[Frontiers in Bioscience 2, d88-125, March 1, 1997]
Reprints
PubMed
CAVEAT LECTOR




Table of Conents
 Previous Section   Next Section

CROSS-TALK SIGNALS IN THE CNS: ROLE OF NEUROTROPHIC AND HORMONAL FACTORS, ADHESION MOLECULES AND INTERCELLULAR SIGNALING AGENTS IN LUTEINIZING HORMONE-RELEASING HORMONE (LHRH)-ASTROGLIAL INTERACTIVE NETWORK

Bianca Marchetti

Department of Pharmacology, Medical School, University of Catania, 95125 Catania, Laboratory of Biotech. Neuropharmacology, OASI Institute for Research and Care (IRCCS) on Mental Retardation and Brain Aging (IRCCS) Troina, (EN), Italy.

Received 8/2/96; Accepted 2/20/97; On-line 3/1/97

TABLE OF CONTENTS

1. Abstract
2. Introduction
2.1. Pathways involved in neuron-glia communication
2.2. Orchestration of neuronal migration by cell surface and extracellular matrix molecules
3. Growth factors and neurotrophic factors are bi-directional signaling molecules involved in neuron-glia dialogue
3.1. Fibroblast growth factors
3.2. Epidermal growth factors
3.3. Insulin and insulin-like growth factors
4. Hormones as mediators of neuron-glia plasticity
4.1. Sex steroids
4.2. Adrenal corticosteroids
5. The second messenger system and transcription factor network
6. Immune system modulation of neuron-glia interactions
6.1. Immunological mediators
7. The LHRH neuron-astroglia network of signals
7.1 LHRH as the primum movens in neuro-endocrine-immune control of reproduction
7.2. The LHRH neuronal system within the central framework of immune signaling systems
7.3. Growth factor and sex steroid sensitivity of the LHRH neurons
8. Lhrh neuron-astroglia interactions during development
9. Lhrh neuron-astroglia interactions in the adult brain
10. Characterization of astroglia influence on the LHRH neuronal system: development of an in vitro system for the study of neuron-glia interactions
10.1 astroglial-derived factors stimulate LHRH neuron differentiation and accelerate the maturation of LHRH secretory potential : age-dependency
10.2. Specificity and regional differences in astroglia modulation of GT1 neuronal differentiation
10.3. Peptidic nature of astroglia-derived factor
11. Astroglia-derived factors stimulate leucocyte proliferation
12. Diffusible molecules and cell-cell contacts participate in LHRH-astroglia interactions
12.1. Role of soluble factors
13. Crosstalk between astroglial-derived immune mediators and intercellular/intracellular signaling agents
14. The role of cell adhesion in neuron-glia interactions
15. Progression and competent factors exert direct neurotrophic and functional effects: presence of a synergism/cooperativity
16. Basic fibroblast growth factor is a candidate signal molecule that collaborates in partnership with LHRH to regulate LHRH neuron-astroglia dialogue
17. Summary and conclusion
18. References
1. ABSTRACT

Neuron-astrocyte interactions play a crucial role during development and in the adult brain. During development, glial cells are involved in the guidance of neuronal precursors and in extending neuronal fiber projections. Astrocytes can promote neurite outgrowth, both "in vitro" and "in vivo". In the central nervous system (CNS), they express receptors for a variety of growth factors (GFs), neurotransmitters and/or neuromodulators. In turn, neuronal cells can respond to astrocyte-derived growth factors and control astrocyte function via a common set of signaling molecules and intracellular transducing pathways. It is also well established that astrocytes are involved with regenerative failure within the CNS following injury. Increasing evidence support the viewpoint that soluble factors from lymphoid/mononuclear cells modulate the growth and function of cells found in the CNS, specifically macroglia and microglia cells. Furthermore, glial cells can secrete immunoregulatory molecules that influence immune cells, as well as the glial cells themselves. In recent years, a bi-directional flow of informational molecules between LHRH neurons, subserving the neuroendocrine control of reproductive function, and astroglia cells has been disclosed. During their maturation and differentiation in vitro, astroglial cells release peptide growth factors that markedly accelerate LHRH neuronal phenotypic differentiation . In addition, these peptides induce the acquisition of mature LHRH secretory potential, with a potency depending on both the "age" and the specific brain localization of the astroglia, as well as the degree of LHRH neuronal differentiation "in vitro". Different experimental paradigms such as co-culture and mixed culture models between the GT1-1 neurons and astroglial cells in primary culture, disclosed the presence of a bi-directional flow of informational molecules regulating both proliferative and secretory capacities of each cell type. Growth factors are key players in LHRH neuron-astroglia crosstalk. In particular, basic fibroblast growth factor (bFGF) was identified as a major differentiation factor for the immortalized hypothalamic LHRH neuronal cell line. A specific synergy/cooperation between bFGF and other growth factors was also revealed at specific stages of LHRH neuron differentiation, indicating that the sequential expression of specific growth factors may participate in the processes of LHRH neuron migration, differentiation and functional regulation. Since bFGF is expressed in GT1-1 neurons and glial cells a possible paracrine/autocrine regulatory loop is suggested. Indeed, neutralization experiments aimed at counteracting endogenous bFGF during neuron-glia interactions dramatically inhibited astroglia neurotrophic effects. On the other hand, the importance of adhesion molecules in cell-to-cell communication was underscored by the significant inhibition of GT1-1 LHRH production and cell proliferation following the counteraction of neuron-neuron/neuron-glia interactions through addition of neuronal cell adhesion molecule (N-CAM) antiserum. Other information came from pharmacological experiments manipulating the astroglial-derived cytokines and/or nitric oxide, which revealed a crosstalk between the neuronal and astroglial compartments. From the bulk of this information, it seems likely that interactions between astroglia and LHRH neurons play a major role in the integration of the multiplicity of brain signals converging on the LHRH neurons that govern reproduction.

2. INTRODUCTION

Neurons have long been thought to represent the sole "information-processing" elements of the central nervous system (CNS). However, the anatomical proximity of the non-neuronal elements, called neuroglia, to the neuronal cells, makes these elements particularly suited for taking active roles in the functions of neural information processing. Knowledge on neuroglia has rapidly accumulated in the last decades, and an extraordinary body of evidence has now been assembled by different investigators from all fields of neuroscience, supporting a key role for the glia in neuronal physiopathology. Indeed, at nearly a century and a half from the time of development of knowledge about neuron-glia interactions (1-5), the possibility of signals passing from neurons to glial cells, and thus to other neurons opens-up many scenarios for intercellular/intracellular crosstalk within single cells of the CNS (Fig. 1).

Figure. 1. The neuronal-astroglial network. Schematic representation of the networks of signals leaving the neuronal cells, signaling the astroglial cell, and finally returning back information to other neuronal cells.

The concept of the existence, in the CNS, of dynamic neuronal-glial signaling processes long thought to be only by virtue of passive transmission of information between these two major cell types (6-9), is now firmly established (10-17). Indeed, in the first description of glia, they were thought to form a connective or ground substance ("Binddesubstanz"), a sort of cement or neural glue (Neuroglia) in which the nerve elements are immersed (1,2). Not consistent with this early idea, the stellate cell "sternformigen Zellen", the star-shaped cell of Golgi, (18), designated as "astrocytes" by Lenhossek (19) and described by Raff and colleagues (20) as the type 2 astrocyte (see Fig. 2), has a neuronal "makeup" in culture.

Figure. 2. Immunohistochemical localization of glial fibrillary acidic protein (GFAP) in pure astroglial cultures and neuron-glial mixed cultures. Primary rat astrocytes were prepared and isolated from cerebral hemispheres and cultured as described (41) during in vitro maturation and differentiation. Immortalized hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons were cultured on the top of 12 day-old astroglial cultures. Cytoplasmatic staining performed on fixed cells with monoclonal antibody to glial-fibrillary protein. A. GFAP staining in astrocyte cultures. B. GFAP staining during neuron-glia interactions.

This resemblance to neurons is further supported by its surface antigens (20) and ion channels (13, 14, 16, 17, 21-23), and in recent decades, this and other evidence, has led to alteration of the idea of a solely supportive role of the astrocytes to the concept of a more central and significant position of astrocytes in the metabolism and functioning of the CNS. Indeed, the anatomical proximity of astrocytes to neuronal synapses and the blood brain barrier (Fig. 3) makes these cells ideally suited for taking an active role in the ion, water and neurotransmitter metabolism of the CNS during both normal and abnormal neuronal function (13, 24-30).

Figure 3. Relationship between astrocytes and other brain elements. The schematic drawing illustrates several possible contacts between the astrocyte and 1. a synaptic cleft; 2. other astrocyte networks; 3. capillary/blood vessel; 4. neuronal cell bodies; 5. Nodes of Ranvier (see 14).

While Kuffler and Nichols first (7) recognized that interactions between neurons and glial cells would necessarily involve diffusible substances within the brain extracellular space, the functional significance of this "transmission" has not been clearly elucidated. Nonetheless, glial cells have been suggested to play a key role in the regulation of neuronal excitability, the modulation of synaptic transmission and neuronal connectivity, as well as the processing of information associated with learning and memory (24, 27, 31). The role of astroglia and infiltrating, inflammatory cells (monocytes and neutrophils) as well as cytokines and growth factors in the dynamics of CNS injury and disease constitute an important chapter of neuron-glia interactions (see 32). In particular, potential biochemical interactions between reactive glial cells (the microglial compartment) and damaged neurons have been hypothesized together with a suspected contribution of the immune system to neuronal death (see 32-40).

In the present work, a brief review on some aspects of the dialogue between the neuronal and glial cells will be presented. The recently disclosed network of interactions between the hypothalamic luteinizing hormone-releasing hormone (LHRH) neuronal system and astroglial cells will be discussed. Different dynamic "in vitro" models together with a number of pharmacological tools are proposed to unravel the LHRH-glial relationship at the biochemical and cellular levels. A key regulatory function of astroglia in the differentiation and maturation of the LHRH neuron is suggested on the basis of such experimental paradigms (41-44).

2.1. Pathways involved in neuron-glia communications

The functioning of the nervous system depends upon a continuos and sophisticated interrelationship between neuronal and glial cells. There are two broad subgroups of glial cells: the macroglia which consists of astrocytes, oligodendrocytes and ependymal cells, and the microglia. In recent years, an array of neurotransmitters, receptors, ion channels, adhesion molecules, and trophic factors have been revealed to be associated with glial cells. An insight about some of the factors that contribute to the neuron-astroglial signaling is presented.

2.2. Orchestration of neuronal migration by cell Surface and extracellular matrix molecules

During development and in the adult brain, astroglia have many different functions . An important facet of neuron-glia interactions concerns the key role of glia in the process of neuronal migration during embryogenesis (see 45-48). Glial-derived neuronal migration is a well recognized phenomenon in different regions of the developing mammalian brain. The migration of neuronal precursors to their final locations and the projection of axons to their appropriate targets are two critical events in neural development that require cell-cell and cell-matrix interactions. Migration of neurons is a remarkable process that relies on chemical communication between many different cells. Axon guidance and target recognition are achieved by highly specific chemical mechanisms using diffusible trophic factors, cell surface and extracellular matrix molecules which allow tropism and cell-cell interactions (46, 48-50). In both the cerebral cortex and the cerebellum, cells have been shown to utilize glial processes as guides in migration (46, 47). Neurons use glial fibers, which radiate from the brain's inner to outer surfaces, as a highway to carry them through the brain and to their final destination. In the neural crest, precursor cells must use a series of cellular and extracellular matrix cues to reach their destinations (see 50, for review). Many factors including genetic mutations, radiation and drugs such as cocaine and alcohol, can interfere with the process of neuron migration, leading to brain abnormalities ranging from epilepsy, to mental retardation and hypogonadism.

Cell adhesion molecules (CAMs) and components of the extracellular matrix (ECM) mediate, at least in part, the neuron-glia interactions. Indeed, astroglia express a number of cell or substrate adhesive molecules along the pathways of developing axonal tracts. Certain populations of astrocytes may also express other extracellular matrix molecules during development, after injury, or during degenerative diseases, that are inhibitory for axonal outgrowth. The majority of cell adhesion molecules described in the CNS can be ascribed to two growing gene families, such as the cadherin superfamily for the calcium-dependent and the immunoglobulin superfamily for the calcium-independent adhesion mechanisms (50). Furthermore, ECM components like laminin , fibronectin or proteoglycans and integrin-type receptors are expressed in developing neural tissues. In certain regions of the CNS during development, pioneering neurites may growth along pre-formed pathways of neuroepithelial cells, which later develop into astroglia. These cells express laminin as well as neural cell adhesion molecule, N-CAM and N-cadherin, on their surface. It is believed that the combined expression of these growth promoting molecules may help to direct growing neurites to specific regions of the developing brain (see 50).

3. Growth and neurotrophic factors are bi-directional signaling molecules involved in neuron-glia dialogue

In the dynamic flow of informational molecules participating in neuron-glia dialogue, the neurotransmitters, peptides and growth factors (GFs), represent the major bi-directional signaling agents. The importance of GFs in the coordination of developmental and adult physiological processes of both neurons and astrocytes is well recognized (36, 41-44, 51-60). A large number of proteins with growth promoting activity has been identified in recent years (Table 1). Substantial data are available for some members of the protein families of neurotrophins, fibroblast growth factors (FGFs), epidermal growth factor (EGF), and insulin-like growth factors (IGFs) (for comprehensive review see 36, 59, 60). A number of GFs have been shown to stimulate survival and differentiation of neurons and are often referred to as neurotrophic factors (36). Most of the characterized actions of growth and neurotrophic factors on brain cells relate to developmental processes, however, GFs also appear to be important in the function of the adult nervous system and for maintenance of structural integrity and regulation of synaptic plasticity (36).

TABLE I: Neuroactive Growth Factors
NeurotrophinsFibroblast & Epidermal GF FamilyInsulin & Insulinlike GFInterleukinsOthers
Nerve Growth Factor (NGF)Acidic Fibroblast Growth Factor (aFGF)InsulinCholinergic Differentiation Factor/Leukemia Inhibitory Factor (CDF/LIF)Choline Acetyltransferase Development Factor
Brain-Derived Neurotrophic Factor (BDNF)Insulin like Growth Factor- I (IGF-I)
Basic Fibroblast Growth Factor (bFGF)Transforming Growth Factor-ß1 (TGF-ß)
Neurotrophin-3 (NT-3)Insulin like Growth Factor-II (IGF-II)Interleukin-1 (IL-1)
Neurotrophin-4 (NT-4)Epidermal Growth Factor (EGF)Interleukin-2 (IL-2)Platelet Derived Growth Factor (PDGF)
Neurotrophin-5 (NT-5)Trasforming Growth Factor-alpha (TGF-alpha)Interleukin-3 (IL-3)
Interleukin-6 (IL-6)Activin
Schwanoma-Derived Growth Factor (SDGF)Heparin-Binding Neurotrophic Factor (HBNF)
Protease Nexin I & II

gamma-Interferon

An important factor in neuron-astroglial cell interactions is that glia in different brain regions express region-specific properties, including ion channels, neurotransmitter uptake and receptor systems, GFs production, and cell-surface adhesion systems (57). Then, the particular nature of the neuronal-glial interaction may depend on the specific neuronal and glial systems involved in a specific brain region (41). Thus, the dynamics of the cross-talk between neurons and astrocytes appears to be very complex. Neurons and glial cells are likely to be exposed to a number of different extracellular signaling molecules that may vary from moment to moment, and as a function of the particular physiological status (sex, age, stressful situation...). Therefore, a sophisticated regulatory network is likely to orchestrate the final appropriate response of both cell types.

3.1 Fibroblast Growth Factors

The family of fibroblast growth factors (FGFs) include bFGF and aFGF, two mitogenic proteins originally purified based on their ability to bind to heparin, FGF-5, FGF-6, keratinocyte growth factor, and the oncogene product int-2 and hst (36, 61-63, for review). These proteins have different lengths but share a core of approximately 120 aminoacids, with a 50% aminoacid identity. Most FGFs, but not aFGF and bFGF, contain hydrophobic signal sequences believed to be necessary for secretion. The location of aFGF to the inner side of the neuronal membrane supports the speculation that it represents a sequestered protein with intracellular function (64). However, export has been observed in fibrosarcoma cells, suggesting a new type of releasing mechanism (65). Three members of a protein family of high affinity FGF receptor proteins have been characterized (61, 62). These proteins contain an intracellular tyrosine kinase domain believed to mediate signal transduction. FGF's bind to proteoglycans, although with a lower affinity than the tyrosine kinase receptor proteins (36). It has be reported that proteoglycans are necessary for binding FGFs to the high affinity receptor, and it has been speculated that they induce a conformational change of FGF necessary for binding to the high affinity receptor (66). FGFs occur in many peripheral tissues, and they are potent mitogens for a large number of cell types. The brain and pituitary are rich sources of bFGF. The biological function of these peptides in the brain is limited to aFGF and bFGF. Basic FGF and its mRNA are extensively distributed in the cells of the circumventricular organs and a neuroendocrine function of this factor in the pituitary-portal system has been suggested (62, 63, 67). Moreover, the association of bFGF mRNA and bFGF-R with specific loci of cells in the hypothalamus, with selective populations of neurons (such as magnocellular neurons (PVN) and the supraoptic neurons (SON) has been recently demonstrated. The median eminence (ME) shows intense hybridization signal for bFGF mRNA, which is mainly associated with neuronal fibers, glial and endothelial cells (67). The ependymal and subependymal cells lining the 3rd ventricle contain bFGF and FGF-R mRNAs (67). Acidic and basic FGF are abundantly expressed in the developing and adult CNS of chick, mouse, rat, monkey, and human (see 59).

