These included the demonstration that modulation of kinase and phosphatase activities or cAMP levels has no effect on DSI, while the relatively rapid onset of IPSC suppression makes a phosphorylation-mediated change in channel activity less likely, since that would typically require several seconds. They confirmed the findings of Lenz et al. that  -conotoxin, but not  -aga-toxin is able to block DSI, which means that the G protein-mediated endocannabinoid actions target only the N-type but not the P/Q-type Ca2 channels in the hippocampus . DSI or the selective Ca2 channel inhibitors never block IPSCs completely, which may be due to a partial reduction of release from all terminals, or to the selective expression of CB1 receptors together with the N-type channels only on a particular subset of interneurons. Wilson et al. provided an elegant resolution to this dilemma using paired recordings, which revealed that interneurons producing IPSCs with distinct kinetics express different presynaptic Ca2 channels, and those that show DSI possess only N-type channels . This finding correlates well with the anatomical observations that CCK-containing basket cells selectively express CB1 receptors, whereas another basket cell type lacks CB1 receptors . The differences in IPSC kinetics observed by Wilson et al. may be due to CCK cells forming synapses that are enriched in 2-subunit-containing GABAA receptors,cannabis grow tent whereas parvalbumin-containing basket cells synapse onto GABAA receptors with five times less 2-subunits .

Taken together, these data suggest that CCK-containing basketcell terminals selectively express N-type Ca2 channels together with CB1 receptors predisposing them to DSI, whereas parvalbumin-containing interneurons may express only the P/Q-type Ca2 channels, lack CB1 receptors, and are therefore unaffected by DSI. This conclusion also suggests that the success of DSI induction in any hippocampal slice preparation depends on the relative contribution of the two basket cell types to the examined spontaneous or evoked IPSCs samples. Carbachol is known to enhance DSI, but the mechanism hasnot been revealed to date . One possibility is that carbachol activates the inositol 1,4,5-trisphosphate system via muscarinic receptors, thereby contributing to the large Ca2 transient required for endocannabinoid release . This sounds unlikely as in most experimental paradigms massive depolarizations or uncaging of calcium has been used; thus it would be difficult to further enhance calcium levels by activation of IP3 receptors on intracellular stores. Furthermore, a recent study showed in sympathetic neuronal cultures that muscarinic receptor-mediated activation of PLC- results in limited if any IP3-mediated intracellular Ca2 release ; thus the major signaling pathway there is the production of DAG. However, under physiological conditions in the hippocampus, a cholinergic activation of PLC may well contribute to endocannabinoid release via both the IP3 cascade and the DAG limb . Another likely explanation for the experimental results with carbachol is that it may suppress IPSCs produced by parvalbumincontaining basket cells via presynaptic m2 receptors, which are selectively expressed by this interneuron type , whereas the spontaneous activity of CCK-containing interneurons may be increased via m1 muscarinic, or perhaps even nicotinic actions of carbachol. The mutually exclusive distribution of CB1 and m2 receptors on two subsets of basket cell terminals is shown in Figure 16 . If this reasoning is correct, DSI could be facilitated via other receptors as well that are selectively present on CCK cells but not on parvalbumin cells, e.g., substance P receptors or 5-HT3 receptors.

Indeed, Ha´jos et al. demonstrated that the increase in the amplitude and frequency of spontaneous IPSCs after bath application of substance P fragment was brought back to near control levels by the coapplication of the CB1 receptor agonist WIN55212–2. Although endocannabinoid-mediated DSE has been convincingly demonstrated in the cerebellum, the existence of this phenomenon in the hippocampus could not be established with the same paradigm used for DSI or DSE in the cerebellum . Cannabinoids do reduce glutamatergic EPSCs in the hippocampus , but the receptor involved is unlikely to be CB1 , since the effect was found to be the same in CB1 knock-out and wild-type animals . However, in a recent study, prolonged depolarization was found to readily induce DSE in hippocampal slices, which was absent in CB1 knock-out mice . This is in conflict with the data of Ha´jos et al. and may be due to age or strain differences. Retrograde endocannabinoid signaling was shown to be responsible for another type of synaptic plasticity of glutamatergic transmission in the striatum. Long-term depression of EPSCs induced by high-frequency stimulation of afferent fibers disappeared in CB1 receptor knock-out animals.Interestingly, recent experiments uncovered that activation of postsynaptic type I mGluR receptors induce LTD in the hippocampus by decreasing glutamate release presynaptically . The striking similarity of induction parameters, as well as the potential role of type I mGluRs inendocannabinoid synthesis , suggests that retrograde signaling via postsynaptic release of endocannabinoids is likely to account for this phenomenon. Thus an important question for future research is to determine how DSE and mGluR-dependent LTD are related, along with the identification of how postsynaptic release of endocannabinoids may contribute to these phenomena. The paragraphs above dealt with cannabinoid signaling phenomena that are, or could be, brought about by endogenously released cannabinoids.

