DSI can be blocked by postsynaptic Ca2 buffers or initiated by activity restricted to the postsynaptic side, and likely involves the opening of voltage-gated Ca2 channels , or release from intracellular stores. Changes in postsynaptic GABAA receptor sensitivity have been excluded, since the response to iontophoretically applied GABA did not change, and DSI had no effect on the amplitude of miniature IPSCs. Despite the clearly postsynaptic site of initiation, numerous experiments demonstrated that DSI is expressed presynaptically, i.e., as a reduction in GABA release. With the use of minimal stimulation, DSI was found to increase failure rate, multi-quantal components were also eliminated, and components of IPSCs were differentially influenced . In the cerebellum, axonal branch point conduction failure was shown to play a role . Furthermore, DSI was reduced by 4-aminopyridine and veratridine, both acting on the presynaptic terminal . Direct evidence for an inhibitory G protein-mediated presynaptic action has been provided by Pitler and Alger , as they showed that DSI was pertussis toxin sensitive. Both laboratories hypothesized from the very beginning that they were dealing with a phenomenon that involves retrograde messengers. Llano et al. stated that “Ca2 rise in the Purkinje cell leads to the production of a lipid-soluble second messenger.” This was a remarkable prediction 10 years before the discovery that, indeed, the lipid-soluble endocannabinoids are these messengers,cannabis grow system although the earlier claim of a retrograde action of arachidonic acid in the presynaptic control of LTP made this assumption rather plausible at that time.
The quest for identifying the chemical nature of this retrograde messenger began with the discovery of DSI. The slow onset , the requirement of a lasting Ca2 rise, and the Ca2 buffer effects were all consistent with a hormone or peptide rather than classical vesicular neurotransmitter. Yet the first substance suggested by direct experimental evidence was glutamate. In the cerebellum, metabotropic glutamate receptor agonists, acting on presynaptic group II mGluRs, were shown to mimic and occlude DSI, whereas antagonists reduced it . Activation of adenylate cyclase by forskolin reduced DSI, which is consistent with the proposed reduction of cAMP levels by mGluR2/3 activation that is known to lead to a reduction of GABA release . In contrast, in the hippocampus, forskolin and group II or III mGluR ligands were without effect on DSI; however, group I agonists occluded, and antagonists reduced it . Pharmacology and the anatomical distribution of the receptors suggested that mGluR5 is likely to be involved in the reduction of GABA release , but it appeared to be confined to the somadendritic compartment of the neurons perisynaptically around glutamatergic contacts , which was difficult to reconcile with the hypothesis of glutamate being the retrograde signal molecule . The long duration of DSI is not due to the dynamics of the Ca2 transient, as it was the same in EGTA and BAPTA , but probably to the slow disappearance of the retrograde messenger molecule from the site of action around the presynaptic terminal. This again is inconsistent with glutamate being the messenger , since this transmitter is known to be rapidly taken up. The fast buffer BAPTA and the slow buffer EGTA reduced DSI to a similar degree, suggesting that the site of Ca2 entry and the site of calcium’s action in DSI induction are relatively far from each other . One possibility is that the target of incoming Ca2 may be an intracellular Ca2 store that is able to produce large Ca2 transients required for the release of the signal molecule. On the other hand, the selective N-type Ca2 channel blocker -conotoxin was able to block DSI , which, according to recent evidence , turned out to be an action on the presynaptic terminals that are sensitive to DSI and selectively express the N-type Ca2 channel.
These data suggest that Ca2 plays a dual role: it is involved in the initiation phase via Ca2 -induced Ca2 release from intracellular stores in the postsynaptic side as well as in the effector phase via N-type Ca2 channels on presynaptic terminals . Obviously, DSI-like phenomena can have a functional role in neuronal signaling only if they can be induced by physiologically occurring activity patterns. In cerebellar Purkinje cells, 100-ms depolarization was required for a detectable reduction in IPSCs , which, under physiological conditions, may correspond to a few climbing fiber-induced complex spikes . Thus a short train of climbing fiber-induced spikes is expected to lead to an increased excitability of the innervated Purkinje cell for tens of seconds. Initiation by very few spikes, occasionally even two if closely spaced, has been reported in the hippocampus. With 100 M BAPTA in the pipette, detectable DSI could be evoked already by depolarization as short as 25 ms, and half-maximal effect was produced by 187 ms, or by 109-ms depolarization in the absence of BAPTA . This suggests a lower threshold, but also a smaller magnitude and shorter time course of DSI compared with the cerebellum. The behavior-dependent electrical activity patterns in the hippocampus that may lead to DSI are discussed in section VD.Recent studies by Kreitzer and Regehr provided evidence that, at least in the cerebellum, excitatory synaptic transmission is also under the control of retrogradely acting signal molecules. Both parallel fiber and climbing fiber-evoked EPSCs were suppressed for tens of seconds by a 50- to 1,000-ms depolarization of the postsynaptic Purkinje cells from 60 to 0 mV. Due to the obvious similarity to DSI, this phenomenon has been termed depolarization-induced suppression of excitation . Paired-pulse experiments, showing that short term plasticity is affected by the depolarization paradigm for both parallel and climbing fiber responses, demonstrated that the site of expression of DSE is presynaptic and involves a reduction in the probability of transmitter release. BAPTA in the recording pipette completely abolishes DSE,cannabis grow lights providing evidence for the requirement of postsynaptic Ca2 rise to trigger the event.Earlier reports are consistent with the lack of DSE in the hippocampus, but a recent study using excessive depolarization for 5–10 s argues for its existence also in this brain region.