Most of the immunohistochemical staining experiments have revealed that aFGF and bFGF are associated with neurons in vivo and vitro. However, aFGF and bFGF and their mRNAs have also been found in astroglial cells. Basic FGF is expressed at high levels by astrocyte and the CA2 hippocampal neurons in rats and mice (68), while aFGF has been shown to be expressed in motor, sensory and retinal neurons (62). Acidic FGF has been localized in a subpopulation of ependymal cells and tanycytes, as well as in some glial cells of adult rat brain. Basic FGF can be internalized by neurons and astrocytes and anterogradely transported by retinal ganglion cells after internalization (69). Both bFGF and aFGF influence development, survival and differentiation of various neuronal populations (70-72). Basic FGF promotes the survival and differentiation of both cultured cholinergic neurons of the rat basal forebrain, mesencephalic dopaminergic neurons (70-75), neostriatal GABAergic neurons (76) and immortalized hypothalamic neurons (44). Acidic FGF stimulates cholinergic, glutamatergic and GABAergic differentiation of spinal cord neurons in culture (77).

In culture, rat and bovine brain astrocytes synthesize bFGF and to a lesser extent aFGF (see 59). Of major importance, the release of endogenous astroglial bFGF has been shown to be influenced by other growths factors and lymphokines (59). In particular, Casper and coworkers (78), have reported that in dissociated cultures of dopaminergic neurons obtained from rat mesencephalon, bFGF mRNA is synthesized during time in culture, and its level increases according to the increased total RNA level and as a result of EGF treatment. These authors have, however, demonstrated that EGF and bFGF exert their trophic actions independently. Moreover, the addition of aFGF or bFGF to astrocytes in culture has been shown to induce them to synthesize and secrete NGF (79). Fibroblast growth factors have shown to elicit strong morphologic effects on astroblast, characterized by a shrinkage of their cell bodies, and give rise to extended processes. Besides other GFs, FGFs have been found to stimulate the proliferation of quiescent astroblasts. The fact that an antisense bFGF oligodeoxyribonucleotide inhibits DNA synthesis in rat astrocytes (80), suggests that bFGF in addition to its classical effect as a growth factor, is also an endogenous growth signal in developing astrocytes (59). Basic FGF can also delay the differentiation of O-2A progenitor cells probably as a result of its mitogenic effect (81). In the olfactory epithelium, FGF appears to promote neurogenesis by delaying terminal differentiation of a committed neuronal precursor and supporting proliferation of a possible stem cell, thereby expanding the initial pool of committed precursors (82). Petroski et al. (83) have shown that bFGF acts on astrocytes, not on the hypothalamic neurons growing on them, arresting the proliferation of embryonic astroblasts and inducing morphological differentiation of astrocytes. Neurons, subsequently differentiate well on astrocytes treated with this growth factor.

3.2. Epidermal growth factor

Epidermal growth factor and transforming growth factors (TGFs) are related acidic proteins of approximately 50 aminoacids, and are members of a larger protein family that includes certain viral proteins (36, 84). Recently discovered growth factors belonging to this family are amphiregulin, heparin binding growth factor, and schwanoma-derived growth factor (SDGF) (see 36). Both EGF and TGF-alpha stimulate a protein kinase receptor structurally related to v-erb-B oncogene (84).

EGF and TGFs are trophic polypeptides that stimulate the proliferation and differentiation of different cell types. EGF is also shown to be a potent stimulator of astrocytic proliferation. EGF is not synthesized by the developing neuronal cells, but its homolog, TGF-alpha , is expressed in the brain. During gliogenesis, immunoreactive EGF is detected in tissues and blood. EGF is also a neurotropic agent in neuronal cell cultures, acting as a neurotrophic factor for a number of neurons including rat embryo mesencephalic dopaminergic neurons in culture (36, 78), and immortalized hypothalamic peptidergic secreting neurons (44). Both EGF and TGFs stimulate the proliferation of primary astrocyte cultures obtained from rat cerebral hemispheres (54, 55). EGF is known to strongly affect the morphology of astrocytes and induce upregulation of the glutamine synthase and of the level of S-100 (see 54, 55, and 59 for review). In the peripheral nervous system (PNS) studies have indicated that individual growth factors act as critical determinants of transmitter type . In the brain, however, the initiation of neurotransmitter specific genes appears to involve more complex mechanisms, requiring the obligatory interaction of multiple signal molecules (see 85, 86).

3.3 Insulin and Insulin-like growth factors

Insulin and the structurally-related insulin-like growth factors I and II (IGF-I and IGF-II), are another family of proteins involved in the regulation of metabolism and cellular growth of different type of cells. They exhibit overlapping receptor specificity, with IGF-I and insulin being the most potent ligands, respectively for the IGF-I receptor and the insulin receptor (87, 88). Studies in "in vivo" and "in vitro", clearly demonstrate that IGF-I is a potent mitogen through interaction with its specific type I IGF receptor, which belongs to the family of transmembrane signal-transducing tyrosine kinases (87). This receptor prefers IGF-I over IGF-II and binds insulin with low affinity. The cellular actions of IGF-II are not clarified. The type II IGF receptor preferentially binds IGF-II over IGF-I and does not bind insulin. IGF-I and IGF-II associate with multiple high affinity binding proteins that can modify peptide-receptor interactions (89). Six distinct IGF-binding proteins (IGFBPs) have been characterized and designated IGFBP-1 to IGFBP-6. These IGFBPs are present in extravascular fluids and are secreted by a variety of cells in culture, suggesting their ability to critically modulate local actions of the IGFs (89, 90).

IGF-I and IGF-II expression in the brain is prominent during early development but its expression is limited decreased in the adult brain (91). In contrast Insulin and type I IGF receptor's proteins seem to be expressed by many neurons in the adult brain, particularly in cortical, hippocampal, and cerebellar structures (see 36). Insulin, IGF-I and IGF-II promote the survival and stimulate neurite outgrowth from cultured central and peripheral neurons, including the forebrain cholinergic and mesencephalic dopaminergic neurons (72, 92), as well as cultured immortalized hypothalamic cells (44). The fact that IGF-I is transiently expressed in projection neurons during synaptogenesis has led to the idea that IGF-I has a functional role in synapse formation or stabilization (36). In support of this idea, IGF-I and its receptor are permanently expressed in the olfactory bulb, where the process of synaptogenesis persists during adult life (91). Since IGF-I stimulates myelin formation, it has been suggested to act as a myelinization signal.

Insulin and the IGFs, have been found to stimulate the proliferation of quiescent astroblasts. Primary astroglial cells possess IGF-R, have the capability of synthesizing IGFs and IGF binding proteins, and exhibit a growth response to IGFs (93, 94). Expression of IGFs is developmentally regulated: IGF is present, at relatively low levels, during fetal growth in rodents, monkeys and humans, and rises post-natally to reach a peak during adolescence (94). Receptors for insulin and IGFs have been characterized both on astrocyte-enriched fractions from rat brain (95), as well as on cultured astrocytes (93, 96). There is also a considerable body of evidence pointing to insulin and IGFs being glial mitogens. Ins and IGFs have been reported to promote precursor incorporation into DNA, or to increase cell number in glial cultures (see 54, 55, 59). Insulin may also be involved in regulating glial differentiation. For example, it has been shown that insulin is capable of influencing the phenotypic appearance of astrocytes and the expression of mRNA for GFAP and its encoded protein in cerebellar organotypic cultures (96). Moreover, Avola and associates have clearly shown that the effect of EGF, IGFs and Ins on astroglial DNA and RNA as well as cytoskeletal protein labeling in primary astroglial cultures may change as a function of the changing micro-environment (see 54, 55).

In peripheral tissues, insulin is well known to be involved in the regulation of cell metabolism. Studies on cultured cells suggest that glial glucose utilization may be similarly controlled by insulin and IGFs, and this metabolic response has been shown to be developmentally regulated (see 59).

4. Hormones as Mediators of Neuron-Glia Plasticity

4.1. Sex Steroids

The role of the sex steroid milieu in glial microenvironment, has been established especially by the work of Garcia-Segura et al., and other investigators (see 97-106), demonstrating that the steroid background is crucial in inducing morphological as well as functional changes of the astroglial cell compartment.

In response to estrogens, astrocytes appear to participate in the remodeling of synaptic contacts on hypothalamic neurons that control the release of pituitary secretions in rodents and primates (99-103). This work is substantiated by the findings indicating that the morphology, immunoreactivity, enzymatic activity, and gene expression of astroglia are sexually dimorphic in several brain areas and/or are modified by different in vivo/in vitro experimental manipulations. Glial cells have been shown to harbor receptors for estradiol and progesterone (102, 105, 106), and estradiol is able to induce the appearance of progesterone receptors. In particular, oligodendrocytes, are known to be capable of synthesizing steroids such as pregnenolone and progesterone, and evidences have been presented for the presence of receptors for these hormones on cultured cells (104-106). Astrocytes were found to possess very few progesterone receptors (PRs); confined to cells derived from female animals (105). In contrast, oligodendrocytes prepared from both male and female animals possessed PRs and, although more abundant in culture from females, receptors in cells from both sexes were increased by exposure to estrogens (105). Estradiol has been shown to induce coordinated modifications in the extension of glial and neuronal processes in the arcuate nucleus of the hypothalamus. This hormonal effect results in natural fluctuations in the ensheathing of the arcuate neurons by glial processes and these glial changes are linked to a remodeling of inhibitory GABAergic synapses during the estrous cycle (see 97, 98). Hormonally induced glial and synaptic changes appear to be dependent on specific recognition or adhesion molecules on the neural and/or glial membranes (see 103). Interestingly enough, the effect of estrogen on astroglial cells has been shown to vary according to the specific CNS region (Fig. 4). Taken together, this information coupled with the finding that astroglial IGF-I-like immune reactivity is affected by the neonatal sex steroid background (99, 107), reinforce the authors view that IGF-I is involved in the hypothalamic control of sexual maturation and in the regulation of neuroendocrine events in adult rats (107, 108).

Figure 4. Regional differences in estrogenic sensitivity of astroglial cells. Primary cultures of astroglial cells were prepared from different brain regions including the hypothalamus, olfactory bulbs, cortex and striatum (43). The effect of estradiol was tested during maturation and differentiation in vitro. Astroglial cell proliferation was determined by the incorporation of [3H]thymidine and results depict a dose-response curve of estradiol 17ß (E2, 10-11 - 10-8 M) on 12 DIV primary rat astrocytes. Note the marked stimulation of DNA labeling in hypothalamic and olfactory bulb astroglia compared to cortical and striatum glia.

4.2. Adrenal corticosteroids

There is abundant evidence that cultured glia possess corticosteroid receptors. Adrenal steroids activate two classes of intracellular receptors, the mineralcorticoid (MR) or type I receptor, and the glucocorticoid (GR) or type II receptor (109). These receptor classes can be distinguished on the basis that the MR displays a higher affinity for corticosterone than does the GR, which preferentially binds synthetic glucocorticoids such as dexamethasone (109). Ligand binding studies have demonstrated the presence of a single population of GRs in both astrocytes and oligodendrocytes (110-112).

Glucocorticoids are known to modulate the expression of a variety of glial proteins, including GFAP, glutamine synthetase (GS), myelin basic protein (MBP), and glycerol phosphate dehydrogenase (110, 113). Low levels of GR mRNA have been detected in white matter cells (114). Using an in vitro model of developing neonatal rat glial cell, we studied developmental expression of GR as a function of time in culture and showed low levels of GR mRNA expressed at 8 days in vitro (DIV) with a progressive increase between 12 and 20 DIV and a plateau reaching thereafter, with the mRNA remaining elevated up to 50 DIV (115). "Young" astroglia respond to dexamethazone with a strong morphologic effect. Astrocytes assume a stellate shape and extend processes (Fig. 5, 115). In the intact brain, glial cells have been shown to respond to glucocorticoids. Adrenalectomy results in increased myelination, while glucocorticoid administration inhibits myelination (see 114, for review), the genesis of oligodendrocytes, and the expression of GFAP (112, 113).

Figure 5. Effect of dexamethazone on the morphologic appearance of rat astroglial cells. Type II astrocytes were cultured as described and the effect of the synthetic glucocorticoid, dexamethazone (DEX, 10-9M) was evaluated after incubation for 24 hr during the differentiation of astroglial cells. A. 12 day-old type II astrocytes stained with the MoAb to GFAP; B. Effect of 24 hr incubation with Dex. Note the stellate appearance and processes extension of astrocytes under glucocorticoid treatment.

5. The Second Messenger System and Transcription Factor Network

The vast majority of the signaling molecules coupled to astrocyte receptors are linked to the stimulation of the protein kinase A (PKA) pathway through activation of adenylyl cyclase and elevation of cAMP levels. A second pathway is represented by the hydrolysis of inositol-containing phospholipids, generating diacylglycerol (DAG).

DAG, can be further metabolized to arachidonic acid (AA), and thus form a substrate for eicoisanoid production (Fig. 6). Indeed, agonist evoked release of unmetabolized AA could be significant not only for substrate supply for further metabolism, but also for "inter-cellular" signaling and cross-talk (see 116-119, for reviews).

Figure 6. Dynamic interaction between the astroglial cell compartment, the endothelial and the neuronal cell. Upon selective stimulation astrocytes may release products able to alter the vascular endothelium. The expression of receptors on astrocytes, their ability to synthesize vasoactive products, and the close spatial relationships of these cells both with neurons and cell of the vasculature implicate astroglial cells in bi-directional signaling processes in the CNS (see 116, 117). PG: prostaglandin, PAF: platelet activating factor, TXA2: tromboxane , NO: nitric oxide, ATP: adenosine triphosphate, ADRF (NO): astrocyte-derived (vaso)-relaxing factor (nitric oxide). The potential interaction between the growths factors released by the astroglial compartment and LHRH released by neuron terminals is also illustrated.

Insulin-like growth factor and other members of the GF family, belong to the family of transmembrane signal-transduction tyrosine kinases. Evidence has accumulated suggesting that the effect of these GFs on a number of cell types is mediated by tyrosine phosphorylation of a variety of cellular proteins including phospholipase C, which leads to the formation of inositol 1, 4, 5-triphosphate (120, 121). In addition to membrane receptors that transduce their biological effects, IGF and other GFs associate with multiple high affinity binding proteins that can modify peptide receptor interactions (89, 90, 122). Moreover, specific cytoskeletal proteins such as actin, vinculin, alpha-actin, and myosin could also serve as substrate for tyrosine phosphorylation (123). Supporting evidence for astroglial cell production of a nitrosyl factor, endothelium-derived relaxing factor (EDRFs), and for its autocrine effect has come from a number of recent reports (see 118, 119). Using an antiserum against arginine, it has been demonstrated that astrocytes are the main store of nitrosyl factor, i.e. NO, in the brain (see 119). Moreover, it was found that norepinephrine (NE) increases astrocyte cGMP by a mechanism dependent upon synthesis of NO (see 116, 117), and that astrocytes contain inducible NO synthase activity (see Murphy et al. 1992). Indeed, endothelial cells, macrophages and astrocytes have been reported to express both constitutive and inducible NOS activity. Since a number of studies support the notion that glial cells can respond to NO via soluble guanylyl cyclase present in astrocytes and also contribute to the production of NO in the brain and in view of the fact that the conditioned medium of astrocytes stimulated by a number substances (calcium ionophores, noradrenaline, and glutamate) may contain NO (see 116), then the intermediacy of astrocyte-derived NO may be claimed in a number of neurotransmitter-induced CNS functions.

6. Immune System Modulation of Neuron-Glia Interactions

6.1. Immunological mediators

One focus of attention in the research on astroglial-neuronal interactions concerns the role of astrocyte-derived immune factors in neuronal pathophysiologylogy. A key compartment is represented by the microglia, which is the resident brain tissue representative of the immune system (32, 33). Astroglia (microglia and astrocytes) in culture can be induced to express major histocompatibility complex (MHC) glycoproteins of class I and II by stimulation with gamma-interferon (tau-IFN), or tumor necrosis factor. They have, then, been proposed as possible antigen-presenting cells, thus influencing immune reactions by their production of various agents that signal the immune system (see 32-38, for reviews). Astrocytes, for example, have been shown to be a major source of clusterin (37). In culture, they can be induced to secrete a variety of cytokines and growth factors including colony stimulating factor 1, which markedly stimulates the proliferation of macrophages (37). The eicosanoids produced by astrocytes may also influence immune regulation. In turn, the interleukin family of growth factors alters powerfully astroglia cell physiology. Interleukin 1 (IL-1) is a potent mitogen for astroglial cells and induces astrocytes to synthesize NGF (124). Interleukin 2 and its receptor occur in the brain and this protein promotes the division and maturation of oligodendrocytes and the survival of peripheral nervous system neurons in culture (125). Interleukin 3 supports the survival of cholinergic neurons in culture and in adult rats with experimental lesions (126). Interleukin 6 promotes survival of cholinergic and dopaminergic neurons developing in culture (127). Finally, tau-IFN stimulates the differentiation of embryonic hippocampal and cortical neurons in culture (128). These findings on interleukins and interferon suggest a close interrelationship between neurotrophic and hemopoietic factors and that mechanisms thought to be specific for the immune system play a role in the CNS (Fig. 7). Recent evidence suggests the participation of the immune system in the communication between the neuronal and astroglial compartments. The activation of astrocytes and microglia may contribute to either the initiation or propagation of intracerebral immune responses (37).

Figure 7. Relationship of brain microglia to other cells of the monocyte phagocytic system. Unifying concept put forward by van Furth (see McGeer and McGeer, 1994), recognizing the relationship between circulating monocytes and tissue histiocytes.

