Presynaptic Cannabinoid Receptors Research Articles

Regulation of neurotransmission in regenerating neuromuscular junctions involving endocannabinoids

The work was dedicated to investigation of the influence of two endocannabinoids – arachidonoylethanolamide (AEA), also known as anandamide, and 2-arachidonoylglycerol (2-AG) on the parameters of miniature endplate potentials (MEPP) and evoked endplate potentials (EPP) of motor synapses at the early stage of regeneration during muscle reinnervation. 2-AG increased the amplitude of MEPP by 35%, and also increased the amplitude of EPP by 37%, without affecting quantal content of EPP or any other parameters of neurotransmitter secretion. This effect was prevented by vesicular acetylcholine transporter inhibitor vesamicol and by inverse agonist of CB1-type cannabinoid receptors AM251. AEA did not change the amplitude or any other parameters of MEPP, but reduced the quantal content of EPP by 27%. The inhibitory effect of AEA was prevented by AM251 and by the L-type Ca2+ channel blocker nitrendipine. Thus, it was established for the first time that in newly formed motor synapses AEA and 2-AG activate the same type of presynaptic cannabinoid receptors, but have different final targets, influence different parameters of quantal ACh secretion and have multidirectional effects on synaptic transmission. The presence of both facilitatory and inhibitory effects of endocannabinoids in regenerating synapses may serve to fine-tune and regulate synaptic transmission during their maturation.

Orexin recruits non-selective cationic conductance and endocannabinoid to dynamically modulate firing of caudal pontine reticular nuclear neurones.

The neuropeptide orexin is involved in motor circuit function. However, its modulation on neuronal activities of motor structures, integrating orexin's diverse downstream molecular cascades, remains elusive. By combining whole-cell patch-clamp recordings and neuropharmacological methods, we revealed that both non-selective cationic conductance (NSCC) and endocannabinoids (eCBs) are recruited by orexin signalling on reticulospinal neurones in the caudal pontine reticular nucleus (PnC). The orexin-NSCC cascade provides a depolarizing force that proportionally enhances the firing-responsive gain of these neurones. Meanwhile, the orexin-eCB cascade selectively attenuates excitatory synaptic strength in these neurones by activating presynaptic cannabinoid receptor type 1. This cascade restrains the firing response of the PnC reticulospinal neurones to excitatory inputs. Intriguingly, non-linear or linear interactions between orexin postsynaptic excitation and presynaptic inhibition can influence the firing responses of PnC reticulospinal neurones in different directions. When presynaptic inhibition is in the lead, non-linear interactions can prominently downregulate or even gate the firing response. Conversely, linear interactions occur to promote the firing response, and these linear interactions can be considered a proportional reduction in the contribution of depolarization to firing by presynaptic inhibition. Through the dynamic employment of these interactions, adaptive modulation may be achieved by orexin to restrain or even gate the firing output of the PnC to weak/irrelevant input signals and facilitate those to salient signals. KEY POINTS: This study investigated the effects of orexin on the firing activity of PnC reticulospinal neurones, a key element of central motor control. We found that orexin recruited both the non-selective cationic conductances (NSCCs) and endocannabinoid (eCB)-cannabinoid receptor type 1 (CB1R) system to pontine reticular nucleus (PnC) reticulospinal neurones. The orexin-NSCC cascade exerts a postsynaptic excitation that enhances the firing response, whereas the orexin-eCB-CB1R cascade selectively attenuates excitatory synaptic strength that restrains the firing response. The postsynaptic and presynaptic actions of orexins occur in an overlapping time window and interact to dynamically modulate firings in PnC reticulospinal neurones. Non-linear interactions occur when presynaptic inhibition of orexin is in the lead, and these interactions can prominently downregulate or even gate firing responses in PnC reticulospinal neurones. Linear interactions occur when postsynaptic excitation of orexin is in the lead, and these interactions can promote the firing response. These linear interactions can be considered a proportional reduction of the contribution of depolarization to firing by presynaptic inhibition.

The Endocannabinoid System as a Potential Therapeutic Target for HIV-1-Associated Neurocognitive Disorder.

