_REF][START_REF] Franzini | Deep brain stimulation of the posteromedial hypothalamus: indications, long-term results, and neurophysiological considerations[END_REF], is also clearly identifiable using high-resolution T13D sequences in 1.5T MRI.

Conclusions

To our knowledge, the present study is the first to show that distinct hypothalamic gray and white structures can be readily and accurately identified and the probabilistic distribution of these hypothalamic subregions determined using 1.5T MRI with specific high-resolution sequences. By providing new insights into the anatomy of the human hypothalamus, this study should be useful both in developing new treatment strategies involving functional stereotactic surgery, as well as in the morphological inspection of the hypothalamus of patients with hypothalamus-related disorders in everyday clinical practice. Table 3. Results of the comparative study between the different sequences for white matter tracts and the preoptic Area. For each given white mater fascicle or the preoptic area, Friedman's test was used to determine whether there was any difference among the scores obtained with the four distinct MRI sequences. Scores were 1 for non-identifiable, 2 for identifiable but poorly delineated, and 3 for identifiable and well-delineated. MRI sequences were then ranked from the most to the least efficient: Sequence 1 > Sequence 2 > Sequence 3 > Sequence 4. A Wilcoxon signed ranks test was used to perform pairwise comparisons between MRI sequences (Sequence 1 vs. Sequence 2, Sequence 2 vs. Sequence 3 and Sequence 3 vs. Sequence 4). MRI sequences used: T1-weighted 3D Fast Field Echo (T13D), T1-weighted inversion-recovery (T1IR), T2-weighted Turbo Spin Echo (T2TSE), T2weighted 2D Fast Field Echo (T2FFE).

Table 4. Results of the comparative study between the different sequences for the Anterior Hypothalamic Area, Tuberal and Mammillary regions. For each given nucleus or area, Friedman's test was used to determine whether there was any difference among the scores obtained with the four distinct MRI sequences. Scores were 1 for non-identifiable, 2 for identifiable but poorly delineated, and 3 for identifiable and well-delineated. MRI sequences were then ranked from the most to the least efficient: Sequence 1 > Sequence 2 > Sequence 3 > Sequence 4. A Wilcoxon signed ranks test was used to perform pairwise comparisons between MRI sequences (Sequence 1 vs. Sequence 2, Sequence 2 vs. Sequence 3 and Sequence 3 vs. Sequence 4). MRI sequences used: T1-weighted 3D Fast Field Echo (T13D), T1-weighted inversion-recovery (T1IR), T2-weighted Turbo Spin Echo (T2TSE), T2weighted 2D Fast Field Echo (T2FFE).

Table 5. Coordinates of the identified hypothalamic areas, nuclei and white matter fascicles in the anatomical MNI space. Coordinates acquired from 20 healthy volunteers are presented as mean  SEM in mm.

  T2 and T13D sequences (Fig 2).

Figure 1 .Figure 2 .

 12 Figure 1. Representative coronal anatomical and histological atlas sections of the anterior hypothalamic area and the preoptic region. A, anatomical section through the anterior commissure and the medial preoptic nucleus. B, Histological section colored with Sudan Black B, which highlights the anterior commissure and the optic tracts. C, Histological section stained with Hoechst through the medial preoptic nucleus. D, Magnification of the area

Figure 3 .

 3 Figure 3. Representative coronal anatomical and histological atlas sections of the tuberal region. A, Anatomical section through the median eminence and the infundibular (arcuate) nucleus. B, Histological section stained with Sudan Black B, which highlights the fornix. C,

Figure 4 .

 4 Figure 4. Representative coronal 1.5T MRI atlas sections of the tuberal region. A, T1-

Figure 5 .

 5 Figure 5. Representative coronal anatomical and histological atlas sections of the mammillary region. A, Anatomical section through the mammillary nucleus and the posterior part of the lateral hypothalamic area. B, Histological section stained with Sudan Black B, which highlights the principal mammillary fasciculus. C, Histological section stained with Hoechst through the mammillary bodies. D, Magnification of the area enclosed in red in C,

Figure 6 .

 6 Figure 6. Representative coronal 1.5T MRI atlas sections of the mammillary region. A, T1-

Figure 7 .

 7 Figure 7. Representative 1.5T MRI atlas sections identifying the diagonal bands (DB) of Broca (A), the retrochiasmatic nucleus (RC) (B), the suprachiasmatic nucleus (SCh) and the medial preoptic nucleus (MPO) (C), the dorsomedial nucleus of the hypothalamus (DMH), the fornix (fx) and the lateral hypothalamic area (LHA) (C,D), the ventromedial nucleus of the hypothalamus (VMH) (E), and the principal mammillary fasciculus (pm) and the mammillothalamic tract (mt) (F). MRI images were obtained from different volunteers. Scale bars = 10

Table 1 .

 1 Gray nuclei and white matter tracts identified by MRI

	Legends :

Table 2 .

 2 Other abbreviations, not listed in table 1.

Acknowledgments

The authors wish to thank M. Demeulaere, G. Lefebvre and F. Stevendart (Laboratory of Anatomy) for their skillful help during dissections and the 20 volunteers who agreed to participate in this study.