A number of brain injuries have been reported to induce proliferation of reactive microglia, including local injection of a variety of neurotoxic agents (such as kainic acid, 6-hydroxydopamine, 5-6, dihydroxitryptamine).Therefore, the contribution of astroglia in a number of neurochemical effects observed following such lesions, should be reconsidered (see 37 for comprehensive review).

7. The LHRH Neuron-Astroglia Network of Signals

7.1. LHRH as the Primum Movens in the Neuro-Endocrine-Immune Reproductive Axis

Neuro-endocrine-immunomodulation (NEI) represents a significant means whereby hormones, growth factors, neuroactive substances and soluble immune mediators convey and translate information to the different neuronal and non-neuronal elements of the CNS. Indeed, evidence, accumulated in the last decades, has clearly documented the vital importance of interacting neuroendocrine-immune networks in the regulation of physiological homeostatic mechanisms (for review see 129-141). In particular, from the early studies of Calzolari (142) almost a century ago, followed by subsequent intuitions of Besedowski (144) and Pierpaoli (143, 144) and more recently others (133, 134, 137-139, 145-153), the brain-pituitary-reproductive axis and the brain thymus-lymphoid axis have been shown to communicate via an array of internal mechanisms of communication that use similar signals (neurotransmitters, peptides, growth factors, hormones) acting on similar recognition targets (the receptors). Moreover, such communication networks form the basis for the controls of each step and every level of reproductive physiology. One such conveying signal is luteinizing hormone-releasing hormone (LHRH), the key reproductive hormone coordinating the major features of mammalian reproduction (Fig. 8).

Figure 8. Schematic representation of the possible interactions between the hypothalamus hypophyseal-gonadal axis and the thymus, with LHRH serving as a major channel of communication. Hypothalamic LHRH governs the release of the pituitary gonadotropins LH and FSH, responsible for gonadal production of the sex steroids. The gonadal hormones in turn, feed back information to the thymus and hypothalamus. At the thymus level, sex steroids act on specific receptors present on the reticulo-epithelial matrix, and induce both up/down regulation of target genes involved in the control of T-cell response. On the other hand, the sex steroid background alters the production of thymic peptides (thymosins) and neuropeptides such as LHRH, with autocrine/paracrine regulatory influence within the thymic microenvironment. The direct neural pathways innervating immune and endocrine organs together with the modulatory influence of glucocorticoids and catecholamines, are also indicated.

Luteinizing hormone-releasing hormone (LHRH), a decapeptide manufactured by highly specialized neuroendocrine cells, is the key regulator of the hypothalamic-hypophyseal-gonadal axis and is essential for reproductive competence (see 137-139, 153, 154). This hormone regulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the gonadotropic cells of the anterior pituitary gland (155).

Hypothalamic LHRH, released into portal capillaries that perfuse the anterior pituitary drives the menstrual cycle by stimulation of pituitary LH and FSH (see 155). Pituitary gonadotropin secretion is finely modulated by classical aminergic neuro-transmitters, the aminoacids, and the neuropeptides (for comprehensive review see 156), regulating the secretion of the "trigger" for the preovulatory surge of pituitary LH secretion on proestrus (Fig. 9).

Figure 9. Schematic representation of hypothalamic peptidergic and aminergic signals together with integrating environmental factors, glial and hypophyseal-mediated mechanisms in the control of the episodic discharge of LHRH. The model includes the LHRH pulse generator, the neural elements (the clock) regulating directly the activity of this generator, and those elements involved in its indirect regulation via the negative feedback action of gonadal steroids. A modulatory influence is represented by the action of sex steroids (estrogens, E2) impinging in this circuitry at both central and peripheral (hypophyseal level) via estrogen receptors, as well as by modifications in the number of pituitary LHRH receptors responsible for alterations in the sensitivity of the gonadotropes to LHRH. Gonadal steroids may also influence astroglial cells to produce and release GFs impinging on the LHRH secretory machinery. The concomitant production of other peptides (i.e. galanine, GAL) together with LHRH and its influence in stimulating the proestrus LH surge (156) is also illustrated.

The episodic manner of LHRH secretion, an intrinsic property of LHRH neuronal networks (157-159) is adjusted by a local hypothalamic network composed of diverse signals including opiates, N-methyl-D-aspartate, t-aminobutyrate and a-adrenergic inputs, the intensity of which may vary according to the sex steroid priming (see 156) (Fig. 9). A further level of control is represented by the ability of the decapeptide to directly modulate its own secretion via an ultra-short feedback mechanism, by exerting both stimulatory and inhibitory actions in LHRH neuronal cells depending on its concentration and duration (153, 159).

7.2. The LHRH Neuronal System within the Central Framework of Immune Signaling Systems

The powerful interaction between the immunologically-derived soluble products (cytokines) and the LHRH system, at the CNS level (140, 141, 160-166) coupled to the immunomodulatory properties of LHRH and its potent analogs (see 147-150, 167-180), lend support to the notion that a commonality of signaling mechanism(s) exists between the immune and neuroendocrine cells. A number of cytokines have been shown to affect LHRH release from the medio-basal-hypothalamus (MBH) either in vivo or in vitro. In particular, interleukin 1 (IL-1), one of the key mediators of immunological responses to stress, infection and antigenic challenge (see 140, 162, 181), has been shown to interfere powerfully with the hypothalamic-hypophyseal-gonadal axis (HHGA). At the CNS level (see 160-162, 164-166), when administrated in an acute fashion, IL-1 has been shown to decrease plasma LH levels, a phenomenon attributed to the inhibition of hypothalamic secretion of LHRH and LHRH gene expression. That IL-1 represents an extremely potent factor inhibiting the activity of the HHGA is supported by several different lines of evidences. Interleukin-1a inhibits pulsatile release of LH via a direct action on the LHRH neurons by suppressing the release of prostaglandin E2 (PGE2) from the MBH (see 164). Moreover, IL-1 administration inhibited of the physiological or experimentally induced afternoon proestrus LH surge follows (161), together with expression early c-fos gene which occurs within the LHRH cell nuclei during this same period of the cycle (166). The ability of endotoxin to induce release of IL-6 from the MBH has been demonstrated by Spangelo and coworkers (182). Moreover, hypothalamic LHRH neurons in culture spontaneously secrete IL-6, and in turn exogenous IL-6 is able to stimulate LHRH release in a dose- and time-dependent fashion (163). The intermediacy of nitric oxide in IL-1-a control of LH in vivo and vitro has been recently established (165). In addition, it was demonstrated that when HHGA is chronically exposed to icv infusion of IL-1ß, a complete disruption of the estrous cycle, decreased biosynthesis/release of hypothalamic LHRH and gonadotropins was accompanied by a block in luteolisis of newly formed corpora lutea (CL) (166).

Interleukin-1 has been shown to be present in the cerebrospinal fluid, IL-1 mRNA is detected in normal brain and IL-1ß-like immunoreactivity in both hypothalamic and extrahypothalamic sites in human brain have been identified (for review see 140, 181). A major compartment of cytokine production is, however, represented by astroglial and microglial cells. It would, then, appear that according to the stage of the estrous cycle, the peptidergic and aminergic background, a number of potential interactions between the cytokines and the central LHRH system, may be envisaged (Fig. 9).

Further evidence for an interaction between LHRH and a central immune network came from the studies of Silverman and collaborators (183) that demonstrated the presence of a population of non-neuronal cells, recognized by LHRH-like immune material present in large numbers in the medial habenula of the ring dove, which presented all the features of mast cells. Therefore, it is possible that mast cell secretion into the brain (and other peripheral organs) may represent an additional delivery system for biologically active substances such as LHRH (183). In many regions, including the CNS, mast cells are innervated or in close proximity to nerve terminals, and can be stimulated to release their granular content by neuropeptides. Of particular interest, is the clinical observation that histamine secretion from mast cells and cutaneous anaphylaxis can be induced with LHRH and LHRH-agonists and antagonists, and that LHRH-agonists (LHRH-A) binding sites are present in mast cells (184, 185).

7.3. Growth Factor and Steroid Sensitivity of LHRH Neurons

The work of Ojeda and other authors, has clearly established a prominent role of polypeptide growth factors with neurotrophic activity in the developmental regulation of the hypothalamus (for extensive review see 186-190). These authors have postulated that the initiation of puberty involves the trans-synaptic stimulation of LHRH neurons by excitatory neurotransmitter system(s) and the facilitatory effects of GFs, that are suspected to act indirectly via activation of glial function (188). TGF-alpha mRNA levels increase gradually in both preoptic area and the MBH after the anestrous phase of puberty, reaching peak values on the afternoon of the first proestrus (186-188). Since parallel changes in hypothalamic astroglial IGF-I like immunoreactivity have been detected (107, 108) an interdependence of the two mechanisms has been suggested. Such findings coupled to the gender differences in astroglial IGF-I immunoreactivity and the reported fluctuations associated with the estrous cycle clearly underline the participation of this growth factor of astroglial origin in the hypothalamic control of sexual maturation (98, 99). Interestingly, an up- and down-regulation of LHRH release from the GT1-1 cell line has been recently measured following activation of growth factor receptors (43). This work suggested that the signaling pathways activated by different GFs (EGF, IGF-I and Ins) may influence the LHRH machinery, possibly via a crosstalk between the protein tyrosine kinase (PTK) and protein kinase C (PKC) transducing pathways.

The protein kinase A (PKA) and the PKC have been implicated in LHRH biosynthesis and secretion (191-197). LHRH release is affected by a number of neurotransmitters that act through the PKA and PKC/calcium second messenger systems. In fact, cAMP and LHRH levels in the hypothalamus vary in concert during the estrous cycle, and both are highly responsive to estrogens (195). Neurotransmitters can stimulate cAMP accumulation, or calcium influx, and their effects can be blocked in the absence of calcium. Moreover, the effects of these neurotransmitters can be mimicked by direct application of PKA or PKC activators (see 195, 196). Forskolin and phorbol esters, such as phorbol myristate acetate (PMA), activators of the PKA and PKC pathways respectively, have been shown to enhance LHRH mRNA steady state levels in the hypothalamus (194-196). In particular, PMA stimulated LHRH release from the GT1 cell line while inhibiting transcription of the pro-LHRH gene and suppressing LHRH messenger RNA (mRNA) levels. Using the GT1-7 immortalized LHRH cell line, Wetsel and coworkers have recently (195) shown that forskolin can produce changes in neuronal morphology while phorbol esters induced decreased neurite formation and cell-cell adhesion (195). Translational efficiency of LHRH mRNA has been also shown to be negatively regulated by phorbol esters in the GT1 cell line (197).

The nitric oxidergic pathway is importantly involved in the dynamic regulation of LHRH expression and peptide release (see 165, 198-199). The source of NO and NO synthase (see 199-201) have been claimed to reside in proximity or in the LHRH neuron itself. For instance, NO has been shown to maintain pulsatile LHRH release, to be involved in NE-induced stimulation of LHRH release, and in cytokine-induced inhibition of LHRH release with the participation of PGE2 (see 140, 165).

8. LHRH Neuron-Astroglia Interactions during Development

During embryonic development, the LHRH neuronal system appears to be unique among all neuropeptide expressing genes in the central nervous system (CNS), to make a migration pathway from the epithelium of the medial olfactory placode into the developing basal forebrain (202-208). Failure of LHRH neuronal migration is responsible for the suppression of the pituitary-gonadal axis in Kallmann's syndrome (209), an inherited migration disorder resulting in hypogonadotropic hypogonadism with anosmia (203-206, 210). The migration of LHRH neurons is in close association with a neural cell adhesion molecule, N-CAM and Ng-CAM enriched fiber bundle (211, 212). In mice (203), monkeys (212) and chicks (207, 208), LHRH is expressed in neurons early in development and continue to be expressed in neurons as they migrate. Vomeronasal nerves have been included as part of a complex of olfactory fibers that participate in LHRH cell migration (205, 206, 213-214). The exact mechanism(s), however, involved in LHRH neuronal migration remain unclear. The fibers associated with LHRH neuronal migration have been demonstrated to express the neural cell adhesion molecule, N-CAM (204, 211), a highly polysialated form of neural cell adhesion molecule, PSA-N-CAM (214), peripherin (213) and the CC2-immunoreactive olfactory glycoconiugate (216). Recently, Yoshida et al. (217) have shown that LHRH neurons migration across the medial olfactory bulb and forebrain is associated with a caudal branch of the vomeronasal nerve. After migration in the forebrain in association with the TAG1+, PSA/N-CAM+ axons, the majority of LHRH neurons continue to migrate laterally and ventrally into the preoptic area/anterior hypothalamus (217) . It seems highly possible that other factors such as soluble chemotropic molecules, extracellular matrix molecules, and adjacent neurons and glial cells, may also be involved in this phenomenon.

9. LHRH Neuron-Astroglia Interactions in the Adult Brain

In the adult rodent, LHRH is synthesized by diffusely organized forebrain neurons which are scattered over a continuum extending from the septal region anteriorly to the premamillary area, with the heaviest concentration in the anterior hypothalamus, the preoptic area and the septum, with fibers projecting not only to the median eminence but also through the hypothalamus and midbrain (218). During this passage, LHRH neurons are known to interact with many types of neurons and glia. Indeed, the architecture of the arcuate nucleus of the hypothalamus is unique in the arrangement of the glial cells within it. The architecture of the arcuate nucleus of the hypothalamus is unique in the arrangement of the glial cells within it. Tanycytes, specialized ependymal cells, line the ventricular wall and send their processes in an arching trajectory toward the surface of the brain (219). Astrocytes of varying morphologies (3) are also located in this region (220, 221). The contribution of glial elements to LHRH axonal targeting was suggested by the early experiments of Kozlowski and Coates (222) demonstrating the existence of ependymal tunnels and their association with LHRH axons. More recently, relationships of glia to LHRH axonal outgrowth from third ventricular grafts in hypogonadal mice have been described by Silverman and coworkers (223). Due to the absence of a functional gene for the neuropeptide LHRH, the hypogonadal (hpg) mice have infantile reproductive tract in adulthood, a condition that can be reversed by the implantation of normal fetal preoptic area tissue that contains LHRH neurons. Interestingly, LHRH axons were found adjacent to glial elements along their entire course from the graft-host interface, and appeared to exit via glial channels (223). Glial processes seem to provide a permissive substrate for LHRH axonal extension and the presence of chemotropic factors specific for the region of the median eminence underlie the accurate navigation of the growing axon as been suggested by Silverman and collaborators (223). The fact that LHRH axons display a remarkable degree of outgrowth during a time of extensive glial hypertrophy and hyperplasia suggests that the glia may play a facilitatory or permissive role in this particular system (223).

Then, in view of the high requirement for signaling environmental conditions to the LHRH neuronal system, and due to the paucity of synaptic inputs to the LHRH neurons , it seems reasonable to hypothesize that LHRH-astroglial interactions may play a key role in the successful decodification and transduction of appropriate signals from the different regions involved in the control of LHRH release. In fact, besides the conventional regulation of LHRH secretion at the level of LHRH cell bodies or terminals at the median eminence (ME), it seems quite apparent that LHRH may locally be modulated by dynamic relationships among neuron terminals, glia and basal lamina (see next sections), as already demonstrated for oxytocin and vasopressin (224) (Fig. 9). Indeed, based on electron microscopic results, King and Letourneau (225) have recently described that LHRH terminals in the median eminence (ME) undergo dramatic changes after gonadal hormone withdrawal, and a possible direct action of "intervening non neuronal (glial) elements", of the ME, has been suggested (225). Kohama and coworkers (226) have recently demonstrated that glial fibrillary acidic protein (GFAP), the main component of the intermediate filaments in cells of astroglial lineage, increases during proestrus in astrocytes of the hypothalamic arcuate nucleus (ARC) (226). Moreover, these changes were associated with altered astrocyte-neuron contacts and synaptic remodeling, during preparation for the preovulatory gonadotropin surge (227). Interestingly, hypothalamic distribution of astrocytes is gender-related (227). The work of Finch and coworkers has also recently found evidence that GFAP in the thalamus and hypothalamus increases with reproductive aging (228), while food restriction delays the age-related increase in GFAP mRNA expression in the hypothalamus (229). Finally, the development of astrocytes immunoreactive for GFAP in the MBH of hypogonadal mice revealed a marked increase for the glial fibrillary protein (230).

Of major importance, the studies of Ojeda and collaborators (186-190) have indicated a key role of astroglia-derived factors in the stimulation of LHRH release and induction of precocious puberty by the lesions of the female rat hypothalamus. Brain injury is known to result in the appearance of various mitogenic and neurotrophic activities in the area surrounding the lesion (see 188). Evidence has been provided that lesions of the preoptic-anterior hypothalamic (POA-AHA) area which induce precocious puberty, result in enhanced expression of the epidermal growth factor receptor (EGF-R) gene in reactive astrocytes (189, 190) surrounding the lesion site. Transforming growth factor a (TGF-alpha) is thought to mediate the puberty advancing effect of POA-AHA lesions on sexual development via its stimulatory effect on LHRH secretion (187-190). These effects have been postulated to require the glial cells as an intermediary, which upon TGF-alpha stimulation produce prostaglandins that act on the LHRH nerve terminals to enhance LHRH release (see 187). Therefore, it has been suggested, that the simultaneous increase in EGFR and TGF-alpha in reactive astrocytes may provide the necessary amplification for TGF-alpha to exert its stimulatory effects on LHRH secretion and cause sexual precocity (for comprehensive review see 190).