Background: Despite the successful introduction of combined antiretroviral therapy, the prevalence of mild to moderate forms of HIV-associated neurocognitive disorders (HAND) remains high. It has been demonstrated that neuronal injury caused by HIV is excitotoxic and inflammatory, and it correlates with neurocognitive decline in HAND. Endocannabinoid system (ECS) protects the body from excitotoxicity and neuroinflammation on demand and presents a promising therapeutic target for treating HAND. Here, we firstly discuss the potential pathogenesis of HAND. We secondly discuss the structural and functional changes in the ECS that are currently known among HAND patients. We thirdly discuss current clinical and preclinical findings concerning the neuroprotective and anti-inflammatory properties of the ECS among HAND patients. Fourth, we will discuss the interactions between the ECS and neuroendocrine systems, including the hypothalamic-pituitary-adrenocortical (HPA) and hypothalamic-pituitary-gonadal (HPG) axes under the HAND conditions. Materials and Methods: We have carried out a review of the literature using PubMed to summarize the current state of knowledge on the association between ECS and HAND. Results: The ECS may be ideally suited for modulation of HAND pathophysiology. Direct activation of presynaptic cannabinoid receptor 1 or reduction of cannabinoid metabolism attenuates HAND excitotoxicity. Chronic neuroinflammation associated with HAND can be reduced by activating cannabinoid receptor 2 on immune cells. The sensitivity of the ECS to HIV may be enhanced by increased cannabinoid receptor expression in HAND. In addition, indirect regulation of the ECS through modulation of hormone-related receptors may be a potential strategy to influence the ECS and also alleviate the progression of HAND due to the reciprocal inhibition of the ECS by the HPA and HPG axes. Conclusions: Taken together, targeting the ECS may be a promising strategy to alleviate the inflammation and neurodegeneration caused by HIV-1 infection. Further studies are required to clarify the role of endocannabinoid signaling in HIV neurotoxicity. Strategies promoting endocannabinoid signaling may slow down cognitive decline of HAND are proposed.

Coordinated Regulation of CB1 Cannabinoid Receptors and Anandamide Metabolism Stabilizes Network Activity during Homeostatic Downscaling.

Neurons express overlapping homeostatic mechanisms to regulate synaptic function and network properties in response to perturbations of neuronal activity. Endocannabinoids (eCBs) are bioactive lipids synthesized in the postsynaptic compartments to regulate synaptic transmission, plasticity, and neuronal excitability primarily through retrograde activation of presynaptic cannabinoid receptor type 1 (CB1). The eCB system is well situated to regulate neuronal network properties and coordinate presynaptic and postsynaptic activity. However, the role of the eCB system in homeostatic adaptations to neuronal hyperactivity is unknown. To address this issue, we used Western blotting and targeted lipidomics to measure adaptations in eCB system to bicuculline (BCC)-induced chronic hyperexcitation in mature cultured rat cortical neurons, and used multielectrode array (MEA) recording and live-cell imaging of glutamate dynamics to test the effects of pharmacological manipulations of eCB on network activities. We show that BCC-induced chronic hyperexcitation triggers homeostatic downscaling and a coordinated adaptation to enhance tonic eCB signaling. Hyperexcitation triggers first the downregulation of fatty acid amide hydrolase (FAAH), the lipase that degrades the eCB anandamide, then an accumulation of anandamide and related metabolites, and finally a delayed upregulation of surface and total CB1. Additionally, we show that BCC-induced downregulation of surface AMPA-type glutamate receptors (AMPARs) and upregulation of CB1 occur through independent mechanisms. Finally, we show that endocannabinoids support baseline network activities before and after downscaling and is engaged to suppress network activity during adaptation to hyperexcitation. We discuss the implications of our findings in the context of downscaling and homeostatic regulation of in vitro oscillatory network activities.

