Innervation of adipocytes is limited in mouse perivascular adipose tissue

Perivascular adipose tissue (PVAT) regulates vascular tone by releasing anticontractile factors. These anticontractile factors are driven by processes downstream of adipocyte stimulation by norepinephrine; however, whether norepinephrine originates from neural innervation or other sources is unknown. The goal of this study was to test the hypothesis that neurons innervating PVAT provide the adrenergic drive to stimulate adipocytes in aortic and mesenteric perivascular adipose tissue (aPVAT and mPVAT), and white adipose tissue (WAT). Healthy male and female mice (8-13 wk) were used in all experiments. Expression of genes associated with synaptic transmission were quantified by qPCR and adipocyte activity in response to neurotransmitters and neuron depolarization was assessed in Adipoq^Cre+;GCaMP5g-tdT^f/WT mice. Immunostaining, tissue clearing, and transgenic reporter lines were used to assess anatomical relationships between nerves and adipocytes. Although synaptic transmission component genes are expressed in adipose tissues (aPVAT, mPVAT, and WAT), strong nerve stimulation with electrical field stimulation does not significantly trigger calcium responses in adipocytes. However, norepinephrine consistently elicits strong calcium responses in adipocytes from all adipose tissues studied. Bethanechol induces minimal adipocyte responses. Imaging neural innervation using various techniques reveals that nerve fibers primarily run alongside blood vessels and rarely branch into the adipose tissue. Although nerve fibers are associated with blood vessels in adipose tissue, they demonstrate limited anatomical and functional interactions with adjacent adipocytes, challenging the concept of classical innervation. These findings dispute the significant involvement of neural input in regulating PVAT adipocyte function and emphasize alternative mechanisms governing adrenergic-driven anticontractile functions of PVAT.NEW & NOTEWORTHY This study challenges prevailing views on neural innervation in perivascular adipose tissue (PVAT) and its role in adrenergic-driven anticontractile effects on vasculature. Contrary to existing paradigms, limited anatomical and functional connections were found between PVAT nerve fibers and adipocytes, underscoring the importance of exploring alternative mechanistic pathways. Understanding the mechanisms involved in PVAT's anticontractile effects is critical for developing potential therapeutic interventions against dysregulated vascular tone, hypertension, and cardiovascular disease.