Neuronal maintenance and neurite extension of adult mouse neurones in non-neuronal cell-reduced cultures is dependent on substratum coating.

Abstract

Adult mouse DRG neurones have been maintained for 14 days in cultures where non-neuronal cell proliferation was inhibited by the inclusion of 5 x 10(-6) microM-cytosine arabinoside (AraC) in the medium from the onset of culture. On uncoated plastic neurone numbers significantly declined in the absence of non-neuronal cell outgrowth compared with uninhibited co-cultures. However, when neurones were maintained in the presence of AraC on certain coated surfaces this decrease in neurone numbers was not observed. Combinations of fibronectin (FN) and laminin (LAM) proved most effective for 7 and 14 days in vitro, although either was beneficial if used separately. Microexudates produced by the fibroblast line, 3T6, also significantly improved neuronal counts for 14 days in vitro. However, a microexudate derived from primary cultures of mouse hepatocytes, although advantageous for 7 days in vitro, was not effective in maintaining neurones over the 14-day culture period, reminiscent of previous observations when synthetic cationic agents were used. Electrophoretic analysis of the fibroblast exudate indicated that fibronectin was present in the substrate-attached material generated by this cell line. The reduction in non-neuronal cell growth facilitated the monitoring of neuronal structural detail by scanning electron microscopy. Examination of neurite extension, indicative of neurone differentiation, was particularly improved. FN/LAM and the fibroblast-derived exudate increased nerve fibre growth, whilst the hepatocyte exudate had little effect on neurite regeneration, and polylysine had a detrimental effect. The data demonstrate that substrata can have a significant effect on maintenance and differentiation of adult neurones in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)