Some thought should be given also to those cannabinoid actions that are unlikely to be reproduced by endogenously released cannabinoids but may still be important for the interpretation of the mechanisms of action of delta-9-THC or synthetic ligands. For example, endogenously released cannabinoids are unlikely to act on LTP in the hippocampus, since 1) DSE could be evoked in this region only by prolonged depolarization , 2) cannabinoids had no effect on LTP or LTD when Mg2 -free solution or pairing with strong postsynaptic depolarization was used , and 3) LTP induction under quasi-physiological conditions may be insufficient stimulation for a detectable endocannabinoid release . Single postsynaptic spikes are able to induce LTP if paired with presynaptic spikes or bursts , and excess endocannabinoid release that would be capable of inhibiting glutamate release is unlikely to occur under these conditions. Thus whether endogenously released cannabinoids are able to influence the efficacy or plasticity of glutamatergic transmission in the hippocampus via a direct action on glutamate release is still to be shown. However, Carlson et al. showed that a weak train of stimuli that normally does not induce LTP will induce NMDA-dependent LTP if given during the DSI period. The simultaneously recorded field EPSPs do not undergo LTP,grow lights for cannabis showing that the weak stimulus train was indeed sub-threshold for LTP induction except in dis-inhibited cells. The single-cell LTP was prevented by pretreatment with AM251, suggesting that locally released endocannabinoids can enhance LTP by causing dis-inhibition of a pyramidal cell.As discussed above, several lines of experimental evidence suggest that rather large increases in intracellular [Ca2] are required for the induction of DSI and DSE via the release of endocannabinoids , and this elevation of Ca2 is essential for the synthesis rather than the release of endocannabinoids . Such profound Ca2 transients may occur only under special physiological conditions, e.g., upon the release of Ca2 from IP3- or ryanodine-sensitive intracellular stores via simultaneous activation of metabotropic receptors and voltage-gated Ca2 channels . Back-propagating action potentials are most likely responsible for the voltage-gated Ca2 influx both in the proximal dendritic and distal dendritic regions , although in small cellular compartments like a spine head, a single NMDA-mediated synaptic event may be sufficient to release Ca2 from the local intracellular stores . In the perisomatic region , type I mGluRs appear to supply IP3 both in pyramidal and Purkinje cells , which may partly explain the apparent involvement of this receptor type in DSI . Indeed, recent papers provide evidence that metabotropic glutamate effects on DSI are mediated by endocannabinoids, as described above. Pairing back-propagating action potentials with mGluR activation increases Ca2 release severalfold compared with spiking alone . The largest amplitude Ca2 transient was observed in the most proximal segment of the apical dendrite, an ideal location for endocannabinergic modulation of GABAergic axon terminals that innervate this region. Electron microscopic studies demonstrate the lack of glutamatergic synapses on the cell bodies and proximal apical shafts of pyramidal cells , which suggests that intracellular Ca2 release in this region has to have a role other than conveying plasticity to glutamatergic synapses.

One possibility is that this Ca2 rise is sufficiently close to the nucleus to trigger transcriptional changes. Alternatively, it may be critically involved in the induction of endocannabinoid release, which results in the down regulation of perisomatic inhibition. Thereby action potentials could better back-propagate into the distal dendrites allowing associative LTP of distal glutamatergic synapses, or would enable the neuron to dissociate itself from the population oscillation maintained by basket cell-mediated inhibition . One problem with this hypothesis, and with the interpretation of the mGluR studies , is the source of glutamate required to activate mGluRs in the somatic/proximal dendritic region, since these parts of pyramidal cells do not receive glutamatergic synapses . Thus, if mGluRs get activated at all in this region under physiological conditions, it either has to involve extrasynaptic mGluRs reached by diffusion of glutamate from distant synaptic sites, or mGluRs may be activated further away from the proximal apical dendrite , and IP3 would have to be able to diffuse very fast to its receptors located on the perisomatic or proximal dendritic endoplasmic reticulum. The latter alternative is possible, since IP3 was calculated to be able to diffuse 50 m in 0.5 s, which is faster than Ca2 diffusion in the cytosol containing Ca2 buffers . Diffusion of synaptically released glutamate, however, is unlikely, since it is limited by the efficient glial and neuronal uptake machinery; a spillover even to the adjacentsynapse is limited . An alternative trigger for IP3 synthesis is muscarinic activation. Indeed, Martin and Alger demonstrated that DSI is enhanced by muscarinic m1 or m3 receptor stimulation. Varicose cholinergic fibers are abundant in all layers of the hippocampus and particularly enriched in stratum pyramidale and near the granule cell layer . Furthermore, principal cells are known to express muscarinic receptors on their perisomatic membrane . Activation of muscarinic receptors induces a profound Ca2 rise in the soma, or Ca2 waves that propagate into the soma, and increases the Ca2 transients evoked by trains of action potentials . Thus it is important to emphasize that, in addition to group I mGluRs, cholinergic transmission may also contribute to the generation of sufficient IP3 levels to trigger large Ca2 transients followed by endocannabinoid release when coinciding with trains of action potentials. However, muscarinic receptor-mediated activation of PLC-_x0007_ in sympathetic neuronal cultures results in limited if any IP3-mediated intracellular Ca2 release; thus the major signaling pathway there is the production of DAG , which, on the other hand, is the precursor of 2-AG synthesis . Whether muscarinic activation uses primarily the DAG limb in hippocampal endocannabinoid signaling remains to be established, although the lack of an antagonist effect on DSI suggests that resting levels of acetylcholine are not involved in the generation of the required DAG pool . The same question arises also for the mechanism of mGluR-mediated endocannabinoid release, since in a recent study in hippo campal cultures, group I mGluR activation was shown to enhance DSI without increasing intracellular calcium signals . This raises the possibility that under some conditions, group I mGluR activation uses the alternative root; it may increase 2-AG synthesis via the DAG limb , and thereby could cooperate with depolarization induced Ca2 transients to enhance endocannabinoid release. The physiologically most relevant question here is which are the behavior-dependent activity patterns that could ensure the coincidence of metabotropic receptor activation and bursts of action potentials that are able to induce sufficiently large Ca2 transients to release endocannabinoids in the hippocampus . Spontaneous or low magnesium-evoked burst potentials that resemble physiological bursts were shown to induce DSI . Hippocampal pyramidal cells typically produce bursts of two to six action potentials at 6-ms intraburst intervals . These bursts were shown to invade parts of the dendritic tree quite efficiently, and therefore their pairing with presynaptic activity readily induces LTP .