We characterized the developmental and regeneration features of a brain circuitry involved in working memory (the ventral tegmental area-substantia nigra [VTA-SN] fibers projecting to the prefrontal cortex [PFC] and the complementary glutamatergic pathways). By utilizing a co-culture system adapted to multi electrode platforms (MEAs), we simultaneously recorded and analyzed from 60 electrodes, at brief (5–7 div) and long-term (15–25 div) conditions, both spikes (bandwidth 250–5000 Hz) and local field potentials (LFP, bandwidth 1–200 Hz) activity. After few days in-vitro (div) the co-cultures show a spontaneous activity in the form of bursts, trains of action potentials, and LFPs lasting ~0.3 and 1.5 s, respectively. We observed that the activity increased in parallel with the growth of new projections from one slice to the other. We observed three types of activities: bursts only in VTA-SN, while PFC was silent, bursts originating in VTA and rapidly reaching PFC (cross-correlated activity), and bursts with synchronous activity in both regions. This correlated activity is completely abolished if the new-born projections between the two areas are cut. Preliminary data suggest a GABAergic and dopaminergic modulation of activity. The co-cultures of VTA-SN/PFC on MEAs represent a good model to study the regenerative processes and how these processes are influenced by detrimental stimuli or implemented by novel neuro-reparative substances.