Dopamine (DA) neurons in the substantia nigra are vital for the control of movement. This fundamental function is encoded by the output signal of DA neurons which switches from spontaneous firing to bursts of action potentials. The latter produces an increase of DA release in targeted areas which is associated to locomotion initiation. The impairment of this process, such as DA neurons degeneration, leads to the motor symptoms of Parkinson’s disease. The activation of the N-methyl-D-aspartate receptors (NMDARs), by the simultaneous binding of glutamate and a co-agonist, is indispensable for the generation of this physiological cue. While both D-serine and glycine can potentially serve as co-agonist, the identity of the main one is correlated to development, synapse specificity, NMDAR subunits and level of synaptic activity. However, in nigral DA neurons the identity of the endogenous co-agonist responsible for the production of bursting activity remains unknown. Identification of the main co-agonist is determined by using glycine and D-serine specific degradation enzymes during whole-cell patch-clamp recordings in brain slices from wild-type Wistar rats (4-7 weeks old). We found that glycine and D-serine affect synaptic and extrasynaptic NMDARs differentially, even though both NMDARs populations are composed of the same subunits (GluN2B-D triheteromers). Interestingly, burst occurrence requires the same co-agonist as the one necessary for extrasynaptic NMDARs. Neither D-serine or glycine removal affected the general pattern of bursts. Furthermore, the specific blockade of synaptic NMDARs doesn't affect generation of burst, indicating that extrasynaptic receptors are responsible for the production of this physiological signal.