Endocannabinoid ligands (Di Marzo et al., 1998b; Mechoulam et al., 1998; Hanus et al., 2001) for CBRs modulate other CNS neurotransmitter systems such as GABA, DA, 5-HT, NE, Ach, opiates, and the glutamate receptors. The ability of these CBRs to interact with multiple systems provides limitless signaling capabilities of cross talk within and possibly between receptor families. It has been suggested that anandamide and, to a lesser extent, 2-arachidonyl glycerol, modulate other CNS neurotransmitter systems in drug addiction or reinforcing properties and memory impairment through presynaptic CBR receptor inputs on neurons releasing different neurotransmitters (for a review, see Onaivi et al., 2002; Schlicker and Kathmann, 2001). Evidence of presynaptic localization of CBRs on native neuron in the CNS has been conrmed through retrograde signaling by endocan-nabinoid action at presynaptic CB1Rs to suppress neurotransmitter release in neurons (Kreitzer and Regehr, 2002; Kano et al., 2002; Trettel and Levine 2003.). The CBR is a member of the slow-acting G-protein-coupled receptors, known to modulate the fast-slow-acting ligand-gated ion channel receptors, and it is just becoming evident that this control is important in diseased states from drug abuse. The existence of the EPCS and an endocannabinoid hypothesis of substance are discussed below.
This section provides information on endocannabinoid modulation of AMPA glutamate recep-tors with implication in learning and memory processes in marijuana habituation. Glutamate is the major excitatory neurotransmitter in the CNS, and the majority of fast glutamatergic synaptic transmissions are mediated by AMPA receptors (Jonas and Sakmann, 1992). NMDA, AMPA, and KA are the 3-receptor subtypes that constitute the excitatory glutamate neurotransmitter system in the CNS. Earlier reports of CBR agonist effects on glutamate receptors were focused on inhibition of NMDA neurotransmission in brain neuronal cultures such as the cerebellum (Hampson et al., 1998), basal ganglia (Glass et al., 1997), hippocampus (Terranova et al., 1995), and forebrain (Nadler et al., 1993). Anandamide is reported to have dual effects on the NMDA receptor by a reduction in NMDA mediated intracellular calcium ux in rat brain slices and potentiation of NMDA intracellular calcium ux in rat brain (cortical, cerebellar, hippocampal slices) in the presence of CB1 antagonists (Hampson et al., 1998).
A direct effect of anandamide in the augmentation of NMDA-stimulated currents was recorded in Xenopus oocytes expressing the recombinant receptor in the same study by Hampson et al., 1998. There is a paucity of data on the physiological effects of the action of endocannab-inoids on glutamatergic neurotransmission beyond their inhibition and potentiation of glutamate-induced LTP and LTD in hippocampal (Rouach and Nicoll, 2003), cerebellar (Brown et al., 2003), and striatal neurons (Ronesi et al., 2004) in relation to learning and memory formation (Chevaleyre and Castillo, 2003) and drug abuse (Derkinderen et al., 2003). A signicant role however, is being ascribed to metabotropic glutamate receptors (mGluR) in the presynaptic
inhibition of glutamate receptors by endocannabinoids (Maejima et al., 2001; Varma et al., 2001;
Ohno-Shosaku et al., 2002; Rouach and Nicoll, 2003). It has now been proposed with ample evidence in the hippocampus that in retrograde signaling in the brain, there is a cooperative production of endocannabinoids by metabotropic glutamate (mGlur) receptor activation, leading to the inhibition of inhibitory postsynaptic currents (IPSCs) in neurons (Varma et al., 2001;
Ohno-Shosaku et al., 2002).
Most of the work on endocannabinoid interaction with glutamatergic system is done in brain slices and to a lesser extent in acutely dissociated neurons and neuronal cultures. There are very few reports in the literature on the effect of endocannabinoids on recombinant glutamate receptors despite the advantages of a well-dened in vitro system free of unknown regulatory factors that may be present in brain slices and neuronal cultures. There is currently no literature cited on the
FIGURE 3.4 Effect of endocannabinoids on AMPA glutamate receptor. In Xenopus oocytes expressing the recombinant rat GluR3 subunit of AMPA receptor, anandamide, 2-arachidonyl glycerol, and noladin ether inhibited kainate-activated currents. The order of potency of the endocannabinoids in inhibiting receptor currents in oocytes was anandamide > 2-arachidonyl glycerol > noladin ether.
GluR3
GluR3
GluR3
ANA(100 µM) 2 mins
KA KA KA
20 nA 60 s
KA KA
KA KA KA
KA
50 nA 90 s NE (100 µM) 2 mins
100 nA 30 secs µ
effect of endocannabinoids on kainate receptors. Few reports of endocannabinoid inhibition of recombinant AMPA receptor currents in vitro has been reported in the Xenopus oocyte expression system (Akinshola et al., 1999b; Onaivi et al., 2002). All the homomeric and heteromeric AMPA receptor subunits expressed in oocytes were reported to be functionally inhibited by anandamide (Akinshola et al., 1999b). This result allows for a denitive conclusion that endocannabinoids profoundly modulate AMPA receptor function in vivo and in vitro (Akinshola et al., 1999).
