Localization of CiCBR in the invertebrate chordate Ciona intestinalis : Evidence of an ancient role for cannabinoid receptors as axonal regulators of neuronal signalling

CiCBR is a G‐protein‐coupled receptor in the sea‐squirt Ciona intestinalis and the first ortholog of vertebrate CB 1 and CB 2 cannabinoid receptors to be identified in an invertebrate (Elphick et al. [2003] Gene 302:95–101). Here we have used Western blotting and immunocytochemistry to examine expression of CiCBR in adult Ciona , employing novel antibodies to the C‐terminal tail of CiCBR. Consistent with the expected mass for CiCBR, a ∼47‐kDa band was detected in Ciona membranes, and immunocytochemical analysis of serial sections of Ciona revealed intense immunoreactivity in the cerebral ganglion localised in a dense meshwork of fibers in the neuropile. Accordingly, Western blot analysis of neural complex homogenates revealed the presence of a ∼47‐kDa band. CiCBR immunoreactivity was also observed in axons exiting the ganglion in the anterior and posterior nerves, and analysis of whole‐mount preparations revealed that these axons project over the interior surface of the oral and atrial siphons. Isolated CiCBR‐immunoreactive axons not associated with the anterior and posterior nerves were observed projecting through the cortical layer of the cerebral ganglion. Central and peripheral CiCBR‐immunoreactive fibers were studded with intensely stained varicosities, indicative of a role for CiCBR in regulation of axonal release of neurotransmitters, neuromodulators, or neurohormones. Collectively, our data suggest that the well‐established role that the CB 1 receptor has as an axonal regulator of neurotransmitter release in mammals may have originated with ancestral‐type cannabinoid receptors in invertebrate chordates before the emergence of CB 1 ‐ and CB 2 ‐type receptors in vertebrates. J. Comp. Neurol. 502:660–672, 2007. © 2007 Wiley‐Liss, Inc.