Susan Wray

Neural crest and Ectodermal cells intermix in the nasal placode to give rise to GnRH-1 Neurons, Sensory Neurons and Olfactory Ensheathing Cells

The origin of GnRH-1 cells and olfactory ensheathing cells has been controversial. Genetic Cre-lox lineage tracing of the neural crest (NC) versus ectodermal contribution to the developing nasal placode was performed using two complementary mouse models, the NC-specific Wnt1Cre mouse line and an ectodermal-specific Crect mouse line. Using these lines we prove that the NC give rise to the olfactory ensheathing cells and subpopulations of GnRH-1 neurons, olfactory and vomeronasal cells. These data demonstrate that Schwann cells and olfactory ensheathing cells share a common developmental origin. Furthermore, the results indicate that certain conditions that impact olfaction and sexual development, such as Kallmann syndrome, may be in part neurocristopathies.

Development of gonadotropin-releasing hormone-1 neurons.

Gonadotropin releasing hormone-1 (GnRH-1) neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain during prenatal development. Once within the brain, GnRH-1 cells become integral components of the CNS-pituitary-gonadal axis, essential for reproductive maturation and maintenance of reproductive function in adults. This review focuses on the lineage and development of the GnRH-1 neuroendocrine system. Although the migration of these cells from nose to brain has been well documented in a variety of species, many questions remain concerning the melecules and cues directing GnRH-1 cell differentiation, migration, axon targeting, and establishment and control of GnRH-1 secretion. These process most likely involve multiple and redundant cues because if these mechanisms fail, reproduction dysfunction will ensue and guarantee that this defect does not remain in the gene pool.

From Nose to Brain: Development of Gonadotrophin‐Releasing Hormone ‐1 Neurones

Gonadotrophin‐releasing hormone‐1 (GnRH‐1) is essential for mammalian reproduction, controlling release of gonadotrophins from the anterior pituitary. GnRH‐1 neurones migrate from the nasal placode into the forebrain during development. Although first located within the nasal placode, the embryonic origin/lineage of GnRH‐1 neurones is still unclear. The migration of GnRH‐1 cells is the best characterised example of neurophilic/axophilic migration, with the cells using a subset of olfactory‐derived vomeronasal axons as their pathway and numerous molecules to guide their movement into the forebrain. Exciting work in this area is beginning to identify intersecting pathways that orchestrate the movement of these critical neuroendocrine cells into the central nervous system, both spatially and temporally, through a diverse and changing terrain. Once within the forebrain, little is known about how the axons target the median eminence and ultimately secrete GnRH‐1 in a pulsatile fashion.

Slice cultures of LHRH neurons in the presence and absence of brainstem and pituitary

Luteinizing hormone releasing hormone (LHRH) neurons from the preoptic area (POA)/hypothalamus of the postnatal rat were cultured for up to 7 weeks using a slice explant roller culture technique. The slices thinned to quasi-monolayers, but maintained organotypic distributions of large numbers of immunocytochemically identifiable LHRH, neurotensin, tyrosine hydroxylase, neurophysin and corticotropin releasing hormone-containing neurons. The distribution, survival and morphology of LHRH cells in co-cultures with brainstem and anterior pituitary was quantitated, and found to be similar to that observed in single cultures. LHRH fibers grew into either pituitary or brainstem tissue, however when all three tissues were co-cultured, LHRH fibers preferentially invaded the pituitary. LH immunoreactive anterior pituitary gonadotropes were maintained only in co-cultures containing POA/hypothalamic slices, and addition of an LHRH antagonist in such cultures, inhibited LH immunoreactivity in the gonadotropes. This slice explant roller culture method effectively maintains the cyto- and chemoarchitecture and functional properties of the LHRH system for long periods in vitro and should provide excellent models for studying the interactive and molecular characteristics of postnatal LHRH neurons.

Direct Action of Estradiol on Gonadotropin-Releasing Hormone-1 Neuronal Activity via a Transcription-Dependent Mechanism

