BACKGROUND: The gonadotrophin-releasing hormone (GnRH) neuronal network is defined as the GnRH neurons and associated assembly of brain cells responsible for controlling GnRH release into the pituitary portal circulation. Among the known neurotransmitters that alter GnRH neurons, the classical excitatory amino-acid glutamate partly through the activation of NMDA receptor, and the recently discovered family of kisspeptins that bind GPR54 receptor, potently stimulate GnRH release and thus luteinizing hormone (LH) secretion from the anterior pituitary. GnRH neurons express GPR54 and are directly stimulated by kisspeptins whereas glutamate may activate GnRH through NMDA receptors in an indirect manner. HYPOTHESIS: The NMDA-induced GnRH/LH release requires the kisspeptin-GPR54 system. PROTOCOL: To define a functional hierarchy for the action of kisspeptins and NMDA on GnRH neurons, we investigated the effect of peripheral injection of NMDA on plasma LH concentration. We used Gpr54-null or Kiss1-null mice of both sexes, and wild-type littermates at 25 days old. Each animal received a single i.p. injection of 0.3mg NMDA, and blood was collected 10 min after the injection, from the the vena cava post mortem. Hormone assays were performed for LH and growth hormone (GH) plasma levels. RESULTS: All wild-type animals positively responded to NMDA injection and exhibited an LH release after NMDA injection (0.45 ± 0.13 ng/ml or 1.19 ± 0.16 ng/ml in females or males respectively) whereas none of the Gpr54-null or Kiss1-null mice exhibited LH release after NMDA injection (individual values for all mice below 0.33 ng/ml; the sensitivity of the LH assay). However, we found that all animals of both genotypes and sexes positively responded to NMDA with an increase in plasma GH levels. The failure of mice lacking a functional GPR54-kisspeptin system to release LH after NMDA stimulation demonstrates that either GPR54 or kisspeptin is required to relay the NMDA signalling that leads to GnRH neuronal activation. Our data also show that the GPR54-kisspeptin system is specifically involved in the NMDA activation of the GnRH/LH release as NMDA still generates the neuroendocrine activation of GH release. CONCLUSION: NMDA-induced GnRH/LH release is dependant of the GPR54-kisspeptin system. Kisspeptin neurons represent primary afferents to the GnRH neurons as NMDA receptors-activating glutamatergic neurons are likely to represent secondary afferents upstream to kisspeptin neurons. The direct activation of kisspeptin neurons by NMDA is under investigation.