Effects of neuronal proteoglycans on activity-dependent growth responses of fetal hippocampal neurons

Excitatory amino-acid (EAA) neurotransmitters act as molecular signals influencing the structure of neurons during development. However, the signal transduction and effector mechanisms responsible for these effects have yet to be fully elucidated. We have previously provided evidence that EAA agonists induce the synthesis and release of proteoglycans (PGs) with neurite-promoting activity from fetal hippocampal neurons. In the present studies exposure of fetal hippocampal neurons to glutamate (100 microM) for 5 min resulted in increases in the neuron-specific growth-associated genes T alpha 1 alpha-tubulin (T alpha 1), microtubule-associated protein-2 (MAP-2) and growth-associated protein-43 (GAP-43). mRNA levels peaked at between 8 and 12 h following exposure as determined by competitive reverse transcription polymerase chain reaction (RT-PCR). Increases in neurite growth as measured by axonal length, the total length of dendrites, the number of branches per axon, the total length of branches per axon and the total neurite length were also observed 48 h after glutamate exposure. The increase in T alpha 1, MAP-2 and GAP-43 mRNA levels following glutamate exposure was mediated via both N-methyl-D-aspartate and metabotropic receptor activation. Heparin, which inhibits the neurite growth-promoting effects of PGs in vitro, and heparitinase, which catalyzes the cleavage of heparan sulphate, also inhibited the glutamate-dependent induction of T alpha 1, MAP-2 and GAP-43 mRNA expression and neurite growth when added to culture medium following glutamate exposure. Chondroitin sulphate and chondroitinase AC had no effects on the mRNA levels tested or on neurite growth. Therefore, these studies suggest that neuronal PGs regulated by activation of EAA receptors mediate neuronal growth responses.