Cardiac vagal neurons (CVNs) in the nucleus ambiguus (NA) are the major determinant of parasympathetic activity to the heart. Spontaneous GABAergic neurotransmission to CVNs is modulated by hypothalamic neuropeptide orexin-A in postnatal days 2-5 (P5) rats, however during early postnatal development orexin expression changes and the role of orexin-A in modulating CVN activity at other stages of development is unknown. In this study we compared changes in GABAergic inhibitory postsynaptic currents (IPSCs) in CVNs evoked by orexin-A in P5, P16-20 (P20), and P27-30 (P30) rats using an in-vitro brain stem slice preparation. Bath-applied orexin-A enhanced GABAergic IPSCs in all CVNs tested in P5 and P30 animals and in the majority of neurons tested in P20 pups. Focal application of orexin-A ejected from a pipette positioned within 30 μm of the patched CVN did not alter GABAergic signaling in P5 pups. In contrast, in both P20 and P30 rats focal application of orexin-A inhibited GABAergic IPSCs and this inhibition persisted in the presence of tetrodotoxin. These results indicate orexin-A facilitates GABAergic IPSCs likely by activating preceding GABAergic neurons that project to CVNs. Orexin-A also likely acts at GABAergic presynaptic terminals surrounding CVNs within the NA to inhibit GABA release. The latter mechanism is absent in P5 pups but occurs in P20 and P30 rats. In conclusion, this study elucidates an important maturation of the parasympathetic cardiac control system. Alterations in these developmental mechanisms may play a role in pathogenesis of disorders related to a specific stage of development maturation.

The parasympathetic control of heart rate arises from premotor cardiac vagal neurons (CVNs) located in the nucleus ambiguus. Previous microinjection studies in nucleus ambiguus show that dopamine evokes a decrease in heart rate, but the underlying mechanisms responsible for these responses were not identified. This study tested whether dopamine modulates inhibitory GABAergic and glycinergic and/or excitatory glutamatergic neurotransmission to CVNs. Retrogradely labeled CVNs were identified in an in-vitro rat brainstem slice preparation and synaptic events were recorded using whole cell voltage clamp techniques. Bath application of dopamine (100 M) had no effect on excitatory synaptic events, but reversibly inhibited the frequency (but not amplitude) of GABAergic inhibitory postsynaptic currents (IPSCs) in CVNs. Similarly, dopamine (10 M & 100 M) inhibited glycinergic IPSC frequency by ~ 50% and 70% respectively. The reduction in inhibitory neurotransmission to CVNs by dopamine was prevented by the sodium channel blocker TTX (1μM) indicating that the dopamine mediated effects were action potential dependent. Dopamine evoked responses were mimicked by the D2-like receptor agonist, Quinpirole but not D1-like receptor agonist, SKF 38393. In addition, the dopamine mediated depression of inhibitory synaptic responses were prevented by the D2 receptor antagonist sulpiride, but not by D1-like or adrenergic or serotonergic receptor antagonists, suggesting that these responses were D2-like receptor mediated and not D1-like or adrenergic or 5-HT receptor mediated. These data suggest that dopamine acts via disinhibition, and diminishes inhibitory GABAergic and glycinergic neurotransmission to CVNs, which would be predicted to increase parasympathetic activity to the heart and evoke a bradycardia.