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Nucleus incertus (NI) is a brainstem structure involved in stress response, arousal, and food intake control. NI is a main source of relaxin-3 in the brain, and relaxin-3 was shown to alter reward and stress related behaviours. Re‑ cently, dopamine receptors were identified in the NI, how‑ ever the effect of their activation on NI neuronal activity was unknown. Therefore, the current study aimed to char‑ acterise the effect of dopamine receptor activation on NI neuronal activity, as well as identify the source of tyrosine hydroxylase (TH)-positive fibres in the structure. Wholecell patch-clamp recordings were used to assess the re‑ sponsiveness of NI neurons to selective D1R and D2R ag‑ onist application. Track-tracing combined with anti-TH immunohistochemical staining was used to define the source of TH immunoreactive fibres in the NI. D1R ago‑ nist SKF-81297 (10 µM) caused depolarization of NI cells by 5.01±0.75 mV (mean change ± SEM). Depolarization persist‑ ed in the presence of tetrodotoxin and glutamate/GABA receptors antagonists which indicates direct postsynaptic action of SKF-81297. Interestingly, activation of D2Rs with quinpirole (20 µM) induced both inhibitory and excitato‑ ry effects on NI neuron activity. In 56% of NI neurons an outward current (13.87±6.55 pA), decrease in action poten‑ tial firing frequency (3.47±1.33 Hz), and hyperpolarization of quiescent cells (2.16±1.01 mV) was observed after quin‑ pirole administration. In 28% of the neurons an increase in inward current amplitude (13.73±2.08 pA) and increase in frequency of action potentials (0.75±0.37 Hz) was recorded after quinpirole application. Both excitatory and inhibitory action of quinpirole persisted in the presence of tetrodo‑ toxin and GABA/glutamate receptors antagonists. Results of track-tracing experiments allowed identification of A11 and A13 cell groups as a source of dopamine innervation in the NI. D1R and D2R are localised postsynaptically on NI neurons. Surprisingly, D2R activation exerted both direct inhibitory and excitatory effects on NI neurons, suggesting a diverse action for dopamine receptor agonists on neuro‑ chemically and/or functionally distinct cell classes in this structure. Identification of A11 and A13 dopamine cells groups as a potential source of TH immunoreactive fibres in the NI allows us to conclude that dopaminergic inner‑ vation of the NI may be involved in the control of alertness and sensorimotor response to salient stimuli.
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