Pulsatile secretion of gonadotropin-releasing hormone-1 (GnRH-1) is essential for reproduction. GnRH-1 induces gonadotropin release and is regulated by 17β-estradiol (E2). Although a subpopulation of GnRH-1 neurons expresses estrogen receptor (ER) β, it is unclear whether E2 acts directly on GnRH-1 neurons or indirectly through interneuronal connections. To test the hypothesis that E2 acts directly on GnRH-1 neurons to regulate neuronal activity, we used calcium imaging to monitor intracellular calcium oscillations in GnRH-1 neurons maintained in nasal explants. TTX was used to minimize synaptic input from other cells. Consistent with previous studies, TTX reduced the activity of individual GnRH-1 neurons to a basal level, while the population of cells maintained synchronized calcium oscillations. Exposure of GnRH-1 cells to TTX plus E2 increased the number of calcium peaks/cell, percentage of cells with ≥10 peaks, mean peak amplitude, and percentage of cells that contributed to each calcium pulse in explants maintained in vitro for 7 d (7 div) compared with TTX alone. These effects were induced within 30 min and were not mimicked by 17α-estradiol, E2 conjugated to BSA (which does not cross the plasma membrane), or seen at 21 div, when the percentage of GnRH-1 cells expressing ERβ transcripts declines. In addition, these effects were inhibited by the ER antagonist ICI 182,780 and prevented by inhibition of gene transcription. These data suggest that, via ERβ, E2 can rapidly act as a hormone-activated transcription complex and are the first to show that E2 directly increases GnRH-1 neuronal activity and synchronization.

Kisspeptin-10 facilitates a plasma membrane-driven calcium oscillator in gonadotropin-releasing hormone-1 neurons.

Kisspeptins, the natural ligands of the G-protein-coupled receptor (GPR)-54, are the most potent stimulators of GnRH-1 secretion and as such are critical to reproductive function. However, the mechanism by which kisspeptins enhance calcium-regulated neuropeptide secretion is not clear. In the present study, we used GnRH-1 neurons maintained in mice nasal explants to examine the expression and signaling of GPR54. Under basal conditions, GnRH-1 cells exhibited spontaneous baseline oscillations in intracellular calcium concentration ([Ca(2+)](i)), which were critically dependent on the operation of voltage-gated, tetrodotoxin (TTX)-sensitive sodium channels and were not coupled to calcium release from intracellular pools. Activation of native GPR54 by kisspeptin-10 initiated [Ca(2+)](i) oscillations in quiescent GnRH-1 cells, increased the frequency of calcium spiking in oscillating cells that led to summation of individual spikes into plateau-bursting type of calcium signals in a subset of active cells. These changes predominantly reflected the stimulatory effect of GPR54 activation on the plasma membrane oscillator activity via coupling of this receptor to phospholipase C signaling pathways. Both components of this pathway, inositol 1,3,4-trisphosphate and protein kinase C, contributed to the receptor-mediated modulation of baseline [Ca(2+)](i) oscillations. TTX and 2-aminoethyl diphenylborinate together abolished agonist-induced elevation in [Ca(2+)](i) in almost all cells, whereas flufenamic acid was less effective. Together these results indicate that a plasma membrane calcium oscillator is spontaneously operative in the majority of prenatal GnRH-1 neurons and is facilitated by kisspeptin-10 through phosphatidyl inositol diphosphate hydrolysis and depolarization of neurons by activating TTX-sensitive sodium channels and nonselective cationic channels.

Development of luteinizing hormone releasing hormone neurones.

This review concentrates on some of the recent discoveries and future questions relevant to the development of the neuroendocrine luteinizing hormone releasing hormone (LHRH) cells. Neuroendocrine LHRH cells originate outside the central nervous system, in the nasal placode, and thereafter migrate into the forebrain during prenatal development. It is this population of LHRH cells that is responsible for reproductive function, becoming integral members of the hypothalamo‐pituitary‐gonadal axis postnatally. Disruption of the development of this system results in reproductive dysfunction. Increasing our understanding of LHRH neuroendocrine cells establishes conditions where we can look with greater precision at the mechanisms controlling reproductive development, both activation and failure. In addition, the ability to manipulate the molecular and cellular biology of the LHRH system opens the route to understanding critical neurobiological issues such as phenotypic commitment, axonal path finding and mechanisms involved in neuronal migration. Each of the topics is discussed in turn and potential mechanisms controlling the development of the neuroendocrine LHRH system are indicated.

Prenatal ontogenesis of pro-opiomelanocortin in the mouse central nervous system and pituitary gland: an in situ hybridization and immunocytochemical study

Pro-opiomelanocortin (POMC) mRNA detected by in situ hybridization and POMC/ACTH (adrenocorticotropin)-containing neurons detected by immunocytochemistry were first observed in the presumptive arcuate nucleus of embryonic mouse brain on gestational day 10.5 (E10.5). Immunostained fibers were also evident on E10.5 in the lateral and dorsal diencephalon. In these areas, a dense network of processes developed by E11.5 and extended into the mesencephalon. Fibers were detected in the myelencephalon at this stage and a day later (E12.5) in the spinal cord. Adult-like patterns of POMC/ACTH fibers were established in the diencephalon, mesencephalon, metencephalon and the myelencephalon between E13.5 and E15.5. POMC-expressing cells in the anterior and intermediate lobes of the pituitary gland appeared on E12.5 and E14.5, respectively. The early expression of POMC and the rapid establishment of dense fiber tracts in the brain is consistent with a role for POMC-derived peptides in the development of the central nervous system.

