[Frontiers in Bioscience 2, d88-125, March 1, 1997]

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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

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).