Whether the mechanisms of DSE are similar in the hippocampus and cerebellum is discussed in the following section.The discovery by Wilson and Nicoll , OhnoShosaku et al. , and Kreitzer and Regehr that DSI/DSE are mediated by endocannabinoids revealed that investigations in both the cannabinoid and DSI/DSE fields have been dealing accidentally with the same subject, i.e., the mechanism of retrograde synaptic signaling via endocannabinoids. Both receptor localization data and identification of the physiological actions of cannabinoids on synaptic transmission confirmed that cannabinoids act on presynaptic axons, reducing transmitter release , whereas endocannabinoids are most likely released from the postsynaptic neuron upon strong stimuli that give rise to large Ca2 transients. Thus the signal molecules, which turned out to be endocannabinoids, travel from the post- to the presynaptic site and thus enable neurons to influence the strength of their own synaptic inputs in an activity-dependent manner. This may be considered as a short definition of retrograde synaptic signaling and perhaps, at the same time, summarizes the function of the endocannabinoid system. However, before trying to correlate the findings of cannabinoid and DSI studies, one should be aware of the major limitations. There are numerous examples of mismatch in receptor/transmitter distribution in the brain; receptors can be found in locations where they hardly ever see their endogenous ligand. Nevertheless, these receptors readily participate in mediating the effects of its exogenous ligands, e.g., during pharmacotherapy. We are facing the same problems with the relative distribution of cannabinoid receptors versus endocannabinoid release sites both at the cellular and subcellular levels. In addition, the distance to which anandamide and 2-AG are able to diffuse is also an important question from the point of identifying the degree of mismatch. Thus correlation of the sites of action of cannabinoid drugs and the sites of expression of DSI should reveal the regional, cellular, and subcellular domains where receptor and endogenous ligand distributions match, i.e., where endocannabinoids are likely to have a functional role in synaptic signaling. Several lines of evidence have been provided that endocannabinoids represent the retrograde signal molecules that mediate DSI both in the hippocampus and cerebellum, as well as DSE in the cerebellum. Antagonists of CB1 receptors fully block and agonists occlude DSI and DSE, whereas DSI is absent in CB1 receptor knock-out animals . In these experiments either single cell or paired recording has been used, and retrograde synaptic signaling has been evoked by the same procedures as described in the original work of Alger’s and Marty’s groups . In addition, Wilson and Nicoll demonstrated that uncaging of Ca2 from a photolabile chelator induces DSI that was indistinguishable from that evoked by depolarization. Thus a large intracellular Ca2 rise is a necessary and sufficient element in the induction of the release of endocannabinoids. As expected from the membrane-permeant endocannabinoids, their release does not require vesicle fusion, since botulinum toxin delivered via the intracellular recording pipette did not affect DSI.
A further crucial question concerns the range to which the released endocannabinoids are able to diffuse. Recordings at room temperature from pyramidal cells at various distances from the depolarized neuron releasing the signal molecules revealed that it is only the adjacent cell, at a maximum distance of 20 m, to which endocannabinoids are able to diffuse in a sufficient concentration to evoke detectable DSI . However, a considerably greater endocannabinoid uptake and metabolism should be expected at physiological temperatures, which likely results in a decreased spread and a more focused action. Earlier data indicating the involvement of glutamate and mGluR receptors in DSI also needed clarification . Varma et al. demonstrated that enhancement of DSI by mGluR agonists could be blocked by antagonists of both group I mGluR and CB1 receptors, whereas the same mGluR agonists were without effect in CB1 receptor knock-out animals. This provides direct evidence that any mGluR effects on DSI published earlier were mediated by endocannabinoid signaling, and glutamate served here as a trigger for the release of endocannabinoids rather than as a retrograde signal molecule as thought earlier. These data were subsequently confirmed by paired recordings from cultured hippocampal neurons . In a recent paper, Maejima et al. demonstrated that mGluR1 activation induces DSE in Purkinje cells even without changing the intracellular Ca2 concentration. This suggests that, at least in the case of cerebellar Purkinje cells, two independent mechanisms may trigger endocannabinoid synthesis ; one involves a transient elevation of intracellular [Ca2], and the other is independent of intracellular [Ca2] and involves mGluR1 signaling. This may imply that, under normal physiological conditions, different induction mechanisms may evoke the release of different endocannabinoids. With the growing number of potential endocannabinoids , the question arises whether they are involved in distinct functions, i.e., by acting at different receptors and/or at specific types of synapses. This question represents one of the hot spots of current endocannabinoid research, and direct measurements of the different endocannabinoid compounds during retrograde signaling should provide an answer. They may induce branch-point failure, decrease action potential invasion of axon terminals, reduce Ca2 influx into the synaptic varicosities via N- or P/Q-type channels, or block the release machinery somewhere downstream from the Ca2 signal. Using Ca2 imaging of single climbing fibers provided evidence that DSE involves a reduction of presynaptic Ca2 influx, which has the same time course as the reduction of the EPSC. Branch-point failure was shown not to contribute to DSE, at least in the case of climbing fibers, as stimulation of the examined single axon evoked a uniform rise of Ca2 throughout its entire arbor. These findings are supported by the fact that cannabinoids are known to block N-type Ca2 channels in neuroblastoma cells and reduce synaptic transmission by inhibiting both N- and P/Q-type channels in neurons . Inhibition of the release machinery is unlikely to play a role, particularly in GABAergic transmission, since CB1 receptor activation has little if any effect on mIPSC frequency in the presence of tetrodotoxin and cadmium . Furthermore, CB1 receptors tend to be localized away from the release sites, having a high density even on preterminal axon segments, which also argues against this possibility . In the hippocampus, evidence has been provided that DSI likely involves a direct action of G proteins on voltage-dependent calcium channels.