Jerome Clasadonte

Pierre Poulain

Naresh Kumar Hanchate

Gabriel Corfas

Sergio R Ojeda
email: ojedas@ohsu.edu

Vincent Prevot
email: vincent.prevot@inserm.fr

Prostaglandin E 2 release from astrocytes triggers gonadotropin-releasing hormone (GnRH) neuron firing via EP2 receptor activation

Keywords: LHRH, glia-to-neuron signaling, gliotransmission, cyclooxygenase, hypothalamus \body

Astrocytes in the hypothalamus release prostaglandin E 2 (PGE 2 ) in response to cell-cell signaling initiated by neurons and glial cells. Upon release, PGE 2 stimulates the secretion of gonadotropin-releasing hormone (GnRH), the neuropeptide that controls reproduction, from hypothalamic neuroendocrine neurons. Whether this effect on GnRH secretion is accompanied by changes in the firing behavior of these neurons is unknown. Using patchclamp recording we demonstrate that PGE 2 exerts a dose-dependent postsynaptic excitatory effect on GnRH neurons. These effects are mimicked by an EP2 receptor agonist and attenuated by protein kinase A inhibitors. The acute blockade of prostaglandin synthesis by indomethacin or the selective inhibition of astrocyte metabolism by fluoroacetate suppresses the spontaneous firing activity of GnRH neurons in brain slices. Similarly, GnRH neuronal activity is reduced in mice with impaired astrocytic PGE 2 release due to defective erbB signalling in astrocytes. These results indicate that astrocyte-to-neuron communication in the hypothalamus is essential for the activity of GnRH neurons and suggest that PGE 2 acts as a gliotransmitter within the GnRH neurosecretory system.

Introduction

It is increasingly clear that astrocytes play an important role in maintaining central nervous system function [START_REF] Gordon | Astrocyte-mediated distributed plasticity at hypothalamic glutamate synapses[END_REF][START_REF] Henneberger | Long-term potentiation depends on release of D-serine from astrocytes[END_REF][START_REF] Panatier | Glia-derived D-serine controls NMDA receptor activity and synaptic memory[END_REF] and controlling key bodily processes, such as breathing (4), sleep [START_REF] Halassa | Astrocytic modulation of sleep homeostasis and cognitive consequences of sleep loss[END_REF], and reproduction [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF]. Because of their perivascular and interneuronal localization, astrocytes are well positioned to sense afferent neuronal and blood-borne signals, and ideally suited for the temporal and spatial propagation of these signals [START_REF] Barres | The mystery and magic of glia: a perspective on their roles in health and disease[END_REF][START_REF] Haydon | Astrocyte control of synaptic transmission and neurovascular coupling[END_REF][START_REF] Iadecola | Glial regulation of the cerebral microvasculature[END_REF]. The activation of astrocytes leads to the release of gliotransmitters [START_REF] Haydon | Astrocyte control of synaptic transmission and neurovascular coupling[END_REF][START_REF] Parpura | Gliotransmission: Exocytotic release from astrocytes[END_REF] that trigger rapid responses in neighboring cells and thus contribute to the region-specific homeostatic regulation of neuronal function.

In the hypothalamus, astrocytes regulate the secretory activity of neuroendocrine neurons [START_REF] Hatton | Function-related plasticity in hypothalamus[END_REF][START_REF] Ojeda | Glia-to-neuron signaling and the neuroendocrine control of female puberty[END_REF][START_REF] Prevot | Function-related structural plasticity of the GnRH system: a role for neuronal-glial-endothelial interactions[END_REF][START_REF] Theodosis | Activity-dependent structural and functional plasticity of astrocyte-neuron interactions[END_REF]. A subset of such neurons secretes the decapeptide gonadotropin hormonereleasing hormone (GnRH), which controls both the initiation of puberty and adult reproductive function. In rodents, GnRH neurons are mostly located in the preoptic region of the ventral forebrain. They project to the median eminence of the hypothalamus, where GnRH is released into the pituitary portal blood for delivery to the anterior pituitary. In the pituitary, GnRH elicits the secretion of luteinizing hormone and follicle-stimulating hormone, which stimulate gametogenesis and gonadal hormone secretion and thus support reproductive function. It is now clear that the secretory activity of GnRH neurons is controlled by both neuronal and glial input [START_REF] Ojeda | Glia-to-neuron signaling and the neuroendocrine control of female puberty[END_REF][START_REF] Prevot | Function-related structural plasticity of the GnRH system: a role for neuronal-glial-endothelial interactions[END_REF][START_REF] Herbison | Physiology of the Gonadotropin-Releasing Hormone Neuronal Network[END_REF][START_REF] Moenter | Mechanisms underlying episodic gonadotropin-releasing hormone secretion[END_REF]. While glutamate is a key neurotransmitter involved in the trans-synaptic activation of GnRH neurons [START_REF] Brann | Excitatory amino acids: evidence for a role in the control of reproduction and anterior pituitary hormone secretion[END_REF][START_REF] Urbanski | A role for N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty[END_REF], prostaglandin E 2 (PGE 2 ) mediates cell-cell communication between astrocytes and GnRH neurons [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF][START_REF] Ma | Hypothalamic astrocytes respond to transforming growth factor-alpha with the secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone[END_REF][START_REF] Rage | Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway[END_REF]. A functional connection between the two systems is demonstrated by the ability of glutamate to elicit PGE 2 release from astrocytes [START_REF] Bezzi | Prostaglandins stimulate calcium-dependent glutamate release in astrocytes[END_REF][START_REF] Dziedzic | Neuron-to-glia signaling mediated by excitatory amino acid receptors regulates ErbB receptor function in astroglial cells of the neuroendocrine brain[END_REF][START_REF] Zonta | Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation[END_REF]. In the hypothalamus, the glutamate-dependent activation of PGE 2 release involves astroglial erbB signaling [START_REF] Dziedzic | Neuron-to-glia signaling mediated by excitatory amino acid receptors regulates ErbB receptor function in astroglial cells of the neuroendocrine brain[END_REF], a crucial component of the cell-cell communication process used by astrocytes to facilitate neuroendocrine reproductive development and adult function [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF][START_REF] Prevot | erbB-1 and erbB-4 receptors act in concert to facilitate female sexual development and mature reproductive function[END_REF]. Whether PGE 2 affects the electrical activity of GnRH neurons is, however, unknown. Here we report a remarkably potent postsynaptic excitatory effect of PGE 2 on GnRH neurons that requires the activation of the EP2 subclass of PGE 2 receptors and of cAMP/PKA-mediated downstream signaling pathways.

