Cannabinoid type 1 receptor-containing axons innervate NPY/AgRP neurons in the mouse arcuate nucleus

Objectives: Phytocannabinoids, such as THC and endocannabinoids, are well known to promote feeding behavior and to control energy metabolism through cannabinoid type 1 receptors (CB₁R). However, the underlying mechanisms are not fully understood. Generally, cannabinoid-conducted retrograde dis-inhibition of hunger-promoting neurons has been suggested to promote food intake, but so far it has not been demonstrated due to technical limitations.

Methods: We applied immunohistochemical labeling of CB₁R for light microscopy and electron microscopy combined with three-dimensional reconstruction from serial sections in CB₁R-expressing and CB₁R-null mice, which served as a negative control. Hunger-promoting neurons expressing Agouti-related protein and neuropeptide Y (AgRP/NPY) in the hypothalamic arcuate nucleus were identified in NPY-GFP and NPY-hrGFP mice.

Results: Using three-dimensional reconstruction from serial sections we demonstrated numerous discontinuous segments of anti-CB₁R labeling in the synaptic boutons and axonal shafts in the arcuate nucleus. We observed CB₁R in the symmetric, presumed GABAergic, synaptic boutons innervating AgRP/NPY neurons. We also detected CB₁R-containing axons producing symmetric and asymmetric synapses onto AgRP/NPY-negative neurons. Furthermore, we identified CB₁R in close apposition to the endocannabinoid (2-arachidonoylglycerol)-synthesizing enzyme diacylglycerol lipase-alpha at AgRP/NPY neurons.

Conclusions: Our immunohistochemical and ultrastructural study demonstrates the morphological substrate for cannabinoid-conducted feeding behavior via retrograde dis-inhibition of hunger-promoting AgRP/NPY neurons.