10. Characterization of Influence of Astroglia on the LHRH Neuronal System: Development of an "In vitro" Model for the Study of Neuronal-Glial Interactions

10.1. Astroglial-derived factors exert potent neurotrophic effects on the immortalized hypothalamic LHRH neuronal (GT1-1) cell line

The immortalized GT1 neuronal cell line derived by targeting the expression of the oncogene, simian virus-40 T-antigen, to the LHRH-expressing hypothalamic neurons of transgenic mice (231) has provided a model system to study the mechanisms involved in LHRH regulation at multiple levels (see 231-236). We have used the GT1 cell line and primary cultures of astroglial cells and assessed different dynamic models (Fig. 10), to investigate LHRH-astroglia interactions (41). Our work shows that in controlled "in vitro" conditions, astroglial cells during their "in vitro" differentiation and maturation, produce factors that significantly accelerate the acquisition of the neuronal phenotype and sharply stimulate the spontaneous release of the decapeptide in the medium (41). While control GT1-1 neuronal cells at 2, 4 and 6 days of culture show a classical morphological pattern (Fig. 11) characterized by a progressive shift from the ovoidal shape after 1-2 days of culture to progressively reach the neuronal phenotype, when GT1-1 neurons are cultured in the presence of astroglial cell conditioned medium (ACM) from immature (10-12 DIV) astroglia, an initial extension of neurite was evident, and extensive neurite outgrowth at 4 and 6 DIV together with manifestation of neurite formation, and establishment of cell-cell contacts (see arrows, Fig. 11).

Figure 10. Schematic illustration showing different dynamic models in the study of neuron-glia interactions. This model takes advantage of a pure hypothalamic neuronal cell line derived by genetically targeted tumorigenesis (231) and primary astroglia cells. Astroglial cells are maintained in vitro and the conditioned medium (CM) prepared at different (8-40 DIV) time intervals during maturation (41). The morphology, proliferative and secretory capacities of LHRH neurons are studied during differentiation and maturation in vitro (2-8 DIV). Different dynamic models (co-culture, mixed culture) permit differentiation between the contribution of soluble mediators and cell-cell contacts in LHRH neuron-astroglia crosstalk.

Figure 11. Immunocytochemistry of LHRH neurons demonstrating the effect of astroglial cell conditioned medium (ACM) on GT1-1 cell morphologic appearance. GT1-1 cells were grown in DMEM (A) or astroglial conditioned medium (ACM, B), and analyzed at different time intervals (2-6 days ). Note neurite extension in GT1-1 cells grown for 8 days in ACM (B), the flattened appearance of the cells, extensive neurite outgrowth, growth cones and cell-cell contacts after 4 (d) days of culture in ACM, compared with GT1-1 neurons grown in DMEM at 8 days of culture (A).

The morphological effects were reflected at a functional level by a sharp stimulation of basal LHRH release into the medium. It should be noticed that the stimulation of spontaneous LHRH release was strictly dependent upon the stage of both glial and LHRH neuron differentiation (41). In fact, the less differentiated stage of astroglia in this "in vitro " maturational profile (8 DIV), is the less active condition for stimulating LHRH release at each time interval during LHRH neuronal differentiation. On the other hand, young glial cells (12 DIV), are highly potent neurotrophic stimuli for the LHRH neuron. Such stimulatory effect is, however, dependent upon the stage of LHRH differentiation. Similarly, at later stages of glial maturation and differentiation (16-40 DIV), glial-derived factors differentially affect LHRH release.

This effect depends on the stage of LHRH neuron differentiation, being highly stimulatory in GT1-1 undifferentiated neurons (2 DIV), and gradually losing this activity with LHRH neuronal differentiation (41). This information suggests a possible different nature of glial factors acting at a particular stage of GT1 neuron differentiation "in vitro", and/or alternatively, the saturation of some intracellular transducing mechanisms responsible for LHRH production (41).

10.2. Specificity and Regional Differences in Astroglial Modulation of GT1-1 Neuronal Differentiation

The specificity of astroglial conditioned medium is further corroborated by a series of information. Addition of a peptidase inhibitor in the different astroglial-conditioned media (ACMs) did not alter the observed effects, thus excluding nonspecific effects due to different degrees of LHRH degradation in the culture medium (41). In addition, ACM from 5 different regions exhibited significantly different degrees of stimulatory activity in both LHRH morphologic appearance and LHRH secretion. Also, CM of oligodendrocyte was unable to modify LHRH output, implying a region-specificity of the glial-derived factors in the modulation LHRH neuron morphology and peptide release from the GT1-1 cell line (Fig. 12). Regional differences in glial-derived factor ability to support axon and dendrite growth, have been also reported by different investigators (237-239). During axonal growth in developing brain, the astroglia present in axonal pathways are relatively immature, and differ from mature astroglia in their cytoskeletal composition and morphology (see 240). In addition, the astroglial support of neurite extension depends on the state of differentiation of astroglial cells (240) and a differential effect of "young" versus "old" glia on LHRH neuronal morphology and morphometry has been recently characterized (41).

Figure 12. Regional differences of glial-derived factors that promote LHRH release from the GT1-1 neuronal cell line. Astroglial conditioned medium from the different regions was prepared as indicated (41) and 12 DIV ACMs or oligodendrocyte CM were tested during in vitro LHRH neuron differentiation (2-8 DIV). LHRH release in the medium is expressed as percentage (%) increase compared to LHRH released from GT1-1 neurons grown in DMEM (control). Results are the mean ± SEM of 2 different experimental manipulations. ** p < 0.01 vs. hypothalamic glia; °° p < 0.01 vs. cortical and olfactory bulb glia.

10.3. Peptidic nature of the astroglial-derived factors

The peptide nature of glial-derived factors was suggested by the fact that boiling ACM completely abolished its activity on both GT1-1 neuron phenotype and peptide release, supporting the protein nature of the trophic factors released during in vitro glial maturation (41). Preliminary observations using SDS-PAGE (sodium dodecyl-sulphate polyacrylamide gel electrophoresis) indicate that glial-derived growth factors are qualitatively and quantitatively different during astroglia differentiation "in vitro " (Avola, Reale, Costa, Gallo and Marchetti, unpublished observation).

11. Astroglial-derived factors stimulate leukocyte proliferation

Since astrocytes and microglial cells produce a variety of cytokines, some of which may have a role during maturation and differentiation of the glial cells (241), we have tested a possible immunological nature of glial soluble factors (44). For this aim thymic lymphocytes were treated with ACM at different stages of glial differentiation. As observed in Fig. 13, thymocytes show a biphasic pattern of response to ACM: 8 and 12 DIV astroglial cell culture medium induced a significant increase in 3H-thymidine incorporation comparable or even greater than the one observed following a subactive dose of the lectin polyclonal mitogen, concanavalin A (Con-A, 0.3 mg/ml). At later stages of glial maturation (40 DIV) ACM produced a sharp inhibition in T-cell proliferation (44).

Figure 13. Astroglial-derived factors modulate lymphocyte proliferation. In order to test the ability of astroglia CM to modulate immune cell proliferation, thymocyte cell preparations were treated with either a subactive dose of the T-cell mitogen, concanavalin A (Con-A, 0.3 mg/ml) and/or ACM of 8, 12 and 40 DIV. Note the marked [3H]thymidine incorporation after treatment of murine thymocyte with 8 and 12 DIV ACM, at 40 DIV however, ACM is no longer stimulatory, and a significant inhibition of proliferative capacity was measured.

12. Diffusible molecules and cell-cell contacts participate in LHRH-astroglia interactions

12.1. Role of soluble factors

As a further step to verify the possible bi-directional communication between astroglial cells and GT1-1 neurons, a co-culture system was established (41). In these conditions, where the two cell-compartments were allowed to communicate with each other, but in the absence of cell contacts, a significant stimulation of basal LHRH release was observed, although GT1-1 proliferative potential was almost doubled, thus resulting in a net decrease of neuronal secretory capacity (Fig. 14).

Figure 14. Effect of astroglial (12 DIV)-GT1-1 neuron co-culture in the maturation of LHRH secretory potential. The technical procedure is described in details (41). Cortical glia was used in this experimental paradigm. LHRH secretion was examined every 2 days for 8 days. For measurement of LHRH release by RIA, the medium was replaced every two days, collected, centrifuged to remove cellular debris, and frozen at -80 °C. Results are the mean ± SEM of 2-3 different experimental manipulations. ** p < 0.01 vs. control.

This experimental paradigm revealed for the first time the presence of a bi-directional flow of informational molecules between the two cell populations, as observed by a doubling of the proliferative potential of each cell population, suggesting that LHRH and GFs released by glia, participate in GT1-1 neuron-astroglia crosstalk (Fig. 15).

Figure 15. Proliferative capacity of GT1-1 neurons and astroglial cells in co-culture conditions. At 2, 4, 6 and 8 days and for each respective cell type, GT1-1 or cortical astroglial cell proliferation were tested in triplicate by incubation of [Methyl-3H]Thymidine (1 mCi/ml of culture medium) for 2 h at 37 °C. Labeled DNA was collected and radioactivity was determined by liquid scintillation spectrophotometry, as described. Results are the mean ± SEM of 2-3 different experimental manipulations. * p < 0.01 vs. control.

In mixed cultures, both spontaneous LHRH release and GT1-1-astroglial cell proliferation were significantly increased. The inability to further stimulate LHRH release in the face of the presence of such a mitogenic effect on the GT1-1 neurons may have different explanations, depending on both 1. the autoregulatory actions of LHRH on its own secretion; 2. the nature of the GFs released by astroglial cells and their coupling to specific intracellular transducing pathways; and 3. the presence of cell-cell contacts interfering with LHRH inter/intracellular dynamics. It is possible that LHRH released in the medium could influence the further production of astroglial-derived factors either directly or indirectly, via receptor-mediated events and/or through second messenger-activated systems delivering signal molecules utilized by the neighboring cells, thus realizing a "cross-talk" between the separated cell compartments (see Fig. 6).

The ability of astrocytes to synthesize and release a number of prostaglandins (PGE2, PGF2alpha) and also tromboxane A2 (TX) in response to arachidonic acid (AA) or calcium ionophore (for review see previous sections) constitute a major link in LHRH-astroglial interactions, since PGE2 is an obligatory component in the phasic discharge of LHRH from the MBH. Another important connection, between the LHRH neuron and astroglia, as previously recalled, is their ability to use and to produce the novel "intercellular" diffusible modulator, NO and to express NO synthase (see previous sections). In the light of the host of receptors present in astrocytes, their ability to synthesize vasoactive products, and close spatial relationship of these cells both with LHRH neurons and cells of the vasculature implicate them in bi-directional signaling processes in the CNS. Signals, in turn, originating from the LHRH neurons could initiate important intracellular changes in astrocytes. The resulting release of prostanoids, and nitrosyl compounds could have profound modulatory effects on the activity of the adjacent (astrocyte/neuronal) cell (Fig. 6).

13. Cross-talk between astroglial-derived immune mediators and intercellular/intracellular signaling agents

In order to establish the ability of astroglial-derived cytokines to modulate LHRH release from the GT cell line, the potent inducer of immune soluble mediators, lipopolysaccharide (LPS) was studied in both co-culture and mixed culture conditions. While in absence of physical contacts (co-culture), LPS treated glia, sharply stimulated LHRH release, a marked inhibition of spontaneous LHRH production followed addition of LPS in the mixed culture condition (Fig. 16). Since LPS is known to stimulate NO production, a possible intermediacy of the nitroxidergic pathway in LPS-induced LHRH release cannot be discounted. On the other hand, the marked inhibition of LHRH in the mixed culture condition in the presence of cell-to-cell contacts may suggest a. the prevalence of inhibitory signals and/or b. the saturation of intracellular transducing pathways leading to the release of LHRH.

Figure 16. Effect of lipopolysaccharide stimulation in LHRH-glia interactions. To test the ability of astroglial-derived cytokines to modulate LHRH output from the GT1 cell line, the potent cytokine inducer, lipopolysaccharide (LPS, 10-100 ng/ml) was added to either the co-culture or mixed culture models. Astroglia proliferation was monitored by DNA labeling. Note the sharp stimulation of LHRH release following 10 and 50 ng/ml LPS and the parallel increase in astroglia proliferation during co-culture. In mixed culture, however, an almost 60 to 70% decrease of LHRH release was measured.

14. The Role of Cell Adhesion in Neuron-Glia Interactions: Effect of Anti-Neural Cell Adhesion Molecule (N-CAM) Antibody (Ab) on LHRH Neuron Morphology, Secretion and Proliferation

When GT1-1 neurons are grown in the presence of astroglial cells, glial tracks begin to build-up diffuse pathways along which LHRH immunoreactive neurons concentrate (Fig. 17A). The quantification of the morphometric features of LHRH-astroglia interactions for process length and branching revealed a significant elongation of the LHRH neuron increasing as a function of time in co-culture, accompanied by a remarkable increase in length and number of LHRH processes per cell (41, 43). "In vitro", astroglia possess neuronal-growth promoting properties, including cell adhesion receptor systems that support neurite extension (see 242-245). Indeed, neuronal-astroglial interactions are believed to be mediated by "adhesion molecules", a heterogeneous group of glycoproteins found either in extracellular matrix, or anchored to the cell membrane (246, 247). Besides other molecules, neural cell adhesion molecule (N-CAM), promotes neurite outgrowth and participates in both kinds of neuron-glia interactions (245). A functional role of N-CAM has been demonstrated during development, with the distribution of the molecule throughout the CNS varying both temporally and spatially (245). During embryogenesis, Schwanzel and Fukuda (see 202-205), have clearly shown that migrating LHRH-immunoreactive cells were never found independent of the N-CAM immunoreactive scaffold, and suggested that: "cells interacting through N-CAM form part of a structure containing a complex of mechanical and chemical cues that guide the LHRH neurons in the brain" (203). Although it was not possible to determine if N-CAM-immunoreactive cells that make up this aggregation were neurons or glia, the importance of N-CAM in LHRH neuronal migration was further supported by disruption analysis (204), where it was shown that injection of an anti-N-CAM serum into the olfactory pit of embryonic mice retarded the migration of the LHRH-immunoreactive neurons. Moreover, among a series of CAMs (including cytotactin, laminins and fibronectin) tested, only fibers immunoreactive for N-CAM were seen along the LHRH migration route (see 204).

Figure 17. Immunocytochemistry and functional capacity of LHRH neurons grown in the absence or the presence of astroglial cells in a mixed culture preparation, with or without the presence of neural cell adhesion molecule antibody (N-CAM Ab) (41). N-CAM antibody (1 mg/ml) was added from the beginning of the experiment (T= 0), and every 2 days, the medium was replaced with fresh medium containing the Ab. Left panel: Maturation of GT1-1 neuron secretory potential. Results are the mean ± SEM of 2-3 different experimental manipulations. * p < 0.05, ** p < 0.01 vs. control GT1-1; °° p < 0.01 vs. Mixed Culture. Right panel A: Control cultures at 4 DIV showing an intense reaction of the LHRH cell bodies sending axons that contact either neighboring LHRH cell bodies; or astroglial cell (for details see text). The glial cells are often surrounded by LHRH neurons and send prolongations to LHRH cells. B: a general atrophy of neurons (see arrows). (Magnification x300).

Figure 18. Effect of astroglia (12 DIV)-GT1-1 neuron mixed-culture in the absence or the presence of neuronal cell adhesion molecule (N-CAM) antibody on the maturation of GT1-1 neuron secretory potential. The N-CAM antibody (1 mg/ml) was added from the beginning of the experiment (T= 0), and every 2 days, the medium was replaced with fresh medium containing the Ab. Results are the mean ± SEM of 2-3 different experimental manipulations. * p < 0.05, ** p < 0.01 vs. control GT1-1; °° p < 0.01 vs. Mixed Culture.

When moderately high doses of N-CAM were added to GT1-1 neurons, an approximately 35% reduction of LHRH secretion was measured. In neuron-astroglial cell cultures, however, the addition of N-CAM Ab resulted in dramatic effects on LHRH morphology (Fig. 17B), and a sharp (almost 95 %) inhibition of both LHRH release and cell proliferation. The effects on LHRH morphologic appearance were striking: a general atrophy and degeneration of GT1-1 neurons followed N-CAM-Ab treatment (see Fig. 17B).

In particular, a sharp reduction of the immunocytochemical reaction together with cyto-plasmatic degeneration, nuclear vacuolization and chromatolysis (tigrolysis) were observed (see arrows in Fig. 17B). The axons that were longer and thinner, were seeking to contact other neurons (see arrows in Fig. 17B). No visible contacts between the GT-1 neurons and glial cells were observed. On the other hand, in control LHRH-astroglial mixed cultures, an intense reaction of the LHRH cell bodies was present together with the establishment of contacts between LHRH neurons, and LHRH neurons with the astroglial compartment, with neurite contacting either neighboring LHRH cell bodies/axons, or astroglial cells (see Fig. 17A). Also, astroglia react to the presence of LHRH neurons with the cell morphology changing from process-bearing to polygonal and flat shapes, in the mixed culture preparation (see Fig. 2). Also from a functional point of view astroglia respond to LHRH signals, since the proliferative capacity of the mixed culture is significantly increased (41).

Polyclonal as well monoclonal N-CAM Abs have already been shown to inhibit cell aggregation and neurite outgrowth depending on the neuronal cell type and the developmental period (245). In PC12 cells and some other neurons, N-CAM appears to stimulate neurite growth through a pertussis toxin-sensitive G protein and activation of Ca2+ channels (245). While we have provided the first documentation that N-CAM Ab exerts a potent inhibition of GT1-1 neuron functional capacity, further studies are required to clarify the mechanisms involved in this phenomenon.

The available results may indicate that diffusible factors regulate glia-LHRH interactions in collaboration with molecules associated with the cell surface matrix. Such findings may suggest that modulation of LHRH secretion may be under local control of interacting (neuron/neuron; neuron/glia) cells. However, the actual contribution of the two phenomena, adhesion per se and/or neuronal-glial interactions, cannot be clarified at present.