Results

PGE 2 activates adult GnRH neurons in a potent and reversible manner

We used whole-cell patch-clamp recording to study the electrical activity of 193 GnRH neurons in brain slices containing the preoptic region from 175 adult transgenic mice (61 males and 114 females) expressing green fluorescent protein (GFP) under the control of the GnRH promoter [START_REF] Spergel | GABA-and glutamate-activated channels in green fluorescent protein-tagged gonadotropinreleasing hormone neurons in transgenic mice[END_REF]. The pipette solution used (ps1) resulted in an average resting potential of -75.54 ± 0.38 mV (n=151) and an input resistance of 1417.76 ± 43.07 M (n=97). In this configuration, 87% of the neurons remained silent at the resting potential (Supplementary results and Suppl. Fig. 1). Bath application of PGE 2 resulted in a striking, dose-dependent depolarizing effect on GnRH neurons (Fig. 1A-C), with an EC 50 of 0.018 µM (Fig. 1C,D). Among all the GnRH neurons treated with 1 µM PGE 2 , 78% (91 out of 116) depolarized rapidly (within 10 to 190 s; mean 41.00 ± 3.21 s) (Fig. 1A-C). PGE 2 -induced (1 µM) depolarization had a mean amplitude of 8.95 ± 0.32 mV (n=91) and was accompanied by the sustained generation of action potentials (Fig. 1A-C) and a decrease in membrane resistance (Fig. 1C). Interestingly, 33 cells out of 91 responded to PGE 2 with bursting activity (Fig. 1B) similar to that which occurred spontaneously in some neurons (Suppl. Fig. 1B,C) and which is thought to be essential for neuropeptide secretion from neuroendocrine cells [START_REF] Leng | The origins and significance of pulsatility in hormone secretion from the pituitary[END_REF]. The PGE 2 excitatory effect was fully reversible in 73 neurons (Fig. 1A-C), whereas 18 neurons did not recover their basal membrane potential after cessation of the PGE 2 treatment. No difference in the response of GnRH neurons to PGE 2 was detected between animals of different sex or stages of the estrous cycle (Suppl. Table 1). Because PGE 2 has previously been shown to stimulate glutamate release from astrocytes [START_REF] Bezzi | Prostaglandins stimulate calcium-dependent glutamate release in astrocytes[END_REF], we next examined the effects of PGE 2 on GnRH-GFP neurons in the presence of 20 µM CNQX and 100 µM DL-AP5 (see methods), to block AMPA/kainate and NMDA receptors, respectively. Under these conditions, the membrane depolarization induced by 1 µM PGE 2 was not altered (10.62 ± 1.20 mV, n=4; Fig. 1E), indicating that glutamate is not an intermediary in the excitatory effects of PGE 2 on GnRH neuronal activity. PGE 2 maintained its potent stimulatory effect on the firing behavior of GnRH neurons recorded using the loose patch-clamp configuration (Fig. 1F). This indicates that the dilution of the intracellular compartment by the patch electrode medium in the whole-cell configuration does not alter the response of GnRH neurons to PGE 2 . In the loose patchclamp configuration, GnRH neurons were spontaneously active and all responded to the addition of 1 µM PGE 2 to the perfusion medium by a transient acceleration of the action potential discharge (0.51±0.15 Hz before treatment vs. 1.35±0.12 Hz with bath application of PGE 2 , t test; p < 0.05; n=5; Fig. 1F). To investigate whether PGE 2 had similarly consistent excitatory effects on the electrical activity of non-GnRH neurons in the preoptic region, 14 non-GFP-labeled neurons located near GnRH-GFP neurons were recorded. While 10 neurons responded to the bath application of 1 µM PGE 2 with either an acceleration (n=6) or an inhibition (n=4, Fig. 1G) of their firing pattern, four were insensitive to the prostaglandin.

PGE 2 elicits membrane depolarization of GnRH neurons via a postsynaptic effect involving activation of an inward current

The effect of PGE 2 was further investigated in the presence of tetrodotoxin (TTX; 0.5-1 µM) to block action potential-dependent synaptic transmission, in addition to the glutamate and GABA A receptor antagonists DL-AP5 (100 µM), CNQX (20 µM) and bicuculline (20 µM) to block ionotropic receptor mediated presynaptic inputs. Under these conditions, PGE 2 (1 µM) consistently depolarized the membrane potential of GnRH neurons (Fig. 2A) with an average depolarization of 7.72 ± 0.48 mV (n=10). The current-voltage relationship before and after applying PGE 2 (1 µM) was obtained by injecting a series of square wave currents from -70 to +70 pA (Fig. 2B1,B2). The input resistance was calculated based on the linear part of the current-voltage curve (Fig. 2B3). PGE 2 (1 µM) decreased the input resistance by 27.21 ± 2.38% (n=10), indicating an increase in conductance. These results suggest that PGE 2 triggers firing in GnRH neurons via a direct postsynaptic mechanism.

