Heparan sulfate proteoglycan and laminin immunoreactivity on cultured astrocytes: Relationship to differentiation and neurite growth

Abstract

Extracellular matrix (ECM) produced by Schwann cells is known to promote growth of several types of neurites (Ard et al., 1987). Whether a similar material produced by astrocytes may be available to promote neurite growth during CNS development is now open to question. The present study was undertaken to define conditions under which cultured astrocytes deposit the ECM components laminin and heparan sulfate proteoglycan (HSPG), and to relate this deposition to the ability of astrocytes to support neurite growth. The use of 2 different culture media permitted the growth of astrocytes either with or without these ECM components. Neonatal rat cortical astrocytes were cultured by the method of McCarthy and de Vellis (1980) and studied by immunocytochemistry and electron microscopy. Astrocytes grown in serum-containing medium for 5 or 9 d after subculturing were shown to have fibrillar patches of ECM containing both HSPG and laminin immunoreactivity. Immunoreactivity for the 2 molecules was usually colocalized. In contrast, astrocytes subcultured for 5 d in defined medium showed no immunocytochemical staining for either laminin or HSPG and had no ECM visible in EM. Formation of stellate processes was increased when cells were grown in defined medium compared with that seen in serum-containing medium, and growth of the population was slower. In 3 other conditions, attainment of stellate morphological differentiation by the astrocytes was correlated with diminution in immunostaining for ECM components. (1) In older cultures (30-42 d after subculturing), stellate, mitotically quiescent cells showed relatively little HSPG or laminin immunoreactivity. (2) Cultures initially maintained in serum-containing medium and then converted to defined medium lost much of their immunoreactivity for ECM components and developed longer processes. (3) When neurites from fetal rat dorsal root ganglion explants grew across monolayers of astrocytes in serum-containing medium, diminution of ECM immunostaining and development of stellate processes were seen in areas directly contacted by the neurites. ECM-containing laminin and HSPG did not appear to be necessary for neurites to interact with astrocytes. In defined medium, in which no ECM was detected, dorsal root ganglion neurites were found in contact with astrocyte surfaces rather than on the rat tail collagen substratum on the culture dish. The 3 major findings of this study are that astrocytes secrete an ECM containing both HSPG and laminin, that expression of these molecules appears to be regulated with morphological differentiation and with contact by neurites, but that the capacity of astrocytes to interact with growing neurites is not dependent on their expression of HSPG and laminin.