15. Progression and competent growth factors exert direct neurotrophic and functional effects on the GT1-1 neuronal cell line: presence of synergism/cooperativity

Stiles and coworkers (248) have allowed the classification of mitogens as "competence" or "progression" factors that cooperate for a full mitogenic response. Competence factors are not able to induce DNA synthesis, but give the "competence" to respond to other hormones ("progression factors") that stimulate "progression" through the cell cycle. Incompetent cells do not respond to progression factors and remain arrested. The category of competence factors include platelet derived growth factor (PDGF) and fibroblast growth factor (FGF), while the progression factors include epidermal growth factor (EGF) and the insulin family of growth factors (IGFs). Since diffusible molecules of peptide nature were previously shown to participate in GT1-1 neuron-astroglia crosstalk, the participation of different growth factors (GFs) was tested by: a. assessment of their ability to directly exert, either alone or in combination, neurotrophic effects during GT1-1 neuronal differentiation; b. perturbation experiments aimed at neutralizing a specific GF during GT1-1 neuron-astroglia interactions. Our recent data (44) provide evidence that bFGF, EGF, IGF-I and Ins can directly exert neurotrophic effects and variously influence cell survival/proliferation and GT1 functional capacity. Moreover, a synergism /cooperativity between the competent factor, bFGF, and the progression factors (EGF, Ins and IGF-I) was measured indicating the possibility that the sequential expression of these factors during development may differentially regulate LHRH neuronal migration, differentiation and modulate the ability of these neurons to elaborate and secrete the neuropeptide.

In rodents, neurogenesis is completed before birth, but gliogenesis occurs in the first week of postnatal life, and it is possible that different mitogenic peptide growth factors may be involved in the regulation of the two processes. Both competence and progression factors were found to differentially affect GT1 neurons according to the specific GF, the schedule of administration, and the stage of GT1-1 neuron differentiation (44).

16. Basic fibroblast growth factor is a candidate signal molecule that collaborates in partnership with LHRH to regulate GT1-1 neuron-astroglia interactions

Basic fibroblast growth factor is known to be present in the telencephalon as early as E9.5 and in the cerebral cortex throughout neurogenesis and into adulthood (70, 249-251). Moreover, bFGF is known to stimulate the division of committed neuronal progenitor cells derived from olfactory epithelium (82). Such findings are of special interest and potential implications in the genesis of LHRH neurons and glia, because both cell types are derived from the olfactory placode. Peptide growth factors released by olfactory bulb glia represent a strong neurotropic stimulus for the GT1 cell line (41). Basic fibroblast growth factor is associated with extracellular matrix and cell membranes and has been suggested as possible candidate mediating cell-cell contacts (251, 252). Our recent study showed that neutralization of bFGF action during GT1-1 neuron-astroglia interactions, produced a significant inhibition of astroglia neurotrophic effect (44). Such result indicates that endogenous bFGF of neuronal and/or astroglial origin as a candidate molecule in such crosstalk. These data are of particular interest in the light of the recent discovery of a paracrine bFGF system, endogenous to the GT1-1 neuron (253). Indeed, using RNAse protection assays and riboprobes specific for murine FGF receptors 1-3, these authors showed that GT1-1 do express FGF receptors, and that occupancy of these receptors by bFGF stimulated the sustained tyrosine phosphorylation of both the 42- and 44-kilodalton mitogen-activated protein kinases (MAPKs) for up to 6 hours (253). In addition, the GT1 cells were also shown to express mRNA for bFGF (261), but at low levels, possibly due to an instability of the bFGF mRNA (253). It should be noted that bFGF lacks a signal peptide (254), and it is unclear if bFGF synthesized by the GT1 cells is released as a biologically active peptide and/or the GF needs further processing. Recent studies have, however, indicated that bFGF-like peptides are released by cultured neuronal and glial cells (255). Moreover, the release of astroglial bFGF may be influenced by other GFs (see 59). The available information raises the possibility that bFGF possibly acting in partnership with LHRH and other GFs of neuronal and/or astroglial origin, may modulate/drive GT1 neuronal differentiation. That growth factors play a prominent role through their cooperation with auxiliary agents has been suggested (see 256-259). Indeed, Iacovitti and coworkers (257, 258) hypothesized that the catecholaminergic (CA) neurons may harbor or have local access to all of the agents necessary for their biochemical differentiation. Induction of the CA-specific gene, tyrosine hydroxylase, has been recently shown to be mediated by a novel mechanism requiring the simultaneous actions of both aFGF and the enzyme's catalytic end products, the CA (257). This could then represent a more general mechanism in the regulation of neuronal phenotype differentiation during development, and in the adult brain.

17. Summary and Conclusion

All together, the presented information supports the concept that astroglia participate in CNS intercellular communication and in the interaction between the nervous and immune systems. In particular, this review has emphasized the commonality of signaling networks shared by LHRH neurons and glial cells. Glial cells play active roles from embryonic development to adulthood. During development, astroglia direct the migration of neurons to the right targets. In the adult brain and spinal cord, the oligodendrocytes in he CNS, and the Schwann cells in the peripheral nervous system, participate in propagation of electrical impulses. The microglia serves as the brain's immune cell. But as pointed out by John Travis (21), the real "stars" in this scenario are the astrocytes, able to manufacture a wide variety of signaling molecules, providing the metabolic support for neuronal function, and involved in memory and information processing. The LHRH neuronal system needs to "navigate" from the epithelium of the medial olfactory pit to the developing basal forebrain. The failure of glial-guided migration is responsible for the suppression of the pituitary-gonadal axis in Kallman's syndrome. Recent findings clearly indicate that besides its well known trophic role, astroglia may play a crucial regulatory function that may vary according to: 1. the specific physiological conditions (i.e.: stage of glia maturation and differentiation), 2. the specific brain region examined, 3. the degree of neuronal differentiation; and 4. the hormonal background. Such control may be exerted through the release of products able to alter LHRH neuronal morphology, the LHRH intracellular secretory machinery and/or proliferation. As a corollary, astroglial cells can respond to GT1-1 neuronal signals, and this mutual trophic and functional interaction is likely to occur via paracrine, and/or autocrine mechanism(s). Our preliminary observations would support the contention that glial-derived, peptide growth factors are involved in LHRH-astroglia crosstalk. On the other hand, other findings support the viewpoint that the hypothalamic decapeptide, LHRH, may act as a growth factor for astroglia. It would, then, appear that astroglia may produce different factors endowed with neurotrophic/differentiating properties, the nature and/or the concentration of which may vary according to the CNS region and the degree of astroglial differentiation. More importantly, glial-derived factors exert different effects according to the degree of GT1-1 neuron differentiation. The study on the role of cell-cell contacts and adhesive mechanisms between LHRH neurons and astroglia highlights the crucial importance of neuron/neuron, neuron/glia juxtaposition for the correct development of LHRH neuronal function in vitro. The dramatic effects on both morphology and secretory capacity of the LHRH neuron clearly indicate that the neural cell adhesion molecule is involved in the dynamic inter-signaling between LHRH neurons, as well as in neurons and astroglia. In view of the signal transduction capabilities of the extracellular matrix (260), a crosstalk between different signal pathways can provide a fine orchestration of cellular processes including growth, differentiation and secretory activity.

The fact that astroglial conditioned medium is able to stimulate the leukocyte proliferation is in line with the view of mutual regulatory mechanisms and shared molecules among neural, glial, endocrine and immune cells. This view is further supported during manipulation of astroglial-derived cytokines and NO induced lipopolysaccharide in LHRH-astroglial mixed cultures, underscoring a potential crosstalk between different mediators in the dynamic control of LHRH release.

LHRH immunoreactive neurons are seen to emerge from the olfactory placode by day 11.5, at 12 and 13 days of gestation cords of LHRH-immunoreactive cells are seen on the nasal septum, and by day 14 LHRH neurons enter the forebrain. Some data also indicate that glial cells arise in the olfactory epithelium and migrate into the olfactory nerve (261, 262). In the X-linked form of Kallmann's disease, the affected gene has recently been found to encode a protein that contains motifs common to several adhesion molecules (263, 264). Basic fibroblast growth factor stimulates both multipotential precursors, which give rise to neurons and astrocytes, and a committed glial precursor. Recently, FGF receptors have been implicated in cell-cell signaling and in cell migration (265-270). Growth factors in collaboration with molecule of the cell surface matrix may then cooperate during embryonic development for the migration and differentiation of the LHRH neuronal system, while in the adult brain they could participate in the neuroendocrine regulation of LHRH secretion. Detailed studies on different aspects of LHRH neuron-glia interactions seem warranted not only because of the clinical and genetic implications for patients with Kallmann's disorder, but might be of importance for the understanding of other migrating disorders.

18. REFERENCES

1. R. Virchow: Ueber das granulierte Ansehen der Wandungen der Gehirnventrikel. Allgem Z Psychiatrrie, Psych Med 3, 242-250 (1846)

2. R. Virchow: Ueber eine im Geehirn und Ruckenmark des Menshen der Cellulose. Arch Pathol Anat Physiolk Klin Med 6, 135-138 (1854)

3. Y. Ramon, S. Cajal: Sobre un nuevo proceder de impregnacion de la neuroglia y sus resultados en los centros nerviosos del hombre y animales. Trab Lab Invest Biol Univ Madrid 11, 219-237 (1913)

4. H. Van der Lons: The history of the neuron and neuronal connectivity. In: The Centennial of the Neuron. Eds: Costa E, Judd LL, Leshner AL, Abstracts, Fidia Res. Foundation, Washington, DC 3, (1991)

5. D.B. Tower: A century of neuronal-glial interactions, and their pathological implications: an overview. In: Neuronal-Astrocytic Interactions. Implications for Normal and Pathological CNS Function. Eds: Yu ACH, Hertz MD, Noremberg E, Sykiova E, Waxman SG. Progr Brain Res, vol 94, 3-18, Elsevier Science Publishers, Amsterdam (1992)

6. R. Galambos: A glial-neural theory of brain function. Proc Natl Acad Sci USA 47, 129-136 (1961)

7. S.V. Kuffler & J.C. Nichols: The physiology of neuroglial cells. Ergeb Physiol 57, 1-90 (1966)

8. L. Hertz & A. Schousboe: Role of astrocytes in compartmentalization of aminoacid and energy metabolism . In: Astrocytes. Vol 2: Biochemistry, Physiology and Pharmacology of Astrocytes. Eds: Federoff S, and Vernadakis A. Academic Press, Orlando, 179-208 (1986)

9. A. Vernadakis: Neuron-glia interrelations. Int Rev Neurobiol 30, 149-224 (1988)

10. A.B. Barres: A new form of transmission? Nature 339, 343-344 (1989)

11. J. Kimelberg, O'Connor: Swelling of astrocytes causes membrane potential depolarization. Glia 1, 219-224 (1988)

12. A.C.H. Yu, L. Hertz, M.D. Noremberg, E. Sykova & S. Waxman: Neuronal-astrocyte Interactions: Implication for Normal and Pathological CNS Function. Progr Brain Res, vol 94, 1992 Elsevier Science Publishers, (1992).

13. S.G. Smith: Do astrocytes process neural information ?. In: Neuronal-Astrocytic Interactions. Implications for Normal and Pathological CNS Function. Eds: Yu ACH, Hertz MD, Noremberg E, Sykiova E, Waxman SG. Progr Brain Res, vol 94, Elsevier Science Publishers, 119-136 (1992)

14. D. Attwell: Glia and neurons in dialogue. Nature 369, 707-708 (1994)

15. G. Blackenfield, K. Enkvist & H. Kettenmann.: Gamma-aminobutyric acid and glutamate receptors. In: Neuroglia. Eds: Ransom BR & Kettenmann H, Oxford University Press, New York Oxford, 335-345 (1995)

16. M.S. Cooper: Intercellular signaling in neuronal-glial networks. Biosystems 34, 65-85 (1995)

17. B. Ransom & H. Kettenmann: Neuroglia, Oxford University Press, New York (1995)

18. C. Golgi: Di una una nuova reazione apparentemente nera delle cellule nervose cerebrali ottenuta col bicloruro di mercurio. Archivio per Scienze Mediche 11, 1-7 (1879)

19. M. Lenhossek: Der Feinere Bau des Nervensystem im Lichster Neuster Forshungen. Ficher/ Kornfeld, Berlin 179-180 (1895)

20. M.C. Raff, E.R. Abney, J. Cohen, R. Lindsay & M. Noble: Two types of astrocytes in culture of developing rat white matter. Differences in morphology, surface gangliosides, and growth characteristics. J Neurosci 3, 1289-1300 (1983)

21. J. Travis: Glia: The brain's other cells. Science 266, 970-972 (1994)

22. H. Sontheimer & J.M. Richie: Voltage-gated sodium and calcium channels. In: Neuroglia. Eds: Kettenmann H, Ransom B.R. Oxford University Press, New York, Oxford, 246-258 (1995)

23. S. Duffy, D.D. Fraser & B.A. MacVicar: Potassium channels . In: Neuroglia. Eds: Kettenmann H, Ranson BR, Oxford University Press, New York, Oxford, 185-201 (1995)

24. B. Ranson: Glial modulation of neural excitability mediated by extracellular pH: a hypothesis In: Neuronal-Astrocytic Interactions. Implications for Normal and Pathological CNS Function. Eds: Yu ACH, Hertz MD, Noremberg E, Sykiova E, Waxman SG Progr Brain Res, vol 94, 1992 Elsevier Science Publishers, 37-46 (1992)

25. E. Sykiova, J. Svoboda, Z. Simonova & P. Jendelova: Role of astrocytes and volume homeostasis in spinal cord during development and injury. In: Neuronal-Astrocytic Interactions. Implications for Normal and Pathological CNS Function. Eds: Yu ACH, Hertz MD, Noremberg E, Sykiova E, Waxman SG Progr Brain Res, vol 94, Elsevier Science Publishers, 47-56 (1992)

26. H.S. White, G.A. Skee & J.A. Edwards: Pharmacological regulation of astrocytic calcium channels: implications for the treatment of seizure disorders. In: Neuronal-Astrocytic Interactions. Implications for Normal and Pathological CNS Function. Eds: Yu ACH, Hertz MD, Noremberg E, Sykiova E, Waxman SG Progr Brain Res, vol 94, 1992 Elsevier Science Publishers, 77-85 (1992)

27. K.T. Ng, M.E. Gibbs, C.L. Gibbs, G. Sedman, E. Sykova, J. Svobova, P. Jendelova, B. O'Dowd, N. Rickard & S.F. Crowe: Ion involvement in memory information: a potential role of astrocytes. In: Neuronal-Astrocytic Interactions. Implications for Normal and Pathological CNS Function. Eds: Yu ACH, Hertz MD, Noremberg E, Sykiova E, Waxman SG Progr Brain Res, vol 94, 1992 Elsevier Science Publishers, 90-109 (1992)

28. A. Schousboe & N. Westergaard: Transport of neuroactive aminoacids in astrocytes. In: Neuroglia. Eds: Kettenmann H, Ransom BR, Oxford University Press, New York, Oxford, 246-258 (1995)

29. S. Murphy & G. Welk: Arachidonic acid evokes inositol phospholipids hydrolysis in astrocytes. FEBS Lett 257, 68-70 (1989)

30. R.P.D. Kraig, Christopher & A. Caggiano: Glial response to brain ischemia. In: Neuroglia. Eds: Kattenmann H, Ransom BR, Oxford University Press, New York, 964-976 (1995)

31. C.M. Muller: Glial cells and activity-dependent central nervous system plasticity. In: Neuroglia. Eds: Kattenmann H, Ransom BR, Oxford University Press, New York, Oxford, 805-814 (1995)

32. E.N. Benveniste: Cytokine production. In: Neuroglia. Eds: Kattenmann H, Ransom BR, Oxford University Press, New York, Oxford 700-716, (1995)

33. E.N. Benveniste: Cytokines: Influence on glial cell gene expression. In: Neuroimmunoendocrinology, Ed: Blalock JE, Chem Immunol. Basel, Karger, Basel vol 52, 106-153 (1992)

34. D. Giulian: Microglia, cytokines, and cytotoxins: modulators of cellular responses after injury to the central nervous system. J Immunol Immunopharmacol 10, 15-21 (1990)

35. D. Giulian: Microglia and disease of the nervous system. Curr Top Neurol 12, 23-54 (1992)

36. F. Hefti: Growth factors and neuron degeneration. In: Neurodegenerative Diseases. Ed: Calne DB, W.B. Saunders Company, Philadelphia, 177-194 (1994)

37. E.G. McGeer & P.L. McGeer: Neurodegeneration and the immune system. In: Neurodegenerative Diseases. Eds: Calne DB, W.B. Saunders Company Press, Harcour Brace, pp. 277-299 (1994)

38. D. Giulian: Microglia and neuronal dysfunction. In: Neuroglia. Eds: Kattenmann H, Ransom BR, Oxford University Press, New York, Oxford, 671-684 (1995)

39. A.H. Cross, B. Cannella, C.F. Brosnam & C.S. Raine: Hypothesis: Antigen-specific T-cells prime central nervous system endothelium for recruitment of non specific inflammatory cells to affect autoimmune demyelination. J Neuroimmunol 33, 237-244 (1991)

40. H. Wekerle: Antigen presentation by central nervous system glia. In: Neuroglia. Eds: Kettenmann H, Ransom BR, Oxford University Press, New York, Oxford, 685-699 (1995)

41. F. Gallo, M.C. Morale, R. Avola & B. Marchetti: Cross-talk between luteinizing hormone-releasing hormone (LHRH) neurons and astroglial cells : developing glia release factors that accelerate neuronal differentiation and stimulate LHRH release from the GT1 cell line and LHRH neurons stimulate astroglia proliferation. Endocrine J 3, 863-874 (1995)

42. F. Gallo, M.C. Morale, Z. Farinella, R. Avola & B. Marchetti: Growth factors released from astroglial cells in primary culture participate in the crosstalk between luteinizing hormone-releasing hormone (LHRH) neurons and astrocytes: Effects on LHRH neuronal proliferation and secretion. Ann NY Acad Sci 784, 513-516 (1996)

43. B. Marchetti: The LHRH-astroglial network of signals as a model to study neuroimmune interaction: assessment of messenger systems and transduction mechanisms at cellular and molecular levels. Neuroimmunomodulation 3, 1-27 (1996).