To identify the conductance involved in the PGE 2 -induced membrane depolarization, we performed voltage-clamp recordings at a holding potential of -70 mV, and observed that 1 µM PGE 2 elicited an inward current of 22.92 ± 5.57 pA (n=8) in the presence of TTX (0.5 µM), DL-AP5 (100 µM), CNQX (20 µM), and bicuculline (20 µM). This current appeared 40.00 ± 13.89 s (10 to 130 s, n=8) after the initiation of PGE 2 -treatment and ended 193.75 ± 32.07 s (n=8) after the removal of PGE 2 from the bath solution (Fig. 2C). We then investigated the current evoked by PGE 2 using a ramp voltage-clamp protocol from -120 mV to -30 mV and a holding potential of -70 mV, in the presence of TTX (0.5 µM), DL-AP5 (100 µM), CNQX (20 µM), and bicuculline (20 µM; Fig. 2D1). PGE 2 (1 µM) increased the current evoked by this protocol (Fig. 2D1). After subtracting control values from the current recorded after applying PGE 2 , a PGE 2 -induced inward current was obtained (Fig. 2D2). This inward current had a linear voltage dependence from -120 to -40 mV and was suppressed at a mean value of -41.12 ± 3.54 mV (n=4; Fig. 2D2), suggesting the activation of a non-selective cation conductance [START_REF] Zhang | Kisspeptin depolarizes gonadotropin-releasing hormone neurons through activation of TRPC-like cationic channels[END_REF][START_REF] Roland | Glucosensing by GnRH Neurons: Inhibition by Androgens and Involvement of AMP-Activated Protein Kinase[END_REF]. This value was also close to the membrane potential measured in current-clamp mode (-45.66 ± 1.68 mV, t test; P > 0.05; n=7; Fig. 2B3), for which the two current-voltage curves obtained in the presence and absence of PGE 2 converged.

EP2 receptors are expressed in GnRH neurons and their activation mimics the effects of PGE 2

To gain insight into the nature of the receptors that mediate the potent postsynaptic excitatory action of PGE 2 on GnRH neurons, we tested the effects of 17-phenyl trinor PGE 2 (17PT-PGE 2 ), sulprostone and butaprost, which are EP1, EP1/EP3 and EP2 receptor agonists [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF], respectively, on GnRH neuronal activity. The bath application of 17PT-PGE 2 or sulprostone at 10 µM, a concentration previously shown to promote GnRH release in GnRHsecreting cell lines [START_REF] Rage | Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway[END_REF], had no effect on GnRH neuronal activity (Fig. 3A,B). However, 10 µM butaprost resulted in membrane depolarization (Fig. 3A,B). Analysis of the voltage-clamp ramps in the presence or absence of butaprost (Fig. 3C1) revealed that this EP2 receptor agonist activated an inward current that was suppressed at a mean value of -48.33±6.23 mV (n=3, Fig. 3C2), close to the value obtained with PGE 2 (Fig. 2D2; t test, P > 0.05). Perfusion of the slices with 30 µM AH 23848, an antagonist of the EP4 receptor [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF], did not modify the response of GnRH neurons to PGE 2 treatment (n = 3). Taken together, these observations strongly suggest that the excitatory effects of PGE 2 on GnRH neuronal activity are mediated by EP2 receptor activation. To confirm the presence of EP2 receptors in GnRH neurons, we used immunohistochemistry. The EP2 receptor was abundantly expressed in the preoptic region; among the 128 GnRH-GFP neurons analyzed, 72 (56 %) displayed EP2 receptor immunostaining (n = 4 animals) (Fig. 3D).

PGE 2 -mediated membrane depolarization in GnRH neurons requires protein kinase A activation

Since EP2 receptors are linked to the Gs-cAMP-protein kinase A (PKA) pathway [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF][START_REF] Sang | Postsynaptically synthesized prostaglandin E2 (PGE2) modulates hippocampal synaptic transmission via a presynaptic PGE2 EP2 receptor[END_REF], we used PKA inhibitors to determine whether the excitatory effect of PGE 2 on GnRH neurons could be inhibited or attenuated. In the presence of 0.5 µM TTX, the bath application of the PKA inhibitors H89 (10 µM, n=5) and KT 5720 (10 µM, n=3), or the competitive PKA antagonist Rp-cAMP (20 µM, n=3) for 30 min significantly attenuated the stimulatory effect of PGE 2 on membrane depolarization in GnRH neurons (Fig. 3E,F). The activation of EP1 and EP3 receptors is coupled to the mobilization of intracellular calcium stores [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF], but did not cause membrane depolarization in GnRH neurons (Fig. 3A,B). Consistent with this observation, the depletion of intracellular calcium stores with thapsigargin (2 µM) did not block the depolarizing effects of PGE 2 on GnRH neurons (n = 2). Taken together, our data suggest that the excitatory effects of PGE 2 on GnRH neuronal activity are exerted via an EP2-Gs-cAMP/PKA signaling pathway.

Blockade of endogenous cyclooxygenase activity inhibits spontaneous firing of GnRH neurons

To monitor spontaneous GnRH neuronal activity, whole-cell patch-clamp recordings were performed using a pipette solution (ps2) that conferred the cells with an average resting potential of -61.50±0.62 mV (n=10) and an input resistance of 1384.27±73.71 M (n=10), as shown previously [START_REF] Clasadonte | Activation of neuronal nitric oxide release inhibits spontaneous firing in adult gonadotropin-releasing hormone neurons: a possible local synchronizing signal[END_REF]. At this resting potential, all neurons exhibited spontaneous activity with a mean discharge of 0.45±0.07 Hz (n=10). To explore the contribution of PGE 2 to this spontaneous activity, we bath-applied indomethacin, an inhibitor of cyclooxygenase, the ratelimiting enzyme in prostaglandin synthesis, to slices of the preoptic region during recording from GnRH neurons (Fig. 4). Bath application of this inhibitor at 50-100 µM either greatly reduced (by 95%; n=3; Fig. 4A) or fully suppressed (n=5; Fig. 4B) the spontaneous discharge of GnRH neurons (Fig. 4C). These effects were either reversible (n=2) or irreversible (n=6).

Lower concentrations of indomethacin (5-10 µM) had no effect on GnRH neuronal activity, except in the case of one cell that exhibited a reversible reduction of its basal firing rate by 57%. At 100 µM indomethacin, the suppression of firing was accompanied by a membrane hyperpolarization (4.5 ± 0.6 mV, n = 6) (Fig. 4B). However, the bath application of PGE 2 (1 µM) remained capable of triggering membrane depolarization (8.12 ± 2.39 mV) as well as action potentials (n=4) (Fig. 4B). These experiments suggest that the endogenous production of PGE 2 contributes to the maintenance of the activity of GnRH neurons in the preoptic region.

