[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

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.