44. F. Gallo, R. Avola, A. Beaudet & B. Marchetti: Basic FGF is a major neurotrophic signaling agent during LHRH neuron-astroglia interactions: bFGF priming sensitizes LHRH neurons to growth factor neurotrophic effects. 26th Ann. Meet. Soc. Neurosci., Washington, 16-21 November, vol 2, Abst. 624.2 (1996)

45. M.E. Hatten, M. Lynch, R.E. Rydel, J. Sanchez, S.J. Joseph, D. Moscatelli & D.B. Rifkin: In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor. Dev Biol 125, 280-289 (1988)

46. M.E. Hatten: Riding the glial monorail: a common mechanism for glial-guided neuronal migration in different regions of the developing mammalian brain. Trends Neurosci 13, 179-184 (1990)

47. M.E. Hatten: The role of neuronal migration in central nervous system neuronal development. Curr Opin Neurobiol 3, 38-44 (1993)

48. P. Rakic: Neuron-glia interactions during brain development. Trends Neurosci 4, 184-187 (1981)

49. B. Voutsinos, L. Chouaf, P. Mertens, P. Ruitz-Flandles, Y. Joubert, M.F. Belin & M. Didier-Bazes: Tropism of serotoninergic neurons towards glial targets in the rat ependyma. Neuroscience 59, 663-672

(1994)

50. A. Faissner & M. Schachner: Tenascin and Janusin: glial recognition molecules involved in neural development and regeneration. In: Neuroglia. Eds: Kattenmann H, Ransom BR, Oxford University Press, New York, 411-426 (1995)

51. F. Hefti: Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transection. J Neurosci 6, 2155-2162 (1986)

52. F. Hefti, J. Hartikka & B. Knusel: Function of neurotrophic factors in the adult and aging brain and their possible use in the treatment of neurodegenerative diseases. Neurobiol Aging 10, 515-533 (1989)

53. H.K. Klimelberg, D. Pang & D.H. Trebble: Excitatory aminoacid-stimulated uptake of 22Na+ in primary astrocyte cultures. J Neurosci 9, 1141-1149 (1989)

54. R. Avola, D.F. Condorelli, S. Surrentino, L. Turpeenoja, A. Costa & A.M. Giuffrida Stella: Effect of epidermal growth factor and insulin on DNA, RNA, and cytoskeletal protein labeling in primary rat astroglial cell cultures. J Neurosci Res 19, 230-238 (1988)

55. R. Avola, S. Reale, A. Costa, L. Insirello, V. Spina-Purrello & A.M. Giuffrida-Stella: Effects of bFGF and IGF-I on polyadenylated RNA and non-histone chromosomal protein labeling in cultured astrocytes. J Neurochem 61, 200-210 (1993)

56. D. F. Condorelli, F. Ingrao, G. Magrì, V. Bruno, F. Nicoletti & R. Avola : Activation of Excitatory aminoacids receptors reduces thymidine incorporation and cell proliferation rate in primary cultures of astrocytes. Glia 2, 67-69 (1989)

57. A. Arenander & J. deVellis: Early response gene induction in astrocytes as a mechanism for encoding and integrating neuronal signals. In: Neuronal-Astrocytic Interactions. Implications for Normal and Pathological CNS Function. Eds: Yu ACH, Hertz L, Norenberg MD, Syková E, Waxman S (Eds) Elsevier Science Publishers B.V., Amsterdam, 177-188 (1992)

58. M. Sensenbrenner: The neurotrophic activity of fibroblast growth factors. Progr Neurobiol 41, 683-704 (1993)

59. G. Labourdette & M. Sensenbrenner: Growth factors and their receptors in the central nervous system. in: Neuroglia. Eds: Kettenman H, Ransom BR, Oxford University Press 441-459 (1995)

60. M. Sendtner: Growth factors and their receptors in the central nervous system. In: Neuroglia. Eds: Kattenmann H, Ransom BR, Oxford University Press, New York, Oxford, 427-440, (1995)

61. W.H. Burges & T. Maciag: The heparin-binding (fibroblast) growth factor family of proteins. Ann Rev Biochem 58, 575-606 (1989)

62. A. Baird & P. Bohlen: Fibroblast growth factors. In: Handbook of Experimental Pharmacology, vol 95, Peptide Growth Factors and their Receptors I. Eds: Sporn MB, Roberts AB. Spring-Verlag Berlin, 369-418 (1990)

63. A. Baird & A.M. Gonzalez: Basic fibroblast growth factor (FGF-2) in the pituitary potential activity and potential significance. In : Molecular and Clinical Advances in Pituitary Disorders. Ed: S. Melmed, Endocrine Research and Education, Inc., Los Angeles, 115-119 (1993)

64. R. Elde, Y. Cao & A. Cintra: Prominent expression of fibroblast growth factor in motor and sensory neurons. Neuron 7, 349-364 (1991)

65. J. Kandell, E. Bossy-Wetsel & F. Radvanyi: Neovascularization is associated with a switch in the export of bFGF in the multistep development of fibrosarcoma. Cell 66, 1095-1104 (1991)

66. E. Rouoshalti & Y. Yamaguchi: Proteoglycans as modulators of growth factor activities. Cell 64, 867-869 (1991)

67. A. M. Gonzalez, A. Logan, W. Ying, D. A. Lappi, M. Berry & A. Baird: Fibroblast growth factor in the hypothalamic-pituitary axis: differential expression of fibroblast growth factor-2 and a high affinity receptor. Endocrinology 134, 2289-2297 (1994)

68. W. R. Woodward, R. Nishi & C.K. Meshul: Nuclear and cytoplasmatic localization of basic fibroblast growth factor in astrocytes and CA2 hippocampal neurons. J Neurosci 12, 142-152 (1992)

69. P.A. Walicke & A. Baird: Internalization and processing of basic fibroblast growth factor by neurons and astrocytes. J Neurosci 11, 2249-2258 (1991)

70. G. Ferrari, M.C. Minozzi, G. Toffano, A. Leon & S.D. Skaper: Basic fibroblast growth factor promotes the survival and development of mesencephalic neurons in culture. Dev Biol 133, 140-147 (1989)

71. B. Knussel, P.P. Michel, J. Scwaber & F. Hefti: Selective and non-selective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, insulin and insulin-like growth factors I and II. J Neurosci 10, 558-570 (1990)

72. J. Engele & M.C. Bohn: The neurotrophic effect of fibroblast growth factors on dopaminergic neurons in vitro are mediated by mesencephalic glia . J Neurosci Res 30, 359-371 (1991)

73. J. Engele, D. Schubert & M.C. Bohn: Conditioned media derived from glial cell lines promote survival and differentiation of dopaminergic neurons in vitro: role of mesencephalic glia. J Neurosci Res 30, 359-371 (1991)

74. E. Mayer, S.B. Dunnet & J.W. Fawcett: Mitogenic effect of basic fibroblast growth factor on embryonic ventral mesencephalic dopaminergic neurone precursors. Dev Brain Res 72, 253-258 (1993)

75. M.M. Bouvier & C. Mytilineou: Basic fibroblast growth factor increases division and delays differentiation of dopamine precursors in vitro. J Neurosci 15, 7141-7149 (1995)

76. D. Zhou & M. Di Figlia: Basic fibroblast growth factor enhances the growth of postnatal neostriatal GABAergic neurons in vitro. Exp Neurol 122, 171-188 (1993)

77. P.M. Sweetman, H.R. Sanon & L.A. White: Differential effects of acidic and basic fibroblast growth factors on spinal cord cholinergic, GABAergic and glutamatergic neurons. J Neurosci 57, 237-249 (1991)

78. D. Casper, C. Mytilneou & M. Blum: EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture. J Neurosci Res 30, 372-381 (1991)

79. M. Spranger, D. Lindholm & C. Bandtlow: Regulation of nerve growth factor (NGF) synthesis in the rat central nervous system: comparison between the effects of interleukin 1 and various growth factors in astrocytes culture in vivo. Eur J Neurosci 2, 69-76 (1990)

80. W. Gerdes, W. Barysh, K.H. Schlingensiepen & W. Seifert: Antisense bFGF oligodeoxynucleotides inhibit DNA synthesis of rat astrocytes. Neuroreport 3, 43-46 (1992)

81. C. Gensburger, G. Labourdette & N. Sensenbrenner: Brain basic fibroblast growth factor stimulates the proliferation of rat neuronal precursor cells in vitro. FEBS Lett 217, 1-5 (1987)

82. M.K. Dehamer, J.L. Guevara, K. Hannon, B.B. Olwin & A.L. Calof: Genesis of olfactory receptor neurons in vitro: regulation of progenitor cell divisions by fibroblast growth factors. Neuron 13, 1083-1097 (1994)

83. R.E. Petrowski, J.P. Grierson, S. Choui-Kwon & H.M. Geller: Basic fibroblast growth factor regulates the ability of astrocytes to support hypothalamic neuronal survival in vitro. Dev Biol 147, 1-13 (1991)

84. G. Carpenter & S. Cohen: Epidermal growth factor. J Biol Chem 265, 7709-7712 (1990)

85. P.H. Patterson & H. Nawa: Neuronal differentiation factors/cytokines and synaptic plasticity. Neuron 10, 123-127 (1993)

86. X. Du, N.D. Stull & L. Iacovitti: Brain-derived neurotrophic factor works coordinately with partner molecules to initiate tyrosine hydroxylase expression in striatal neurons. Brain Res 680, 229-233 (1995)

87. P. Nissley & Y. Lopaczynski: Insulin-like growth factor receptor. Growth factors 5, 29-43 (1991)

88. P.H. Daughaday & P. Rotwen: Insulin-like growth factor I and II. Peptide mRNA and gene structures, serum and tissue concentrations. Endocr Rev 10, 68-91 (1989)

89. D.R. Clemmons: Insulin-like growth factor binding proteins. Trends Endocrinol Metab 1, 412-417 (1990)

90. C.A. Conover, J.T. Clarkson & L.K. Bale: Insulin-like growth factor II enhancement of human fibroblast growth via a non-receptor-mediated mechanism. Endocrinology 135, 76-82 (1994)

91. C.A. Bondy: Transient IGF-I gene expression during the maturation of functionally related central projection neurons. J Neurosci 11, 3442-3455 (1991)

92. E. Recio-Pinto, M.M. Rechel & D.N. Ishi: Effects of insulin, insulin-like growth factor and nerve growth factor on neurite formation and survival in cultured sympathetic and sensory neurons. J Neurosci 6, 1211-1219 (1986)

93. V.K.M. Han, A. Smith, W. Myint, K. Nygard & S. Bradshaw: Mitogenic activity of epidermal growth factor on newborn astroglia: Interaction with insulin-like growth factors. Endocrinology 131, 1134-1142 (1992)

94. V.K.M. Han, J.M. Lauder & E.J. D'Ercole: Characterization of somatomedin/insulin-like growth factor receptors and correlation with biological actions in cultured rat astroglial cells. J Neurosci 7, 501-511 (1987)

95. R. Ballotti, F.C. Nielsen, N. Pringle, A. Kowalski, W.D. Richardson, E. Van Oberghen & S. Gammeltoft: Insulin-like growth factor I in cultured rat astrocytes expression of the gene and receptor tyrosine kinase. EMBO J 6, 3633-3639 (1987)

96. C.D. Torran-Allerand, W. Bentham, R.C. Miranda & J.P. Anderson: Insulin influences astroglial morphology and glial fibrillary acid protein (GFAP) expression in organotypic cultures. Brain Res 1, 558-296-304 (1991)

97. L.M. Garcia-Segura, S. Luquin, A. Parduez & F. Naftolin: Gonadal hormone regulation of glial fibrillary acid protein immunoreactivity and glial ultrastructure in the rat neuroendocrine hypothalamus. Glia 10, 59-69 (1994)

98. L.M. Garcia-Segura, J.A. Chowen, M. Duenas, I. Torres-Aleman & F. Naftolin: Gonadal steroids as promoters of neuro-glial plasticity. Psychoneuroendocrinology 19, 445-453 (1994)

99. L.M. Garcia-Segura, I. Torres-Aleman & F. Naftolin: Astrocytic shape and fibrillary acidic protein immunoreactivity are modified by estradiol in primary rat hypothalamic cultures. Dev Brain Res 47, 298-302 (1989)

100. C.D. Torran-Allerand, L. Ellis & K.H. Pfenninger: Estrogen and insulin synergism in neurite growth enhancement in vitro: mediation of steroid effects by interactions with growth factors ? Dev Brain Res 41, 87-100 (1988)

101. G. Olmos, F. Naftolin, J. Perez, P.A. Tranque & L.M. Garcia-Segura: Synaptic remodeling in the rat arcuate nucleus during the estrous cycle. Neuroscience 32, 663-667 (1989)

102. M.C. Langub Jr & R.E. Watson Jr: Estrogen receptor-immunoreactive glia, endothelia, and ependima in guinea pig preoptic area and median eminence: electron microscopy. Endocrinology 130, 364-372 (1992)

103. F. Naftolin, C. Leranth, J. Perez & L.M. Garcia Segura: Estrogen induces synaptic plasticity in adult primate neurons. Neuroendocrinology 57, 935-939 (1993)

104. I. Yung-Testas, Z.Y. Hu, E.E. Beaulieu & P. Robel: Neurosteroids: biosynthesis of pregnenolone and progesterone in primary cultures of rat glial cells. Endocrinology 125, 2083-2091 (1989)

105. I. Yung-Testas, J.M. Renoir, J.M. Gasc & E.E. Beaulieu: Oestrogen-inducible progesterone receptor in primary cultures of rat glial cells. Exp Cell Res 193, 12-19 (1991)

106. I. Yung-Testas, J.M. Renoir, H. Brugnard, G.L. Greene & E.E. Beaulieu: Demonstration of steroid hormones receptor and steroid action in primary cultures of rat glial cells. J Steroid Biochem Mol Biol 41, 3-8 (1992)

107. M. Duenas, S. Luquin, S. Chowen, I. Torres-Aleman, F. Naftolin & L.M. Garcia-Segura: Gonadal hormone regulation of insulin-like growth factor I-like immunoreactivity in hypothalamic astroglia of developing and adult rats. Neuroendocrinology 59, 528-538 (1994)

108. J.K. Hiney, S.R. Ojeda & W. Lees Dees: Insulin-like growth factor I: A possible metabolic signal involved in the regulation of female puberty. Neuroendocrinology 54, 420-423 (1991)

109. J.M.H.M. Reul & E.R. DeKloet: Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117, 2505-2511 (1985)

110. S. Kumar & J. deVellis: Glucocorticoid-mediated functions in glial cells. In: Glial Cell Receptors. Ed: Kimelberg H.K., Raven Press, New York, 243-264 (1988)

111. Y. Chou, W.G. Luttge & G. Sumnersa: Expression of mineralcorticoid type I and glucorticoid type II receptors in astrocyte glia as a function of time in culture. Dev Brain Res 61, 55-61(1991)

112. B. Pearce & G.P. Wilkin: Eicosanoids, purine, and hormone receptors. In: Neuroglia. Eds: H. Kattenmann and B.R. Ransom, Oxford University Press, New York, Oxford, 377-386 (1995)

113. J.P. O'Kallagan, R. Brinton & B.S. McEwen: Glucorticoids regulate the concentration of glial fibrillary acidic protein throughout the brain. Brain Res 494, 159-161

114. M. Aronsson, K. Fuxe, Y. Dong, L. Agnati, L.F. Ockret & J.A. Gustafsson: Localization of glucocorticoid receptor mRNA by in situ hybridization. Proc Natl Acad Sci USA 85, 9331-9335 (1988)

115. B. Marchetti, F. Gallo, M.C. Morale, M. Cioni, Z. Farinella & R. Avola: Glucocorticoid-growth factor interactions during maturation and differentiation of astroglial cell in primary culture. 25th Annual Meeting Society for Neuroscience, San Diego, California, Abs. 128.10, 1, 305 (1995)

116. S. Murphy, G. Bruner & M.L. Simmons: The role of polyphosphoinositides in agonist-evoked release of vasoactive factors from astrocytes. In: Neuronal-Astrocytic Interactions. Eds: Yu ACH, Hertz L, Norenberg MD, SykovŠ E, Waxman S, Elsevier Science Publishers BV, Amsterdam, 153-162 (1992)

117. B. Pearce & G. P. Wilkin: Eicosanoieds, purine and hormone receptors. In : Neuroglia. Eds: Kettenmann H Ransom BR, Oxford University Press, Oxford, 377-386 (1995)

118. S. Murphy, R.L. Minor, G. Welk & D.G. Harrison: CNS astroglial cells release nitrogen oxides with vasorelaxant properties. J Cardiovasc Pharmacol 17, S265-S268 (1991)

119. S. Murphy, M.L. Simmons, L. Agullo, A. Garcia, D.L. Feinstein, E. Galea, D.J. Reis, D. Minc-Colomb & J.P. Schwartz: Synthesis of nitric oxide in CNS glial cells. Trends Neurosci 16, 323-328 (1993)