Astrocytic PGE 2 regulates GnRH neuronal activity in situ

Hypothalamic astrocytes are a source of PGE 2 [START_REF] Ma | Hypothalamic astrocytes respond to transforming growth factor-alpha with the secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone[END_REF], which is required for the normal release of GnRH [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF][START_REF] Prevot | erbB-1 and erbB-4 receptors act in concert to facilitate female sexual development and mature reproductive function[END_REF]. To determine whether astrocytes contribute to the regulation of GnRH neuronal firing, we pre-treated hypothalamic slices with the glial toxin fluoroacetate (5 mM; 60-120 min) [START_REF] Henneberger | Long-term potentiation depends on release of D-serine from astrocytes[END_REF][START_REF] Fonnum | Use of fluorocitrate and fluoroacetate in the study of brain metabolism[END_REF][START_REF] Okada-Ogawa | Astroglia in medullary dorsal horn (trigeminal spinal subnucleus caudalis) are involved in trigeminal neuropathic pain mechanisms[END_REF][START_REF] Parsons | ATP-sensitive potassium channel-mediated lactate effect on orexin neurons: implications for brain energetics during arousal[END_REF]. Fluoroacetate significantly impaired astrocyte function as assessed by the inability of astrocytes to take up sulforhodamine 101 (SR101) (Fig. 4D), a fluorescent dye that is selectively taken up by astrocytes both in vivo [START_REF] Nimmerjahn | Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo[END_REF] and in living brain slices [START_REF] Kafitz | Developmental profile and properties of sulforhodamine 101--Labeled glial cells in acute brain slices of rat hippocampus[END_REF].

The number of astrocytes capable of taking up the dye in the vicinity of GnRH neurons was reduced by 78 ± 1% when compared to untreated slices (n=4; t-test, p < 0.01). The spontaneous firing of GnRH neurons (detected using ps2 conditions) was strikingly reduced in fluoroacetate-treated slices (9 out of 12) (Fig. 4C,E). The remaining neurons (3 out of 12) either exhibited a sporadic pattern of action potential discharge (0.01 Hz, n=2), or fired in repetitive bursts (0.15 Hz, n=1). In contrast, fluoroacetate treatment did not affect the basal membrane properties of GnRH neurons (resting potential of -74.29 ± 1.44 mV and input resistance of 1123.50 ± 130.18 MΩ in ps1, n=7; t-test for treated vs. control slices; p > 0.05).

Importantly, in most GnRH neurons, fluoroacetate treatment did not affect the membrane depolarizing effect of PGE 2 (1 µM; 6 out of 9 neurons; 7.92 ± 0.42 mV; Fig. 4E). The treatment also failed to affect the latency of the membrane depolarization upon initiation of PGE2 treatment (50.00 ± 13.36 s in ACSF vs. 31.25 ± 6.10 s in FA, n = 8 for each treatment, t-test, p > 0.05), the duration of the effect (234.28 ± 36.70 s in ACSF vs. 377.14 ± 74.08 s in FA, n = 7 for each treatment, t-test, p > 0.05) or the ability of the prostaglandin to trigger action potentials (5 out of 6 neurons, Suppl. Fig. 2).

We next sought to investigate the firing activity of GnRH neurons in mice with deficient astrocytic PGE 2 production. We used transgenic mice expressing a dominant-negative form of the erbB4 receptor (DN-erbB4) specifically targeted to astrocytes by means of the human GFAP promoter [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF]. This mutated receptor blocks the ligand-dependent activation of erbB2 and erbB4 receptors without affecting signaling through other receptors, such as erbB1 or Notch1 [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF][START_REF] Patten | Notch1 signaling regulates radial glia differentiation through multiple transcriptional mechanisms[END_REF]. GnRH secretion is deficient in GFAP-DN-erbB4 mice due to the inability of astrocytes to respond to erbB4 activation by producing PGE 2 [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF]. To visualize GnRH neurons in these animals we crossed GFAP-DN-erbB4 mice with GnRH-GFP animals. GnRH neurons recorded from four double-transgenic mice under ps2 conditions were either silent (n=5; Fig. 4F) or displayed a sporadic low-frequency (0.02 Hz) pattern of action potential discharge (n=1), in contrast to GnRH-GFP neurons recorded from wild-type controls (Fig. 4C). These results suggest that the excitability of GnRH neurons is reduced in GFAP-DN-erbB4 mice due to the failure of PGE 2 production by erbB4-deficient astrocytes. We tested this assumption by determining whether exogenous PGE 2 could restore GnRH membrane depolarization in GFAP-DN-erbB4 mice. The bath application of PGE 2 (1 µM) elicited membrane depolarization equally strongly in hypothalamic slices from wild-type and GFAP-DN-erbB4 mice (8.67±1.32 mV in DN-erbB4 mutant mice vs. 8.72 ± 067 mV in wild-type controls, n = 6 and 10, respectively, t-test, p > 0.05) (Fig. 4F).

Discussion

The disruption of astrocytic erbB4 receptor signaling by the overexpression of a dominantnegative erbB4 receptor leads to diminished astrocytic PGE 2 release in response to liganddependent erbB4 activation, leading in turn to reduced GnRH release, delayed puberty and disrupted adult reproductive function [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF][START_REF] Prevot | erbB-1 and erbB-4 receptors act in concert to facilitate female sexual development and mature reproductive function[END_REF]. The present results show that GnRH neuronal activity is decreased in these animals, and that this deficiency is mimicked by the bath application of either fluoroacetate, an inhibitor of astrocyte metabolism [START_REF] Henneberger | Long-term potentiation depends on release of D-serine from astrocytes[END_REF][START_REF] Fonnum | Use of fluorocitrate and fluoroacetate in the study of brain metabolism[END_REF][START_REF] Parsons | ATP-sensitive potassium channel-mediated lactate effect on orexin neurons: implications for brain energetics during arousal[END_REF], or the cyclooxygenase blocker indomethacin, to slices of the preoptic region from wild-type animals.

