Cultured cerebellar granule cells and cerebellar slices from neonatal rats have been widely used to examine the biochemistry of
excitatory amino acid-induced cell death mediated in part by the activation of
NMDA receptors. However, the
NMDA subunit stoichiometry, producing functional
NMDA receptors is different in cultured granule cells, neonatal and adult rat cerebellum as compared to the
NMDA receptors in forebrain regions. We have used the L-2-chloropropionic
acid (L-CPA) (750 mg/kg) model of
NMDA-mediated selective cerebellar granule cell
necrosis in vivo to examine the role of the
glycine binding site and possible effect of the NR2C subunit (which is largely expressed only in the cerebellum) on granule cell
necrosis. The abilities of various
NMDA receptor antagonists were examined in vivo to determine the relative contribution of both
glutamate and
glycine sites involved in the L-CPA-induced neurotoxicity. The potent neuroprotective, non-competitive
NMDA receptor antagonist
dizocilpine (MK-801) was compared with
glutamate and
glycine site
NMDA antagonists. We have examined a number of markers for the L-CPA-induced granule cell
necrosis. The L-CPA-induced reduction in cerebellar
aspartate and
glutamate concentrations were used as markers of granule cell
necrosis. We also measured the cerebellar water content and
sodium concentrations as measures of the L-CPA-induced cerebellar
edema that accompanies the granule cell
necrosis. Finally the ability of the
NMDA antagonists to attenuate the L-CPA-induced reductions in
body weight gain and the prevention of the loss in hindlimb function using a behavioral measure of hindlimb retraction were examined. The potent
glutamate antagonists,
CPP and CGP40116 and
dizocilpine prevented the L-CPA-induced locomotor dysfunction and granule cell
necrosis as measured by their ability to prevent L-CPA-induced reduction in
aspartate and
glutamate concentrations.
CPP, CGP40116 and
dizocilpine also prevented the appearance of cerebellar
edema following L-CPA administration. In addition,
dizocilpine,
CPP and CGP40116 were able to partially prevent the L-CPA-induced loss in
body weight over the 48 h experimental period. In contrast, none of the
glycine partial agonists or antagonists, namely (+/-)
HA-966, D-
cycloserine, MDL-29,951, DPCQ,
MNQX or L-701 252 were able to prevent the L-CPA-induced loss in
body weight, L-CPA-induced granule cell
necrosis and behavioral disturbances when administered to rats. None of the
NMDA antagonists had any effect on the cerebellar neurochemistry when injected alone or had any effect on animal behavior except for
dizocilpine,
CPP, CGP40116 and (+/-)
HA-966 which resulted in a transient sedation for between three and five hours immediately following their administration. In conclusion, we demonstrated that
NMDA open channel blockade and
glutamate antagonists can provide full neuroprotection against the L-CPA-induced granule cell
necrosis. The failure of the
glycine partial agonist and antagonists to provide any neuroprotection against L-CPA-induced neurotoxicity in the cerebellum contrast with their neuroprotective efficacy in other animal models of
excitatory amino acid-induced cell death in forebrain regions in vivo. We therefore suggest that the
glycine site plays a lesser role in modulating
NMDA receptor function in the cerebellum and may explain why cells expressing
NMDA receptors composed of NR1/NR2C subunits are particularly resistant to
excitatory amino acid-induced neurotoxicity.