A follow-up question is whether the other endocannabinoids, — 2-AG and noladin ether — have similar effects to those that anandamide has on glutamate receptors in vivo and in vitro.
Neuronal data suggest that 2-AG produces effects to those similar to those of anandamide in the modulation of LTD and retrograde signaling. In Xenopus oocytes expressing the recombinant rat GluR3 subunit of AMPA receptor, anandamide, 2-AG and noladin ether inhibited kainate-activated currents as seen in Figure 3.4. The order of potency of the endocannabinoids in inhibiting receptor currents in oocytes was anandamide > 2-AG > noladin ether. In the gure, 2-AG appears to temporarily reverse the GluR3 inward current in oocytes. The endocannabinoid effects were not blocked by rimonabant and therefore were independent of a CB1 effect. A similar inhibitory effect of 2-AG and the synthetic endocannabinoid methanandamide is shown in oocytes expressing homomeric GluR1 and GluR3 AMPA receptor subunits in Figure 3.5. The effects of the endocannabinoids were compared to arvanil and olvanil (vanilloid agonist) effects in the AMPA receptors and expressed as percentage inhibition of receptor currents in oocytes. Arvanil and 2-AG had similar potencies, just as olvanil and methanandamide — all at concentrations of 100 !M — exhibited similar potencies in inhibiting GluR1 and GluR3 receptor currents in oocytes. Whereas capsazepine, the vanilloid antagonist, also inhibited AMPA receptor currents in oocytes, SR141716A, the CB1 receptor antagonist, has no effect on AMPA receptor currents or endocannabinoid inhibition of receptor currents in vitro (Akinshola et al., 2002). It can be concluded from these in vitro reports that endocannabinoids can directly modulate glutamatergic neurotransmission in vitro via CB1R independent mechanism.
The amino acid sequence alignments (Figure 3.6) and construction of the phylogenetic tree of known CBRs (Figure 3.7A) and tree depicting closely related lyosophosphatidic and vanilloid receptors (Figure 3.7B) indicate some similarities and signicant divergence between CB1 and closely related CB2, LPA, and VR1 receptors. Thus, there is indication of a natural variation in gene expression, which is extensive in human and other organisms.
FIGURE 3.5 Percent inhibition of kainate activated current. The similar inhibitory effects of 2-AG and the synthetic endocannabinoid methanandamide are shown in oocytes expressing homomeric GluR1 and GluR3 AMPA receptor. The effects of the endocannabinoids were compared to arvanil and olvanil (vanilloid agonist) effects in the AMPA receptors and expressed as percentage inhibition of receptor currents in oocytes.
60 50 40 30 20 10 0
Glu R1 Glu R3
Arvanil Olvanil mAna 2AG
158 1-1RNCh 2-1RNCh fosi-1RNCh 1-1RNCm 2-1RNCm 1RNCr 1RNCc 1RNCgt a1RNCrf b1RNCrf 1RNCb 2RNCh 1-2RNCm 2-2RNCm 2RNCr susnesnoC
68071 1-1RNCh 2-1RNCh fosi-1RNCh 1-1RNCm 2-1RNCm 1RNCr 1RNCc 1RNCgt a1RNCrf b1RNCrf 1RNCb 2RNCh 1-2RNCm 2-2RNCm 2RNCr susnesnoC K K f K K K K r n f ~ ~ ~ ~ ~ .
171552 1-1RNCh 2-1RNCh fosi-1RNCh 1-1RNCm 2-1RNCm 1RNCr 1RNCc 1RNCgt a1RNCrf b1RNCrf 1RNCb 2RNCh 1-2RNCm 2-2RNCm 2RNCr susnesnoC S S S S S S S S S S ~ S S S S S
FIGURE 3.6Amino acid sequence alignments of CB1 and CB2 CNRs. hCNR1, mCNR1, and mCNR2 have different entries in the Genbank. The plurality used is 4 and the consensus sequence is shown at the bottom of the alignments. Identical amino acid residues are in black, closely related ones in dark gray, less-closely related ones in light gray, and unrelated in white. The seven putative transmembrane domains™ are indicated at the top of the alignments. The Genbank accession numbers are U73304 (hCNR1, human), AF107262 (hCNR1-2, human), X81121, U22948 (mCNR1-1, mouse), U17985 (mCNR1-2, mouse), X55812 (rCNR1, rat), U94342 (cCNR1, cat), AF181894 (tgCNR1, Taricha granulosa), X94401 (frCNR1a, type a from F. rubripes), X94402 (frCNR1b, type b), U77348 (bCNR1, partial bovine sequence), X74328 (hCNR2), X93168 (mCNR2-1), X86405 (mCNR2-2), and AF176350 (rCNR2).
MARIJUANA USE IN CELL SURVIVAL, NEUROGENESIS,