Our findings that GnRH neurons respond to PGE 2 with an enhancement of the firing rate, and that PGE 2 rescues GnRH neuronal activity in brain slices with reduced astrocytic PGE 2 output indicate that glial PGE 2 is an important component of the homeostatic mechanism controlling GnRH neuronal activity.

The presence of EP2 receptors in GnRH neurons ((38), present study), and the finding that the selective EP2 receptor agonist butaprost mimics the effect of PGE 2 , suggest that the excitatory effect of PGE 2 on GnRH neuronal activity involves the activation of EP2 receptors. This inference is supported by an earlier report showing that the stimulation of GnRHproducing GT1-7 cells with butaprost results in GnRH release [START_REF] Moenter | Mechanisms underlying episodic gonadotropin-releasing hormone secretion[END_REF]. In addition, the attenuation of the effect of PGE 2 by the bath application of PKA inhibitors or biologically inactive cAMP is consistent with earlier findings demonstrating the coupling of the EP2 receptor to the cAMP/PKA pathway [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF] and the involvement of cAMP in the intracellular mechanism underlying the stimulatory effect of PGE 2 on GnRH secretion [START_REF] Ojeda | Stimulation of cyclic adenosine 3',5'-monophosphate production enhances hypothalamic luteinizing hormonereleasing hormone release without increasing prostaglandin E2 synthesis: studies in prepubertal female rats[END_REF]. A previous study has reported that native GnRH neurons also express EP1 receptors [START_REF] Rage | Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway[END_REF], which are coupled to the mobilization of intracellular calcium stores [START_REF] Coleman | International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes[END_REF] and to GnRH release [START_REF] Rage | Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway[END_REF]. Our electrophysiological data show that the EP1 receptor agonists 17PT-PGE 2 and sulprostone had no effect on GnRH neuronal activity, and that the depletion of intracellular calcium stores with thapsigargin did not alter the depolarizing response of GnRH neurons to PGE 2 . These results, together with earlier findings demonstrating that PGE 2 -induced GnRH release from median eminence explants requires the mobilization of intracellular calcium stores [START_REF] Ojeda | Prostaglandin E2-induced luteinizing hormonereleasing hormone release involves mobilization of intracellular Ca+2[END_REF], suggest that the EP1 receptor-dependent stimulation of GnRH release is exerted at the level of GnRH nerve terminals rather than GnRH cell bodies. Thus, PGE 2 may stimulate both GnRH neuron firing and GnRH release by acting at the cell soma and nerve terminal levels, respectively.

An intriguing finding in this study is that the cyclooxygensase (COX) inhibitor indomethacin failed to affect spontaneous GnRH neuronal activity at doses lower than 50 µM, which is > 1700 times the EC50 of COX-1 (0.028 µM), but only ~ 25 times the EC50 of COX-2 (1.68 µM) [START_REF] Mitchell | Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase[END_REF]. This finding suggests that eicosanoids sustaining spontaneous GnRH neuronal activity are mostly COX-2 derived products, a conclusion consistent with evidence that COX-2 is the most abundant COX form expressed in astrocytes [START_REF] Koyama | Endothelins stimulate expression of cyclooxygenase 2 in rat cultured astrocytes[END_REF][START_REF] Luo | Transforming growth factor beta1 regulates the expression of cyclooxygenase in cultured cortical astrocytes and neurons[END_REF]. Because endocannabinoids, such as 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (AEA or anandamide) were recently shown to be substrates for COX-2 (44) and because endocannabinoids modulate GABAergic excitatory inputs to GnRH neurons [START_REF] Farkas | Retrograde endocannabinoid signaling reduces GABAergic synaptic transmission to gonadotropin-releasing hormone neurons[END_REF], we cannot rule out that part of the effects of indomethacin on the spontaneous activity of GnRH neurons may be due to inhibition of endocannabinoid oxygenation by COX-2 [START_REF] Prusakiewicz | Differential sensitivity and mechanism of inhibition of COX-2 oxygenation of arachidonic acid and 2arachidonoylglycerol by ibuprofen and mefenamic acid[END_REF].

Equally intriguing is the apparent lack of sex and estrous cycle effects of PGE 2 on GnRH neuronal activity. Previous studies have shown that GnRH firing activity is modified by estradiol in a diurnal dependent manner [START_REF] Christian | Diurnal and estradiol-dependent changes in gonadotropin-releasing hormone neuron firing activity[END_REF], and that COX-2 expression in the preoptic region is greater in males than females [START_REF] Amateau | Induction of PGE2 by estradiol mediates developmental masculinization of sex behavior[END_REF]. Perhaps the lack of changes in GnRH firing activity is related to the fact that all recordings were made at the same time of the day and using a saturating dose of PGE 2 . In addition to its postsynaptic effect on GnRH neurons, PGE 2 may also modulate the activity of steroid-sensitive afferent neuronal populations [START_REF] Christian | The neurobiology of preovulatory and estradiolinduced gonadotropin-releasing hormone surges[END_REF], including kisspeptin neurons that ensure coordinated progression of neuroendocrine events sustaining ovulatory cyclicity [START_REF] Mayer | Timing and completion of puberty in female mice depend on estrogen receptor alpha-signaling in kisspeptin neurons[END_REF]. The recent development of a mouse model that enables identification of kisspeptin neurons using fluorescent reporter genes (51) now renders these neurons amenable to scrutiny. PGE 2 has long been known to play a role in the control of GnRH neuronal function [START_REF] Ojeda | Effect of inhibitors of prostaglandin synthesis on gonadotropin release in the rat[END_REF][START_REF] Ojeda | Prostaglandins in reproductive neuroendocrinology. Handbook of Pharmacologic Methodologies for the Study of the Neuroendocrine System[END_REF]. More recent work points to PGE 2 as a mediator of astrocyte-to-GnRH-neuron communication initiated by the activation of erbB signaling in astrocytes [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF][START_REF] Ma | Hypothalamic astrocytes respond to transforming growth factor-alpha with the secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone[END_REF][START_REF] Prevot | erbB-1 and erbB-4 receptors act in concert to facilitate female sexual development and mature reproductive function[END_REF][START_REF] Ma | Neuregulins signaling via a glial erbB-2-erbB-4 receptor complex contribute to the neuroendocrine control of mammalian sexual development[END_REF].

