Cannabinoid Mechanisms of Pain Suppression 525the antibody employed by Tsou and colleagues (Sanudo-Pena et al. 1999) addi-
tionally labeled cell bodies. Tsou and colleagues, using an antibody raised against
the first 77 residues of the N terminus of CB1R, identified beaded immunoreactive
fibers throughout the spinal dorsal horn and in lamina X surrounding the central
canal (Tsou et al. 1998a). Further work by this group also revealed the presence
of lightly stained cells throughout the spinal cord gray matter (Sanudo-Pena et
al. 1999). Farquhar-Smith and colleagues, using an antibody directed against the
C-terminal 13 amino acids of CB1R, demonstrated immunoreactivity for CB1Rs
in fibers and terminals with no consistent immunoreactivity observed in any cell
bodies (Farquhar-Smith et al. 2000).
3.3.3
Evidence for CB1Rs on Spinal Interneurons
There is considerable support for localization of CBRs in rat spinal cord postsy-
naptic to primary afferents at both light and electron microscope levels. Direct
evidence for postsynaptic localization of CB 1 in spinal dorsal horn is derived
from the observation that intrinsic excitatory interneurons in lamina IIi that ex-
pressed protein kinase C isoformγshowed high levels of colocalization with CB 1
(Farquhar-Smith et al. 2000); this pattern may suggest an anatomical basis for
the efficacy of cannabinoids in ameliorating inflammatory and neuropathic pain
(Bridges et al. 2001; Fox et al. 2001; Herzberg et al. 1997; Malmberg et al. 1997; Mao
et al. 2000).
CB1R immunoreactivity has also been localized to dorsal horn interneurons
containingγ-aminobutyric acid (GABA) (Salio et al. 2002b). GABA presynaptically
inhibits primary afferent input to the spinal cord. The observation of GABAergic
dendrites postsynaptic to primary afferents also suggests that primary afferents
are anatomically positioned to activate GABAergic inhibitory circuits. GABAergic
interneurons can also synapse directly on dorsal horn neurons to reduce exci-
tatory input. The demonstrated colocalization of CB1R with GABA is consistent
with functional studies demonstrating a CB1R-mediated presynaptic inhibition of
GABAergic and glycinergic transmission in recordings performed in rat medullary
dorsal horn in vitro (Jennings et al. 2001). By contrast, postsynaptic effects on
medullary substantia gelatinosa neurons were not observed (Jennings et al. 2001).
These data suggest that cannabinoids act through a disinhibitory action on lam-
ina II neurons by inhibiting GABAergic transmission.
Immunoreactivity for CB1R andμ-opioid receptors (MOR) is also colocalized
on lamina II interneurons at the ultrastructural level (Salio et al. 2001). In this work,
CB1R was predominantly localized postsynaptically in dendrites and cell bodies,
but immunoreactive axons and axon terminals were also observed (Salio et al.
2001). Both species showed rare labeling of the plasma membrane. Since MOR1 is
not colocalized with GABA (Gong et al. 1997; Kemp et al. 1996), these data support
the presence of CB1R in distinct populations of intrinsic spinal neurons (Salio et
al. 2001). By contrast, colocalization of CB1R with MOR1 in thin primary afferent
terminals could not be convincingly demonstrated in this work (Salio et al. 2001).