Dopaminergic (DA) midbrain neurons within the substantia nigra (SN) display an autonomous pacemaker activity that is crucial for dopamine release and voluntary movement control. Their progressive degeneration is a hallmark of Parkinson's disease. Their metabolically demanding activity-mode affects Ca2+ homeostasis, elevates metabolic stress, and renders SN DA neurons particularly vulnerable to degenerative stressors. Accordingly, their activity is regulated by complex mechanisms, notably by dopamine itself, via inhibitory D2-autoreceptors and the neuroprotective neuronal Ca2+ sensor NCS-1. Analyzing regulation of SN DA neuron activity-pattern is complicated by their high vulnerability. We studied this activity and its control by dopamine, NCS-1, and glucose with extracellular multi-electrode array (MEA) recordings from midbrain slices of juvenile and adult mice. Our tailored MEA- and spike sorting-protocols allowed high throughput and long recording times. According to individual dopamine-responses, we identified two distinct SN cell-types, in similar frequency: dopamine-inhibited and dopamine-excited neurons. Dopamine-excited neurons were either silent in the absence of dopamine, or they displayed pacemaker-activities, similar to that of dopamine-inhibited neurons. Inhibition of pacemaker-activity by dopamine is typical for SN DA neurons, and it can undergo prominent desensitization. We show for adult mice, that the number of SN DA neurons with desensitized dopamine-inhibition was increased (~60–100%) by a knockout of NCS-1, or by prevention of NCS-1 binding to D2-autoreceptors, while time-course and degrees of desensitization were not altered. The number of neurons with desensitized D2-responses was also higher (~65%) at high glucose-levels (25 mM), compared to lower glucose (2.5 mM), while again desensitization-kinetics were unaltered. However, spontaneous firing-rates were significantly higher at high glucose-levels (~20%). Moreover, transient glucose-deprivation (1 mM) induced a fast and fully-reversible pacemaker frequency reduction. To directly address and quantify glucose-sensing properties of SN DA neurons, we continuously monitored their electrical activity, while altering extracellular glucose concentrations stepwise from 0.5 mM up to 25 mM. SN DA neurons were excited by glucose, with EC50 values ranging from 0.35 to 2.3 mM. In conclusion, we identified a novel, common subtype of dopamine-excited SN neurons, and a complex, joint regulation of dopamine-inhibited neurons by dopamine and glucose, within the range of physiological brain glucose-levels.