EFFECTS OF INSULIN-LIKE GROWTH FACTORS I AND II AND INSULIN ON THE IMMORTALIZED HYPOTHALAMIC GTI-7 CELL LINE

Insulin and insulin-like growth factor I (IGF-I) participate in energy metabolism, regulate cellular growth and differentiation, and are thought to act locally in a paracrine manner through specific receptors. Systemic levels of these peptides in humans and primates are directly associated with levels of activity of the reproductive axis. To date, it is unclear whether these peptides participate in reproductive function by acting at the level of the GnRH neuron. In this study we examined the effects of IGF-I, IGF-II and insulin on immortalized GnRH-secreting neurons, the GTI-7 cell line. The GTI-7 cells expressed all three members of the insulin receptor family as determined by analysis of 125I-IGF-I, 125I-IGF-II and 125I-insulin binding sites. Insulin receptors bound insulin, IGF-II and IGF-I with a ratio of potency of 1:5:20. IGF-I and IGF-II receptors bound both IGF-I and IGF-II. The ratio of potency of IGF-I/IGF-II was 1:5 for the IGF-I receptor and 100:1 for the IGF-II receptor. The binding characteristics of the growth factors at 22 degrees C suggested the possibility that these cells may secrete IGF binding proteins. To ensure that changes in GnRH levels in the media were due to secretion and not to changes in cell number, the mitogenic effect of these peptides on GTI cells was evaluated. Both insulin and IGF-I were strong mitogens (48-hour incubation), restoring cell number to that of serum-replete cultures at a dose of 0.1 ng/ml. A 100-fold higher dose of IGF-II was required to produce a similar level of mitogenicity, implicating an action through the IGF-I and/or insulin receptor. Due to these mitogenic effects, the effect of insulin, IGF-I and IGF-II on GnRH secretion was studied after short-term exposure. Insulin and IGF-I did not affect GnRH secretion, but IGF-II had a biphasic effect on GnRH release after 2 h of incubation (a maximum stimulatory effect occurred with a 0.1 ng/ml dose). In order to examine the signal transduction mechanism, the role of cytoplasmic calcium mobilization in IGF-II-induced GnRH secretion was examined in single cells using calcium imaging. The effect of IGF-II on GnRH secretion appeared to operate via a calcium-independent mechanism. The studies document an insulin/IGF system in the GTI-7 neuronal cell line and show that insulin and IGFs can exert direct effects on the immortalized GnRH neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholecystokinin Modulates Migration of Gonadotropin-Releasing Hormone-1 Neurons

Expression of the brain–gut peptide cholecystokinin (CCK) in the developing olfactory–gonadotropin-releasing hormone-1 (GnRH-1) neuroendocrine systems was characterized, and the function of CCK in these systems was analyzed both in vivo and in vitro. We present novel data demonstrating that CCK transcript and protein are expressed in sensory cells in the developing olfactory epithelium and vomeronasal organ, with both ligand and receptors (CCK-1R and CCK-2R) found on olfactory axons throughout prenatal development. In addition, migrating GnRH-1 neurons in nasal regions express CCK-1R but not CCK-2R receptors. The role of CCK in olfactory–GnRH-1 system development was evaluated using nasal explants, after assessing that the in vivo expression of both CCK and CCK receptors was mimicked in this in vitro model. Exogenous application of CCK (10-7 m) reduced both olfactory axon outgrowth and migration of GnRH-1 cells. This inhibition was mediated by CCK-1R receptors. Moreover, CCK-1R but not CCK-2R antagonism caused a shift in the location of GnRH-1 neurons, increasing the distance that the cells migrated. GnRH-1 neuronal migration in mice carrying a genetic deletion of either CCK-1R or CCK-2R receptor genes was also analyzed. At embryonic day 14.5, the total number of GnRH-1 cells was identical in wild-type and mutant mice; however, the number of GnRH-1 neurons within forebrain was significantly greater in CCK-1R–/– embryos, consistent with an accelerated migratory process. These results indicate that CCK provides an inhibitory influence on GnRH-1 neuronal migration, contributing to the appropriate entrance of these neuroendocrine cells into the brain, and thus represent the first report of a developmental role for CCK.