120. D. Aharoni, A. Dantes & A. Amsterdam: Cross-talk between adenylate cyclase activation and tyrosine phosphorylation leads to modulation of the actin cytoskeleton and to acute progesterone secretion in ovarian granulosa cells. Endocrinology 133, 1426-1436 (1993)

121. R. Asakai, Y. Akita, K. Tamura, N. Kenmotsu & Y. Aoyama: Protein kinase C-dependent down-regulation of basic fibroblast growth factor (FGF-2) receptor by phorbol ester and epidermal growth factor in porcine granulosa cells. Endocrinology 136, 3470-3479 (1995)

122. R.C. Baxter & J.L. Martin: Binding proteins for the insulin-like growth factors: structure, regulation and function. Prog Growth Factor Res 1, 49-68 (1989)

123. L. Kornberg & R.L. Juliano: Signal transduction from the extracellular matrix: the integrin-tyrosine kinase connection. Trends Pharmacol Sci 13, 93-95 (1992)

124. M. Spranger, D. Lindholm & C. Bandtlow: Regulation of nerve growth factor (NGF) synthesis in the rat central nervous system: comparison between the effects of interleukin 1 and various growth factors in astrocytes culture in vivo. Eur J Neurosci 2, 69-76 (1990)

125. P.K. Haugen & P.C. Letourneau: Interleukin 2 enhances chick and rat sympathetic, but not sensory neurite outgrowth. J Neurosci Res 25, 443-452 (1990)

126. M. Kamegai, K. Niikima & T. Kunishta: Interleukin 3 as a trophic factor for central cholinergic neurons in vitro and in vivo. Neuron 2, 429-436 (1990)

127. T. Hama, Y. Kushima & M. Miyamoto: Interleukin 6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from post-natal, two-week-old rats in cultures. Neuroscience 40, 445-452 (1991)

128. M.E. Barish, N.B. Mansdof & S.S. Raissdana: Gamma interferon promotes differentiation of cultured cortical and hippocampal neurons. Dev Biol 144, 412-429 (1991)

129. J.E. Blalock: Neuroimmunoendocrinology. Ed: Blalock JE, Chem Immunol Karger Basel, 52 (1992)

130. J.E. Blalock: Shared ligands and receptors as a molecular mechanism for communication between the immune and neuroendocrine systems. Ann NY Acad Sci 741, 292-298 (1994)

131. W. Pierpaoli & N.H. Spector: Neuroimmunomodulation: Interventions in Aging and Cancer, Ann NY Acad Sci 719, (1994)

132. W. Pierpaoli: The pineal gland as ontogenetic scanner of reproduction, immunity, and aging. The aging clock. Ann NY Acad Sci 741, 46-49

133. B. Marchetti, M.C. Morale, V. Guarcello, N. Cutuli, F. Raiti, N. Batticane, G.Jr. Palumbo, Z. Farinella & U. Scapagnini: Crosstalk communication in the neuroendocrine-reproductive axis: age-dependent alterations in the common communication networks. Ann NY Acad Sci USA 594, 309-325 (1990)

134. B. Marchetti, M.C. Morale, V. Guarcello, N. Cutuli, F. Gallo & U. Scapagnini: The neuroendocrine-immune connections in the control of reproductive functions. In: Major advances in human female reproduction. (E.Y. Adashi and S. Mancuso, Eds). Serono Symposia Publications from Raven Press 73, 279-289 (1990)

135. B. Marchetti, A. Peiffer, M.C. Morale, N. Batticane, F. Gallo, & N. Barden: Transgenic animals with impaired type II glucocorticoid receptor expression: a model to study aging of the neuroendocrine immune system. Ann NY Acad Sci 719, 308-327 (1994)

136. M.C. Morale, N. Batticane, F. Gallo, N. Barden & B. Marchetti: Disruption of hypothalamic-pituitary-adrenocortical system in transgenic mice expressing type glucocorticoid receptor antisense ribonucleic acid permanently impairs T-cell functions: effects on T-cell trafficking and T-cell responsiveness during post-natal maturation. Endocrinology 136, 3949-3960 (1995)

137. B. Marchetti, F. Gallo, Z. Farinella, R. Avola & M.C. Morale: Neuroendocrineimmunology (NEI) at the turn of the century: towards a molecular understanding of basic mechanisms and implications for reproductive physiopathology. Endocrine 3, 845-861 (1995)

138. B. Marchetti, F. Gallo, Z. Farinella & M.C. Morale: Unique neuroendocrine-immune (NEI) interactions during pregnancy. In : The Physiology of Immunity., M. Kendal and J. Marsh, Eds, CRC Press, London, 297-328 (1996)

139. B. Marchetti, F. Gallo, C. Romeo, Z. Farinella & M.C. Morale: The luteinizing hormone-releasing hormone (LHRH) receptors in the neuroendocrine immune network: biochemical bases and implications for reproductive physiopathology. Ann NY Acad Sci 784, 209-236 (1996)

140. S.M. McCann, L. Milenkovic, M.C. Gonzalez, K. Lyson, S. Karnth & V. Rettori: Endocrine aspects of neuroimmunomodulation: Methods and overview In: Neurobiology of Cytokines, Part A, Vol 16, Methods in Neurosciences, E.B. de Souza, Ed, San Diego, Academic Press, 187-210 (1993)

141. S.M. McCann, S. Karanth, A. Kamat, W. LesDees, K. Lyson, M. Gimeno & V. Rettori: Induction by cytokines of the pattern of pituitary hormone secretion in infection. Neuroimmuno-modulation 1, 2-13 (1994)

142. A. Calzolari: Recherches experimentales sur un rapport probable entre la fonction du thymus et celle des testicules. Arch Ital Biol (Tor) 307, 71-76 (1989)

143. H.O. Besedowski & E. Sorkin: Thymus involvement in sexual maturation. Nature 249, 356-359 (1974)

144. W. Pierpaoli & H.O. Besedowski: Interdependence of the thymus in programming neuroendocrine functions. Clin Exp Immunol 20, 323-329 (1975)

145. C.J. Grossman: Regulation of the immune system by sex steroids. Endocr Rev 5, 435, 1984.

146. Farookhi, R., Wesolowski, E., Trasler, J. M. and Robairg, B.: Modulation by neonatal thymectomy of the reproductive axis in male and female rats during development. Biol Reprod 27, 1267 (1988)

147. B. Marchetti, V. Guarcello & U. Scapagnini: Luteinizing hormone-releasing hormone agonist (LHRH-A) binds to lymphocytes and modulates the immune response. In: Biology and Biochemistry of Normal and Cancer Cell Growth, Eds: Castagnetta L, and Nenci I, Harwood Academic Press, London, 149-152 (1988)

148. B. Marchetti: Involvement of the thymus in reproduction. Progr Neuroendocrin Immunol 2, 64-69 (1989)

149. B. Marchetti, V. Guarcello, G. Triolo, M.C. Morale, Z. Farinella & U. Scapagnini: Luteinizing Hormone-Releasing Hormone (LHRH) as Natural Messenger in Neuro-Immune-Endocrine Communications. In: Interactions among CNS, Neuroendocrine and Immune Systems. Eds: Hadden J W, Masek K, and NisticÚ G, Pythagora Press, Rome-Milan, 127-146, (1989)

150. B. Marchetti, V. Guarcello, M.C. Morale, G. Bartoloni, G.jr. Palumbo, F. Raiti, N. Cutuli, Z. Farinella & U. Scapagnini: A physiological role for the neuropeptide luteinizing hormone-releasing hormone (LHRH) during the maturation of thymus gland function. Intern J Neurosci 51, 287-289 (1990)

151. C.J. Grossman: Are there underlying immune-neuroendocrine interactions responsible for immunological sexual dimorphism? Progr Neuroendocrinimmunol 3, 75-80 (1990)

152. M.C Morale, N. Batticane, G. Bartoloni, V. Guarcello, Z. Farinella, M.G. Galasso & B. Marchetti: Blockade of central and peripheral luteinizing hormone-releasing hormone (LHRH) receptors in neonatal rats with a potent LHRH-antagonist inhibits the morphofunctional development of the thymus and maturation of cell-mediated and humoral immune responses. Endocrinology 128, 1073-1085 (1991)

153. C. Rivier: Luteinizing hormone-releasing hormone, gonadotropins, and gonadal steroids in stress. Ann NY Acad Sci 771, 187-191 (1995)

154. S.S. Stojilkovic, J. Reinhart & K.J. Catt: Gonadotropin-releasing hormone receptors: structure and signal transduction pathways, Endocr Rev 15, 462-498 (1994)

155. E. Knobil & J. Hotchkiss: The menstrual cycle and its neuroendocrine control. In: The Physiology of Reproduction. Eds: Knobil E, and Neill J, Raven Press, New York, 1971-1994 (1988)

156. S.P. Kalra: Mandatory neuropeptide-steroid signaling for the preovulatory luteinizing hormone-releasing hormone discharge. Endocr Rev 14, 507-538 (1993)

157. R.I. Weiner, W. Wetsel, P. Goldsmith, G. Martinez De La Escalera, J. Windle, C. Padula, A. Choi, A. Negro-Vilar & P. Mellon: Gonadotropin-releasing hormone neuronal cell lines. Front Neuroendocrinol 13, 95-119 (1992)

158. G. Martinez De La Escalera, A.L.H. Choi & R.I. Weiner: Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT1-1 GnRH neuronal cell line. Proc Natl Acad Sci USA 89, 1852-1855 (1992)

159. L.Z. Krsmanovic, S.S. Stojilkovic, L.M. Mertz, M. Tomic & K.J. Catt: Expression of gonadotropin-releasing hormone receptors and autocrine regulations of neuropeptide release in immortalized hypothalamic neurons. Proc Natl Acad Sci USA 90, 3908-3912 (1993)

160. C. Rivier & W. Vale: In the rat interleukin acts at the level of the brain and the gonads to interfere with gonadotropin and sex steroid secretion. Endocrinology 124, 2105-2109 (1989)

161. S.P. Kalra, A. Sahu & S.P. Kalra: Interleukin-1 inhibits the ovarian steroid-induced luteinizing hormone surge and release of hypothalamic luteinizing hormone-releasing hormone in rats. Endocrinology 126, 2145-2152 (1990)

162. C. Rivier: Role of endotoxins and interleukin-1 in modulating ACTH, LH, and sex steroid secretion. In: Circulating Regulatory Factors and Neuroendocrine Function. Eds: Porter JC, and Jezova D, New York, Plenum, vol 274, 295-301 (1990)

163. M. Yamaguchi, K. Koike, Y. Yoshimoto, N. Matsuzaki, A. Miyake & O. Tanizawa: Interleukin-6 stimulates gonadotropin-releasing hormone secretion from rat hypothalamic cells. Horm Res 35, 252-256 (1991)

164. V. Rettori, M.F. Gimeno, A. Karara, M.C. Gonzalez & S.M. McCann: Interleukin-1a inhibits prostaglandin E2 release to suppress pulsatile release of luteinizing hormone but not follicle-stimulating hormone Proc Natl Acad Sci USA 88, 2763-65 (1991)

165. V. Rettori, N. Belova, A. Kamat, K. Lyson & S.M. McCann: Blockade by interleukin-1 a of the nitricoxidergic control of luteinizing hormone-releasing hormone in vivo and in vitro. Neuroimmunomodulation 1, 86-91 (1994)

166. S. Rivest, S. Lee, B. Attardi & C. Rivier: The chronic intracerebroventricular infusion of interleukin-1ß alters the activity of the hypothalamic-pituitary-gonadal axis of cycling rats. Effect on LHRH and gonadotropin biosynthesis and secretion, Endocrinology 133, 2424-28 (1993)

167. K.M. Ataya, W. Sakr, C.M. Blacker, M.A. Mutchnick & Z.A. Latif: Effect of GnRH agonists on the thymus in female rats. Acta Endocrinol 121, 833-838 (1989)

168. B.D. Greenstein, F.T.A. Fitzpatrick, M.D. Kendall & M.J. Wheeler: Regeneration of the thymus in old male rats treated with a stable analogue of LHRH. J Endocrinol 112, 345-348 (1987)

169. B. Marchetti, V. Guarcello, M.C. Morale, G. Bartoloni, Z. Farinella, S. Cordaro & U. Scapagnini: Luteinizing Hormone-Releasing Hormone (LHRH) binding sites in the rat thymus: Characteristic and biological function. Endocrinology 125, 1025-1036 (1989)

170. B. Marchetti, V. Guarcello, M.C. Morale, G. Bartoloni, F. Raiti, G.Jr. Palumbo, Z. Farinella, S. Cordaro & U. Scapagnini: Luteinizing Hormone-Releasing Hormone (LHRH) agonist restoration of age associated decline of thymus weight, thymic LHRH receptors, and Thymocyte proliferative capacity, Endocrinology 125, 1037-1045 (1989)

171. N.V. Emanuele, M.A. Emanuele, J. Tentler, L. Kirsteins, N. Azad & A.M. Lawrence: Rat spleen lymphocytes contain an immunoactive and bioactive luteinizing hormone releasing hormone. Endocrinology 126, 2482-2486 (1990)

172. N. Azad, N.V. Emanuele, M.M. Halloran, J. Tentler & M.R. Kelley: Presence of luteinizing hormone-releasing hormone (LHRH) mRNA in rat spleen lymphocytes. Endocrinology 128, 1679-1681 (1991)

173. N. Batticane, M.C. Morale, F. Gallo, Z. Farinella & B. Marchetti: Luteinizing Hormone-Releasing Hormone Signaling at the Lymphocyte Involves Stimulation of Interleukin-2 Receptor Expression, Endocrinology 128, 277-286 (1991)

174. J.D. Glass: A sequence related to the human gonadoliberin precursor near the N-termini of HIV and SIV gag polyproteins. J Theor Bio 150, 489-496 (1991)

175. C.C. Maier, B. Marchetti, R.D. LeBoeuf & J.E. Blalock: Thymocytes express a mRNA that is identical to hypothalamic Luteinizing Hormone-Releasing Hormone mRNA. Cell Mol Neurobiol 12, 447-450 (1992)

176. L.V. Rao, R.P. Cleveland & K.M. Ataya: Sequential changes in functional lymphocyte subpopulations during long term administration of GnRH agonist in postpubertal female mice. Endocrine J 1, 451-459 (1993)

177. N. Azad, N. La Paglia, K. Abel, J. Jurgens, L. Kirsteins, N.V. Emanuele, M. Kelley, A.M. Lawrence & N. Mohagheghpour: Immunoactivation enhances the concentration of luteinizing hormone-releasing hormone peptide and its gene expression in human peripheral T-lymphocytes. Endocrinology 133, 215-223 (1993)

178. F. Standaert, B.P. Chew, D. De Avila & J.J. Reeves: Presence of luteinizing hormone-releasing hormone binding sites in cultured porcine lymphocytes. Biol Reprod 46, 997-1000 (1992)

179. J.D. Jacobson, B.C. Nisula & A.D. Steinberg: Modulation of the expression of murine lupus by gonadotropin-releasing hormone analogs. Endocrinology 134, 2516-2523 (1994)

180. T.M. Wilson, L. Yu-Lee & M.R. Kelly: Coordinate gene expression of luteinizing hormone-releasing hormone (LHRH) and the LHRH-receptor following prolactin stimulation in the rat Nb2 T cell line: Implications for a role in immunomodulation and cell-cycle gene expression. Mol Endocrinol (1995)

181. T. Takao & E. De Souza In: Receptors: Model Systems and Specific Receptors. P.M. Conn (Ed), Methods Neurosci Vol 11, Academic Press, Inc. 29-42.

182. B. L. Spangelo, A.M. Judd, R.M. MacLeod, D.W. Goodman, & P.C. Isaakson: Endotoxin-induced release of interleukin-6 from rat medial basal hypothalami. Endocrinology 127, 1779-1785 (1990)

183. R.C. Silverman, M.J. Gibson & A.J. Silverman: Relationship of glia to GnRH axonal outgrowth from third ventricular grafts in hpg hosts. Exp Neurol 1991, 114, 259-274.

