Endocannabinoids (eCBs) have emerged as putative retrograde intercellular messengers in the mammalian CNS (Kreitzer and Regehr, 2001; Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001). Two major eCBs have been characterized; both are derivatives of arachidonic acid [arachidonoyl ethanolamide (AEA or anandamide) and 2-arachidonoylglycerol] that act at the G-protein-coupled receptor CB1 (for review, see Freund et al., 2003; Iversen, 2003). Mobilization of eCBs can occur through Ca2+-dependent or relatively Ca2+-independent path-ways (Maejima et al., 2001; Kim et al., 2002), with different down-stream effects. Ca2+-dependent eCB mobilization causes a reversible, short-term depression of synaptic transmission, a process termed depolarization-induced suppression of inhibition (DSI), in which excitation of a hippocampal CA1 pyramidal neuron (Pitler and Alger, 1992) or cerebellar Purkinje cell (Llano et al., 1991) leads to a transient reduction of GABA release from presynaptic terminals of inhibitory interneurons. Hitherto, insights into eCB action have been derived primarily from pharmacological experiments (Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001), because intrinsic properties of eCBs have hampered direct physiological study of eCB signaling at CNS synapses. The hydrophobicity of eCBs severely limits their penetration into brain tissue, and eCBs are rapidly degraded by abundant endogenous lipases.
To circumvent these obstacles, Kao and colleagues designed a highly water-soluble caged AEA that is inert to lipases. When perfused into hippocampal slice preparations, the caged AEA serves as a latent eCB pool, and focal photolysis rapidly liberates highly hydrophobic AEA in situ to activate CB1 receptors. They have used whole-cell voltage-clamp recording and intracellular Ca2+ measurement in combination with photorelease of caged AEA and Ncm-Glu (Cai et al., 2004) to probe the dynamics of eCB signaling in the hippocampal CA1 region (Heinbockel et al., 2005). Photorelease of AEA transiently suppressed spontaneous IPSCs with a time course comparable with that of DSI (Fig. 4), supporting the role of eCB as mediator of the process. These experiments also allowed them to determine the time for synthesis and release of eCB from the postsynaptic neuron, which was estimated to be 75-190 ms at room temperature, comparable with the timescale of metabotropic signaling and at least an order of magnitude faster than previously supposed. These findings suggest that, far from simply serving long-term neuromodulatory functions, eCB signaling is sufficiently fast to exert moment-by-moment control of synaptic transmission.
Whereas neuronal processes in the CNS and their integrative functions have been extensively studied by both optical and electrophysiological methods, most peripheral sensory nerve terminals, being embedded in opaque tissue, are inaccessible to experimental manipulation. A notable exception is the cornea, which has the highest density of peripheral sensory nerve innervation of any tissue and which is entirely transparent and thus ideal for study by optical methods. Corneal nerve terminals reside in the epithelial (outer-most) layer of the cornea (Zander and Weddell, 1951; MacIver and Tanelian, 1993), are almost exclusively nociceptive A and C fibers bearing the vanilloid receptor (TRPV1), and originate from the ophthalmic branch of the trigeminal ganglion (Marfurt, 2000). Kao and colleagues have developed a novel preparation that permits fluorescence imaging of [Ca2+]i in corneal nerve terminals (Gover et al., 2003). They have also developed methods to stimulate individual nerve terminals by photorelease of N-(2-nitroveratryl)-N-vanillyl-nonanoylamide (Nv-VNA), a caged vanilloid. Because of the high density of nerve endings in the 50-µm-thick corneal epithelium (>106 in 0.5 cm2 area), selective stimulation of individual nerve terminals is extremely difficult even with standard uncaging techniques. This is because above and below the focal plane, the defocused UV light beam can still photorelease some vanilloids, which can inadvertently excite nearby nerve terminals. Therefore, they resorted to two-photon photolysis, in which 120 fs pulses of 720 nm (near-infrared) light from a titanium:sapphire laser are focused onto the target nerve terminal. The extremely high light intensity thus achieved within the subfemtoliter focal volume permits Nv-VNA to capture two 720 nm photons "simultaneously." This is equivalent to absorption of a single 360 nm (UV) photon and leads to uncaging. Light intensity outside the focal volume is too low to permit two-photon absorption; therefore, photorelease can be achieved with very high spatial resolution. Selective stimulation of a corneal nerve terminal by two-photon uncaging of Nv-VNA is shown in Figure 5.