Neuroanatomical Relationship Between Nucleus Accumbens A_2A R Neurons and Orexin Neurons in the Mouse Brain

To explore morphological evidence for neuronal circuits between adenosine A_2A receptor (A_2A R) neurons in the nucleus accumbens (NAc) and orexin neurons. The utilized adeno-associated virus (AAV) encoding humanized Renilla green fluorescent protein (hrGFP) as a tracer to visualize the expression of the A2AR neurons in the NAc and their axon distributions in orexin neurons. The Cre-dependent AAV was injected into the NAc in adenosine A_2A R-Cre mice. Immunohistochemistry was then used to visualize hrGFP and their projections in orexin neurons. The data revealed that NAc A_2A R neurons, mainly expressed in γ-aminobutyric acidergic (GABAergic) projection neurons, projected to the hypothalamus, with apparent contacts of hrGFP containing boutons onto orexin-immunoreactive (IR) soma and dendrites. We also observed strong staining of orexin-IR varicose terminals in the NAc, with apparent contacts of orexin-IR terminals onto A_2A R neurons. Furthermore, in the NAc, orexin-IR varicosities contained vesicular glutamate transporter 2 (VGluT₂) and synaptophysin (Syp). These data provide morphological evidence for exploring the function between NAc A_2A R neurons and orexin neuron in the hypothalamus, particularly in regulating sleep processes.