184. K. Sundaram, A. Didolkar, R. Thau, M. Chandhuri & F. Schmidt: Antagonists of luteinizing hormone releasing hormone bind to rat mast cells and induce histamine release. Agents Actions 25, 307-310, (1988)

185. A. Phillips, D.W. Hahn, J.L. McGuire, D. Ritchie, R.J. Capetola, C. Bowers & K. Folkers: Evaluation of the anaphylactoid activity of a new LHRH antagonist. Life Sci 43, 883-886 (1988)

186. S.R. Ojeda, H.F. Urbanski, M.E. Costa, D.F. Hill & M. Moholt-Siebert: Involvement of transforming growth factor a in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus. Proc Natl Acad Sci USA 87, 9698-9702 (1990)

187. Y.J. Ma, M.P. Junier, M.E. Costa & S.R. Ojeda: Transforming growth factor a gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation. Neuron 9, 657-670 (1992)

188. S.R. Ojeda, G.A. Dissen & M.P. Junier: Neurotrophic factors and female sexual development. Front Neuroendocrinology 13, 120-162 (1993)

189. M.P. Junier, F.D. Hill, M.E. Costa, S. Felder & S.R. Ojeda: Hypothalamic lesions that induce female precocious puberty activate glial expression of epidermal growth factor receptor gene: Differential regulation of alternative spliced transcripts. J Neurosci 13, 703-71311 (1993)

190. S.R. Ojeda & H.F. Urbanski: Puberty in the rat. In: The Physiology of Reproduction, 2nd Edition. Eds: Knobil E, Neill JD, Raven Press, New York, 363-409 (1994)

191. A. Negro-Vilar, D. Conte & M. Valenca: Transmembrane signals mediating neural peptide secretion: role of protein kinase C activators and arachidonic acid metabolites in luteinizing hormone-releasing hormone. Endocrinology 119, 2796-2802 (1986)

192. S.R. Ojeda, H.F. Urbanski, K.H. Katz, M.E. Costa & P.M. Conn: Activation of two different but complementary biochemical pathways stimulates release of hypothalamic luteinizing hormone-releasing hormone. Proc Natl Acad Sci USA 83, 4932-4936 (1986)

193. W.C. Wetsel & A. Negro-Vilar: Testosterone selectively influences protein kinase-C-coupled secretion of proluteinizing hormone-releasing hormone-derived peptides. Endocrinology 125, 538-547 (1989)

194. J.M. Bruder, W.D. Krebs, T.M. Nett & M.E. Wierman: Phorbol ester activation of the protein kinase C pathway inhibits gonadotropin-releasing hormone gene expression. Endocrinology 131, 2552-2558 (1992)

195. W.C. Wetsel, S.A. Eraly, D.B. Whytw & P.L. Mellon: Regulation of Gonadotropin-Releasing Hormone by protein kinase-A and -C in immortalized hypothalamic neurons. Endocrinology 132, 2360-2370 (1994)

196. K.L. Yu, T.T.S. Yeo, K.W. Dong, M. Jakubowski, C. Lackner-Arkin, M. Blum & J.L. Roberts: Second messenger regulation of mouse gonadotropin-hormone releasing hormone gene expression in immortalized mouse hypothalamic GT1-3 cells. Mol Cell Endocrinol 102, 85-92 (1994)

197. A.C. Gore, A. Ho & J.L. Roberts: Translational efficiency of gonadotropin releasing hormone messenger ribonucleic acid is negatively regulated by phorbol esters in GT1-7 cells. Endocrinology 136, 1620-1625 (1995)

198. M. Moretto, F.J. López & A. Negro-Vilar: Nitric oxide regulates Luteinizing Hormone-Releasing Hormone secretion. Endocrinology 133, 2399-2402 (1993)

199. J.J. Bonavera, A. Sahu, P.S. Kalra & S.P. Kalra: Evidence that nitric oxide may mediate the ovarian steroid-induced luteinizing hormone surge: involvement of excitatory amino acids. Endocrinology 133, 2481-2487 (1993)

200. S.R. Viscent & H. Kimura: Histochemical mapping of nitric oxide synthase in rat brain. Neuroscience 46, 755-784 (1992)

201. S.H. Snyder: Nitric oxide: first in a new class of neurotransmitters? Science 257, 494-496 (1992)

202. M. Schwanzel-Fukuda & D.W. Pfaff: Origin of luteinizing hormone-releasing neurons, Nature 338, 161-164 (1987)

203. M. Schwanzel-Fukuda & D.W. Pfaff: The migration of luteinizing hormone-releasing hormone (LHRH) neurons from the medial olfactory placode into the medial basal forebrain. Experientia 46, 956-962 (1990)

204. M. Schwanzel-Fukuda, S. Abraham, K.L. Crossin, G.M. Edelman & D.W. Pfaff: Immunocytochemical demonstration of neural cell adhesion molecule (NCAM) along the migration route of luteinizing hormone-releasing hormone (LHRH) neurons in mice. J Comp Neurology 321, 1-18 (1992)

205. M. Schwanzel-Fukuda, K.L. Jorgenson, H.T. Bergen, G.D. Weesner & D.W. Pfaff: Biology of normal luteinizing hormone-releasing hormone neurons during and after their migration from olfactory placode. Endocrine Rev 13, 623-634 (1992)

206. H. Kaikoku-Ischido, Y. Okamura, N. Yanahihara & S. Daikoku: Development of luteinizing hormone-releasing hormone-containing neuron system in the rat: In vivo and in transplantation studies. Dev Biol 140, 374-387 (1990)

207. R.B. Norgren & M.N. Lehman: Neurons that migrate from the olfactory epithelium in the chick express luteinizing hormone-releasing hormone. Endocrinology 128, 1676-1678 (1991)

208. R.B. Norgren, C. Gao, Y. Ji & B. Fritzsch: Tangential migration of luteinizing hormone-releasing hormone (LHRH) neurons in the medial telencephalon in association with transient axons extending from the olfactory nerve. Neurosci Lett 202, 1-4 (1995)

209. F.J. Kallmann, W.A. Schoenfeld & S.E. Barrera: The genetic aspects of primary eunuchoidism. Am J Ment Defic 48, 203-236 (1994)

210. W.F. Crawley & J.L. Jameson: Clinical counterpoint: gonadotropin-releasing hormone deficiency : Perspectives from clinical investigations. Endocr Rev 13, 635-640 (1992)

211. R.B. Norgren & R. Brackenbury: Cell adhesion molecules and the migration of LHRH neurons during development. Dev Biol 160, 377-387 (1993)

212. O.K. Ronnekleiv & J.A. Resko: Ontogeny of gonadotropin releasing-hormone -containing neurons in early fetal development of Rhesus macaques. Endocrinology 126, 498-511 (1990)

213. S. Wray, A. Nieburgs & S. Elkabes: Spatiotemporal cell expression of luteinizing hormone releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode. Dev Brain Res 46, 309-318 (1989)

214. S. Murakami, T. Sekim, K. Wakabayashi & Y. Arai: The ontogeny of luteinizing hormone-releasing hormone (LHRH) producing neurons in the chick embryo : possible evidence for migrating LHRH neurons from the olfactory epithelium expressing a highly polysialylated neural cell adhesion. Mol Neurosci Res 12, 421-431 (1991)

215. S. Daikoku, I. Koide, M Chikamori-Aoyama & Y. Shimomura: Migration of LHRH neurons derived from the olfactory placode in rats. Arch Histol Cytol 56, 353-370 (1993)

216. S.A. Tobett, J.E. Crandall & G.A. Schwarting: Relationship of migrating luteinzing hormone-releasing hormone neurons to unique olfactory system glycoconiugates in embryonic rats. Dev Biol 155, 417-482 (1992)

217. K. Yoshida, S.A. Tobet, J.E. Crandall, T.P. Jimenez & G.A. Schwarting: The migration of luteinizing hormone-releasing hormone neurons in the developing rat is associated with a transient, caudal projection of the vomeronasal nerve. J Neurosci 15, 7769-7777 (1995)

218. A.J. Silverman: The Gonadotropin-releasing hormone (GnRH) neuronal systems: immuno-cytochemistry. In: The Physiology of Reproduction. Eds: Knobil E and Neill JD, Raven Press, New York, 1283-1304 (1988)

219. I. Basco, P.L. Woodhams, F. Haljos & R. Balasz: Immunocytochemical demonstration of glial fibrillary acidic protein in mouse tanycytes. Anat Embryol 162, 217-222 (1981)

220. E. Horstmann: Die faserglial des selachiergehirns. Z Zellforsh 39, 588-617 (1954)

221. O.E. Millhouse: Light and electron microscopic studies of the ventricular wall. Z Zellfosch 127, 149-174 (1972)

222. G.P. Kozlowski & P.W. Coates: Ependymo-neuronal specializations between LHRH fibers and cells of the cerebroventricular system. Cell Tiss Res 242, 301-311 (1985)

223. R.C. Silverman, M.J. Gibson & A.J. Silverman: Relationship of glia to GnRH axonal outgrowth from third ventricular grafts in hpg hosts. Exp Neurol 114, 259-274. (1991)

224. G.I. Hatton, L.S. Perlmutter, A.K. Salm & C.D. Tweedle: Dynamic neuronal-glial interactions in hypothalamus and pituitary: implications for control of hormone synthesis and release. Peptides 5, 121-138 (1984)

225. J.C. King & R.J. Letourneau: Luteinizing hormone-releasing hormone terminals in the median eminence of rats undergo dramatic changes after gonadectomy, as revealed by electron microscopic image analysis. Endocrinology 134, 1340-1351 (1994)

226. S.G. Kohama, J.R. Goss, T.H. McNeill & C.E. Finch: Glial fibrillary protein increases at proestrus in the arcutae nucleus of mice. Neurosci Lett 183, 164-166 (1995)

227. P. Collado, C. Beyer, J.B. Utchison & S.D. Holman: Hypothalamic distribution of astrocytes is gender-related in Mongolian gerbils. Neurosci Lett 184, 86-89 (1995)

228. S.G. Kohama, J.R. Goss, C.E. Finch & T.H. McNeill: Increases of glial fibrillary acidic protein in the aging female mouse brain. Neurobiol Aging 16, 59-67 (1995)

229. N.R. Nichols, C.E. Finch & J.F. Nelson: Food restriction delays the age-related increase in GFAP mRNA in rat hypothalamus. Neurobiol Aging 16, 105-10 (1995)

230. J.K. McQueen & H. Wilson: The development of astrocytes immunoreactive for glial fibrillary acidic protein in the medio-basal hypothalamus of hypogonadal mice. Mol Cell Neurosci 5, 623-631 (1994)

231. P. Mellon, J. Windle, P. Goldsmith, C. Padula, J.L. Roberts & R.I. Weiner: Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5, 1-10 (1990)

232. G. Martinez De La Escalera, F. Gallo, A.L.H. Choi & R.I. Weiner: Dopaminergic regulation of the GT1 gonadotropin releasing hormone (GnRH) neuronal cell lines: stimulation of GnRH release via D1-receptors positively coupled to adenylate cyclase. Endocrinology 131, 2965-2971 (1992)

233. G. Martinez De La Escalera, A.L.H. Choi & R.I. Weiner: ß1-Adrenergic regulation of the GT1 GnRH neuronal cell lines: stimulation of the GnRH release via receptors positively coupled to adenylate cyclase. Endocrinology 131, 1397-1402 (1992)

234. Z. Liposits, I. Merchenthaler, W.C. Wetsel, J.J. Reid, P.L. Mellon, R.I. Weiner & A. Negro-Vilar: Morphological characterization of immortalized hypothalamic neurons synthesizing luteinizing hormone-releasing hormone. Endocrinology 129, 1575-1583 (1991)

235. Zhou Jie, D.M. Holtzman, R.I. Weiner & W.C. Mobley: Expression of TrkA confers neuron-like responsiveness to nerve growth factor on an immortalized hypothalamic cell line. Proc Natl Acad Sci USA 91, 3824-3828 (1994)

236. G. Martinez de La Escalera, A.L.H. Choi & R.I. Weiner: Signaling pathways involved in GnRH secretion in GT1 cells. Neuroendocrinology 61, 310-317 (1995)

237. J. Quian, M.S. Bull & P. Levitt: Target derived astroglia regulate axonal outgrowth in a region specific manner. Dev Biol 149, 278-294 (1992)

238. P.D. Le Roux & T.A. Reh: Regional differences in glial-derived factors that promote dendritic outgrowth from mouse cortical neurons in vitro. J Neurosci 14, 4639-4655. (1994)

239. P.D. LeRoux & T.A. Reh: Astroglia demonstrates regional differences in their ability to maintain primary dendritic outgrowth from mouse cortical neurons in vitro. J Neurobiol 27, 97-112 (1995)

240. L.C. Wang, D.H. Baird, M.E. Hatten & C.A. Mason: Astroglial differentiation is required for support of neurite outgrowth. J Neurosci 14, 3195-3207 (1994)

241. T. Minizuma, M. Sawada, A. Suzumura & T. Marunouchi: Expression of cytokines during glial differentiation. Brain Res 656, 141-146 (1994)

242. M. Noble, J. Fok-Seang & J. Cohen: Glia are unique substrate for the in vitro growth of central nervous system neurons. J Neurosci 4, 1892-1903 (1984)

243. M.E. Hatten: Riding the glial monorail: a common mechanism for glial-guided neuronal migration in different regions of the developing mammalian brain. Trends Neurosci 13, 179-184 (1990)

244. M.E. Hatten: The role of neuronal migration in central nervous system neuronal development. Curr Opin Neurobiol 3, 38-44 (1993)

245. P. Doherty, D.E. Moolenaar, S.V. Ashton, R.J. Michalides & F.S. Walsh: Morphoregulatory activities of NCAM and N-cadherin can be accounted by G-protein-dependent activation of L- and N-type neurite Ca2+ channels. Cell 67, 21-33 (1991)

246. J.R. Fallon: Preferential outgrowth of central nervous system neurites on astrocytes and Schwann cells as compared with non glial cells in vitro. J Cell Biol 100, 198-207 (1985)

247. K.J. Tomaselli, K.M. Neugebauer, J.L. Bixby, J. Lilien & L.F. Reichardt: N-Cadherin and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surface. Neuron 1, 33-43 (1988)

248. C.D. Stiles, G.T. Cappone, C.D. Scher, H.N. Antoniades, J.J. Van Wyk & W. J. Pledger : Dual control of cell growth by somatomedins and platelet-derived growth factor. Proc Natl Acad Sci USA 76, 1279-1283 (1979)

249. T.J. Killpatrick & P.F. Bartlett: Cloned multipotential precursors from the mouse cerebrum require FGF-2, whereas glial restricted precursors are stimulated with either FGF-2 or EGF. J Neurosci 15, 3653-3661 (1995)

250. T.J. Killpatrick & P.F. Bartlett: Cloning and growth of multipotential neural precursors : requirements for proliferation and differentiation. Neuron 10, 255-265 (1993)

251. S. Temple & X. Qian: bFGF, neurotrophins, and the control of cortical neurogenesis. Neuron 15, 249-252 (1995)

252. R.B. Campenot: NGF and the local control of nerve terminal growth. J Neurobiol 25, 599-611 (1994)

253. P.S. Tsai, S. Werner & R.I. Weiner: Basic fibroblast growth factor is a neurotropic factor in GT1 gonadotropin-releasing hormone neuronal cell lines. Endocrinology 136, 3831-3838 (1995)

254. R. Westerman, C. Grothe & K. Unsicker: Basic fibroblast growth factor (bFGF), a multifunctional growth factor in the developing and adult brainstem. J Cell Sci Suppl 13, 97-117 (1990)

255. D.M. Araujo & C.W. Cotman: Basic FGF in astroglial, microglial and neuronal cultures: characterization of binding sites and modulation of release by lymphokines and trophic factors. J Neurosci 12, 1668-1678 (1992)

256. P.H. Patterson & H. Nawa: Neuronal differentiation factors /cytokines and synaptic plasticity. Neuron 10, 123-127 (1993)

257. X. Du & L. Iacovitti: Synergy between growth factors and transmitters required for catecholalamine differentiation in brain neurons. J Neurosci 15, 5420-5427 (1995)

258. X. Du, N.D. Stull & L. Iacovitti: Novel expression of the tyrosine hydroxylase gene requires both acidic fibroblast growth factor and an activator. J Neurosci 14, 7688-7694 (1994)

259. N.Y. Ip, T.G. Boulton, Y. Li, J.M. Verdi, S.J. Birren, D.J. Anderson & G.D. Yacopulos: CNTF, FGF and NGF collaborate to drive the terminal differentiation of MAH cells into postmitotic neurons. Neuron 13, 443-455 (1994)

260. L. Kornberg & R.L. Juliano: Signal transduction from the extracellular matrix: the integrin-tyrosine kinase connection. Trends Pharmacol Sci 13, 93-95 (1992)

261. M.I. Chuah & C. Au: Olfactory Schwann cells in olfactory nerve are derived from precursors cells in the olfactory epithelium. J Neurosci Res 29, 172-180 (1991)

262. R.B. Norgren, N. Ratner & Brackenbury: Development of olfactory nerve glia defined by a monoclonal antibody specific for Schwann cells. Develop Syn 194, 231-238 (1992)

263. B. Franco, S. Guioli, A. Pragliola, B. Incerti, R. Tonlorenzi, R. Carrozzo, E. Maestrini, M. Pieretti, P. Taillon-Miller, C.J. Brown, H.F. Willard, C. Lawrence, M.G. Persico, G. Camerino & A. Ballabio: A gene deleted in Kalmann's syndrome shares homology with neural cell adhesion and axonal path-finding molecules. Nature 353, 529-536 (1991)

264. R. Legouis, J.P. Hardelin, J. Levilliers, J.M. Claverie, S. Compain, V. Wunderle, P. Milassean, D. LePaslier, D. Caterina, L. Bougueleret, H. Delemarre-Van de Waal, G. Lutfalla, G. Weissenbach & C. Petit: The candidate gene for the X-linked Kalmann's syndrome encodes a protein related to adhesion molecules. Cell 67, 423-435 (1991)

265. Y. Kinoshita, C. Kinoshita, J.G. Heuer & M. Bothwell: Basic fibroblast growth factor promotes adhesive interactions of neuroepithelial cells from chick neural tube with extracellular matrix proteins in culture. Development 119, 943-956 (1993)

266. Tessier-Lavigne: Axon guidance by diffusible repellents and attractants. Curr Opin Genet Dev 4, 596-601 (1994)

267. F.M. Reichman, B. Dickinson, E. Hafen & B.Z. Shilo: Elucidation of the role of breathless, a Drosophila FGF receptor homolog, in tracheal cell migration. Genes Dev 8, 428-439 (1994)

268. S. MacFarlane, L. McNeill & C.E. Holt: FGF signaling and target recognition in the developing xenopus visual system. Neuron 15, 1017-1028 (1995)

269. E.J. Williams, J. Furness, F.S. Walsh & P. Doherty: Activation of the FGF receptor underlines neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. Neuron 13, 583-594 (1994)

270. D. Zhengshan & H.B. Peng: Presynaptic differentiation induced in cultured neurons by local application of basic fibroblast growth factor. J Neurosci 15, 5466-5475 (1995)