Astrocytes, which are sophisticated sensors of neuronal activity [START_REF] Barres | The mystery and magic of glia: a perspective on their roles in health and disease[END_REF][START_REF] Haydon | Astrocyte control of synaptic transmission and neurovascular coupling[END_REF], release PGE 2 at active synapses in response to glutamate [START_REF] Bezzi | Prostaglandins stimulate calcium-dependent glutamate release in astrocytes[END_REF][START_REF] Zonta | Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation[END_REF][START_REF] Zonta | Glutamate-mediated cytosolic calcium oscillations regulate a pulsatile prostaglandin release from cultured rat astrocytes[END_REF]. Interestingly, a ligand-dependent increase in erbB signaling appears to mediate glutamate-stimulated PGE 2 release in hypothalamic astrocytes [START_REF] Dziedzic | Neuron-to-glia signaling mediated by excitatory amino acid receptors regulates ErbB receptor function in astroglial cells of the neuroendocrine brain[END_REF]. Within the hypothalamus, both glutamate and glial erbB signaling play physiological roles in promoting GnRH release at the onset of puberty [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF][START_REF] Urbanski | A role for N-methyl-D-aspartate (NMDA) receptors in the control of LH secretion and initiation of female puberty[END_REF][START_REF] Ma | Neuregulins signaling via a glial erbB-2-erbB-4 receptor complex contribute to the neuroendocrine control of mammalian sexual development[END_REF][START_REF] Ojeda | Involvement of transforming growth factor alpha in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus[END_REF] and during adult reproductive life [START_REF] Brann | Excitatory amino acids: evidence for a role in the control of reproduction and anterior pituitary hormone secretion[END_REF][START_REF] Prevot | erbB-1 and erbB-4 receptors act in concert to facilitate female sexual development and mature reproductive function[END_REF]. The present results identify PGE 2 as a potent excitatory regulator of GnRH neuronal activity, and indicate that the production of PGE 2 by astroglial cells plays a hitherto unappreciated role in the homeostatic regulation of GnRH neuronal excitability.

Materials and Methods

Animals. Electrophysiological recordings were performed on adult GnRH-GFP and GFAP-DN-erbB4/GnRH-GFP transgenic mice. The generation of GFAP-DN-erbB4 mice has been described previously [START_REF] Prevot | Normal female sexual development requires neuregulin-erbB receptor signaling in hypothalamic astrocytes[END_REF].

Electrophysiological experiments. GnRH-GFP neurons were recorded as described in a previous study [START_REF] Clasadonte | Activation of neuronal nitric oxide release inhibits spontaneous firing in adult gonadotropin-releasing hormone neurons: a possible local synchronizing signal[END_REF]. Pipette solution 1 (ps1) contained (in mM) K-gluconate, 125; HEPES, Fluorescent staining. Detection of EP2 receptor (rabbit polyclonal antibody, Cayman Chemical) and SR101 uptake were performed using protocols described previously in references ( 6) and [START_REF] Kafitz | Developmental profile and properties of sulforhodamine 101--Labeled glial cells in acute brain slices of rat hippocampus[END_REF], respectively. Detailed methods are provided in SI Materials and Methods.

10; CaCl 2 , 1 ;

 21 MgCl 2 , 1; ATP-Mg, 2, EGTA, 11; GTP, 0.3; NaCl, 15 (pH: 7.3 with KOH, osmolarity: 270-280 mOsm). Pipette solution 2 (ps2) contained (in mM): K-gluconate, 140; HEPES, 10; ATP-Mg, 2; EGTA, 1; KCl, 10 (pH: 7.3 with KOH, osmolarity: 270-290 mOsm). Drugs were applied to the perfusing system (bath application) to obtain the final concentrations indicated. The drugs used were: PGE 2 , Butaprost, Sulprostone, 17-phenyl trinor PGE 2 (Cayman chemical); 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-Amino-5-phosphonopentanoic acid (DL-AP5), Tetrodotoxin citrate (TTX), Thapsigargin (Tocris); Sodium fluoroacetate, Indomethacin, AH 23848, H-89, KT 5720, Rp-cAMPS and Bicuculline methiodide (Sigma). Detailed methods are provided in SI Materials and Methods.

Figure 1 .

