Endogenous cannabinoids are considered to function as diffusible and short-lived modulators that may transmit signals retrogradely from postsynaptic to presynaptic neurons. To evaluate this possibility, we have made a paired whole-cell recording from cultured hippocampal neurons with inhibitory synaptic connections. In about 60% of pairs, a cannabinoid agonist greatly reduced the release of the inhibitory neurotransmitter GABA from presynaptic terminals. In most of such pairs but not in those insensitive to the agonist, depolarization of postsynaptic neurons and the resultant elevation of intracellular Ca2+ concentration caused transient suppression of inhibitory synaptic currents, which is mainly due to reduction of GABA release. This depolarization-induced suppression was completely blocked by selective cannabinoid antagonists. Our results reveal that endogenous cannabinoids mediate retrograde signals from depolarized postsynaptic neurons to presynaptic terminals to cause the reduction of transmitter release.

No abstract

We report a type of synaptic modulation that involves retrograde signaling from postsynaptic metabotropic glutamate receptors (mGluRs) to presynaptic cannabinoid receptors. Activation of mGluR subtype 1 (mGluR1) expressed in cerebellar Purkinje cells (PCs) reduced neurotransmitter release from excitatory climbing fibers. This required activation of G proteins but not Ca2+ elevation in postsynaptic PCs. This effect was occluded by a cannabinoid agonist and totally abolished by cannabinoid antagonists. Depolarization-induced Ca2+ transients in PCs also caused cannabinoid receptor-mediated presynaptic inhibition. Thus, endocannabinoid production in PCs can be initiated by two distinct stimuli. Activation of mGluR1 by repetitive stimulation of parallel fibers, the other excitatory input to PCs, caused transient cannabinoid receptor-mediated depression of climbing fiber input. Our data highlight a signaling mechanism whereby activation of postsynaptic mGluR retrogradely influences presynaptic functions via endocannabinoid system.

Endocannabinoids work as retrograde messengers and contribute to short-term and long-term modulation of synaptic transmission via presynaptic cannabinoid receptors. It is generally accepted that the CB1 cannabinoid receptor (CB1) mediates the effects of endocannabinoid in inhibitory synapses. For excitatory synapses, however, contributions of CB1, "CB3," and some other unidentified receptors have been suggested. In the present study we used electrophysiological and immunohistochemical techniques and examined the type(s) of cannabinoid receptor functioning at hippocampal and cerebellar excitatory synapses. Our electrophysiological data clearly demonstrate the predominant contribution of CB1. At hippocampal excitatory synapses on pyramidal neurons the cannabinoid-induced synaptic suppression was reversed by a CB1-specific antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and was absent in CB1 knock-out mice. At climbing fiber (CF) and parallel fiber (PF) synapses on cerebellar Purkinje cells the cannabinoid-dependent suppression was absent in CB1 knock-out mice. The presence of CB1 at presynaptic terminals was confirmed by immunohistochemical experiments with specific antibodies against CB1. In immunoelectron microscopy the densities of CB1-positive signals in hippocampal excitatory terminals and cerebellar PF terminals were much lower than in inhibitory terminals but were clearly higher than the background. Along the long axis of PFs, the CB1 was localized at a much higher density on the perisynaptic membrane than on the extrasynaptic and synaptic regions. In contrast, CB1 density was low in CF terminals and was not significantly higher than the background. Despite the discrepancy between the electrophysiological and morphological data for CB1 expression on CFs, these results collectively indicate that CB1 is responsible for cannabinoid-dependent suppression of excitatory transmission in the hippocampus and cerebellum.

ADP-ribosylation is a ubiquitous post-translational addition of either monomers or polymers of ADP-ribose to target proteins by ADP-ribosyltransferases, usually by interferon-inducible diphtheria toxin-like enzymes known as PARPs. While several PARPs have known antiviral activities, these activities are mostly independent of ADP-ribosylation. Consequently, less is known about the antiviral effects of ADP-ribosylation. Several viral families, including Coronaviridae, Togaviridae, and Hepeviridae, encode for macrodomain proteins that bind to and hydrolyze ADP-ribose from proteins and are critical for optimal replication and virulence. These results suggest that macrodomains counter cellular ADP-ribosylation, but whether PARPs or, alternatively, other ADP-ribosyltransferases cause this modification is not clear. Here we show that pan-PARP inhibition enhanced replication and inhibited interferon production in primary macrophages infected with macrodomain-mutant but not wild-type coronavirus. Specifically, knockdown of two abundantly expressed PARPs, PARP12 and PARP14, led to increased replication of mutant but did not significantly affect wild-type virus. PARP14 was also important for the induction of interferon in mouse and human cells, indicating a critical role for this PARP in the regulation of innate immunity. In summary, these data demonstrate that the macrodomain is required to prevent PARP-mediated inhibition of coronavirus replication and enhancement of interferon production.