 1 Figure 1. PGE 2 powerfully activates GnRH neurons. A-B, Whole-cell current-clamp recordings showing the effect of bath application of PGE 2 on two GnRH neurons. Note that in these two silent GnRH neurons, which had a resting membrane potential of -74 mV (A) and -68 mV (B) respectively, PGE 2 induced a reversible membrane depolarization that led to the initiation of spike firing. The effect was short-lived in A and long-lasting in B. Note that in B, the spike firing started with a bursting pattern (asterisks). C, Whole-cell current-clamp recording of a single GnRH neuron showing that PGE 2 (0.01-1 µM) depolarized the membrane in a dose-dependent manner. Note that in this cell, 0.01 µM of PGE 2 did not trigger spike firing. In this and the following figures, the downward deflections correspond to voltage responses to 300 ms hyperpolarizing pulses used to test the membrane input resistance. D, Dose-response curve of the PGE 2 -induced membrane depolarization. The numbers of neurons tested at each dose is given in parentheses. Error bars indicate SEM. The EC 50 for the PGE 2 -induced membrane depolarization was 0.018 µM, based on a logistic equation fitted to the data points. E, The excitatory effect of PGE 2 on GnRH-neurons persisted in the presence of the AMPA/kainate and NMDA receptors antagonists CNQX (20 µM) and DL-AP5 (100 µM) respectively. F, Loose patch-clamp recording showing the excitatory effect of PGE 2 on a GnRH neuron, characterized by a reversible acceleration of firing. Note that the effect was reproduced by a second application of PGE 2 to the same neuron. G, Loose patch-clamp recording showing the inhibitory effect of PGE 2 on a non-GnRH neuron located in the vicinity of GnRH-GFP neurons. The effect was characterized by a reversible slowing down of firing.

Figure 2 .

 2 Figure 2. The PGE 2 -induced activation of GnRH neurons is direct and involves an inward current. A, Whole-cell current-clamp recording showing that PGE 2 depolarized GnRH neurons in the presence of TTX (0.5 µM), CNQX (20 µM), DL-AP5 (100 µM), and bicuculline

Figure 3 .

 3 Figure 3. The PGE 2 -induced activation of GnRH neurons is mediated by the EP2 receptor and requires the cAMP-PKA pathway. A, In a single GnRH neuron recorded with a whole-cell current clamp, the EP2 receptor agonist Butaprost evoked a membrane depolarization similar to that induced by PGE 2 whereas the EP1 receptor agonist 17-phenyl trinor PGE 2 (17PT-PGE 2 ) and the EP1-3 receptor agonist Sulprostone had no effect. Note that the discharge elicited by the Butaprost-induced membrane depolarization led to bursts of action potentials (asterisks). B, A bar graph illustrating the membrane depolarization in GnRH neurons induced by PGE 2 , Butaprost, 17PT-PGE 2 and Sulprostone (*, p < 0.05 when compared with the membrane depolarization induced by PGE 2 , one-way ANOVA; n=4-10 neurons). Error bars indicate SEM. C1-C2, Current traces evoked by voltage ramps (duration: 12.5 s) from -120 mV to -30 mV at a holding potential of -70 mV in GnRH neurons. C1, Traces showing the current response to the voltage ramp in GnRH neurons before

Figure 4 .

 4 Figure 4. Astrocytic prostaglandin production sustains the electrical activity of GnRH neurons. Recordings of GnRH neurons were performed under whole-cell current-clamp using pipette solution 2 (ps 2, see Materials and Methods) to obtain a background of spontaneous activity from GnRH neurons. A, GnRH neurons showed spontaneous activity (Control), which was reduced after 10 min of perfusion with indomethacin (INDO, 50 µM), an inhibitor of the cyclooxygenases, enzymes responsible for prostaglandin production. B, INDO at a higher concentration (100 µM) strongly attenuated the spontaneous activity of GnRH neurons, accompanied by membrane hyperpolarization. Note that PGE 2 reversed the inhibitory effect of INDO. C, Bar graph illustrating the firing rate of GnRH neurons recorded in brain slices from wild-type mice (WT) exposed to INDO or fluoroacetate (FA), and from DN-erbB4 mice. (*, p < 0.05 when compared with the firing rate of GnRH neurons recorded in brain slices from WT mice, one-way ANOVA; n=6-12 neurons). Error bars indicate SEM. D, Pretreatment of brain slices with fluoroacetate (FA, 5 mM, 60-120 min), a glial toxin, impaired the astrocytic uptake of sulforhodamine101 (SR101, red). Arrows show cells that took up SR101

Acknowledgments

This research was supported by the Agence National pour la Recherche (ANR, France) grants ANR-07-NEURO-026-03 and ANR-09-BLAN-0267, the Fondation pour la Recherche Médicale (Equipe FRM 2005, France) and the Institut Fédératif de Recherche 114 (IFR114, France) electrophysiological platform (VP), in addition to National Institute of Health (NIH, USA) grants HD25123 and RR000163 for the operation of the Oregon National Primate Research Center (SRO). J.C. was supported by a doctoral fellowship from the Inserm and the Région Nord Pas de Calais. We thank Julien Devassine (Animal facility, IFR114) for his brilliant management of our mouse colony.

Data analysis. Statistical analysis was performed using Sigma-Stat software (Jandel, San Raphael, CA). Differences between several groups were analyzed by one-way ANOVA, followed by a Student-Newman-Keuls multiple comparison test. The Student's t test was used to compare two groups. The data points of the dose-response curve were fitted with a four-parameter logistic curve using Sigma Plot 2001 (SPSS, Chicago, IL). A p-value of < 0.05 was considered to indicate a statistically significant difference. Values are reported as the mean ± the standard error of the mean (S.E.M.).

in the vicinity of GnRH neurons (GFP, green) under control conditions (left panel). After FA treatment, very few cells were labeled with SR101 (right panel, arrowhead). Images were acquired at the level of the organum vasculosum of the lamina terminalis (OVLT). Scale bar: 100 µm. E, FA strongly reduced the firing rate of GnRH neurons. In this example, the slice was pre-treated with FA for 60 min and action potentials could be driven by a brief injection of a depolarizing current (asterisk). Note that PGE 2 retained its depolarizing effect. F, In DN-erbB4 mice, in which astrocytic PGE 2 release is diminished, most GnRH neurons were silent.

In this recording, action potentials could be driven by a brief injection of depolarizing current (asterisk). Note that PGE 2 retained its depolarizing effect as well as reducing the membrane input resistance (downward deflections).