Acute cocaine exposure occludes long-term depression in ventral tegmental area GABA neurons

The ventral tegmental area (VTA) in the midbrain is essential in incentive salience of reward behavior. Drugs of abuse increase midbrain dopamine cell activity and/or dopamine levels, and can alter endogenous VTA glutamate plasticity, leading to addiction or dependence. VTA dopamine cells are regulated by local inhibitory GABA cells, which exhibit a form of pre-synaptic cannabinoid receptor 1-dependent long-term depression of their glutamatergic inputs. Our current aim was to determine cocaine's influence on VTA GABA cell glutamate plasticity and circuity. Using whole cell voltage-clamp electrophysiology in VTA slices of GAD67-GFP knock-in mice, we recorded excitatory inputs on VTA GABA cells. Acute and chronic injections of cocaine were sufficient to occlude long-term depression. The plasticity could be reversed to the naïve state however, as long-term depression was again observed following a 7-day abstinence from acute cocaine exposure. Furthermore, chronic cocaine decreased AMPA/NMDA ratios at glutamate synapses onto VTA GABA cells, compared to vehicle injection controls, the opposite change noted in dopamine cells. Collectively, our data suggest the cellular mechanism of cocaine-mediated synaptic modification that may result in dependence/withdrawal could involve changes in glutamate input to VTA GABA circuitry in addition to VTA dopamine cells. Therefore VTA GABA cells may also play a role, possibly in a synergistic manner with the dopamine circuit, in cocaine-induced changes to the VTA reward pathway than previously known.

Cannabinoid receptor‐1 expressing interneurons require postsynaptic dystroglycan for targeting during postnatal brain development

Dystroglycan is a transmembrane glycoprotein highly expressed in muscle and brain that links the extracellular matrix to the actin cytoskeleton through its two subunits, a heavily glycosylated extracellular subunit alpha dystroglycan (a‐DG), and the membrane spanning beta‐dystroglycan (b‐DG). Glycosylation of dystroglycan by at least 18 enzymes is essential for its ability to bind extracellular ligands throughout development. Mutations in genes encoding glycosyltransferases such as B4gat1 and ISPD reduce dystroglycan function and cause a spectrum of congenital muscular dystrophies characterized by muscle weakness, brain malformations, and cognitive deficits. In the brain, dystroglycan is expressed by both glia and excitatory neurons, but its specific function in neurons has remained elusive. Recently, neuronal dystroglycan was discovered to be important for the formation of presynaptic cannabinoid receptor 1 (CB1R) synapses derived from cholecystokinin (CCK) interneurons, a population of inhibitory cells in the forebrain. However, the mechanism by which dystroglycan regulates the development of CCK/CB1R+ inhibitory synapses remains unknown. In the present study, we use multiple mouse lines to determine how dystroglycan regulates CB1R+ synapse development, including mouse models of dystroglycanopathy (B4gat1, ISPD), conditional genetic deletion of dystroglycan from neurons and glia (Dag1‐cKO: Nex‐Cre, Emx‐Cre, Nestin‐Cre), and a mouse lacking the intracellular signaling domain of dystroglycan (Dag1ICD). We found that deletion of dystroglycan specifically from excitatory neurons resulted in the loss of nearly all presynaptic CB1R+ terminals within the first week after birth. Surprisingly, deletion of dystroglycan from both neurons and glia using Nestin‐Cre or Emx‐Cre resulted in abnormal targeting of CB1R+ terminals. We also find that maintenance of CB1R+ terminals in adulthood does not require full glycosylation of a‐DG or the intracellular signaling domain of b‐DG. Collectively, these results suggest that dystroglycan may be required for the proper targeting of CB1R+ synapses during postnatal brain development.Support or Funding InformationNINDS R01 NS091027 (KMW)NINDS F31 NS108522 (DSM)ARCS Fellowship (DSM)Tartar Fellowship (DSM)

Cyclic AMP-dependent protein kinase and D1 dopamine receptors regulate diacylglycerol lipase-α and synaptic 2-arachidonoyl glycerol signaling.

Brain endocannabinoids serve as retrograde neurotransmitters, being synthesized in post-synaptic neurons "on demand" and released to bind pre-synaptic cannabinoid receptors and suppress glutamatergic or GABAergic transmission. The most abundant brain endocannabinoid, 2 arachidonoyl glycerol (2-AG), is primarily synthesized by diacylglycerol lipase-α (DGLα), which is activated by poorly understood mechanisms in response to calcium influx following post-synaptic depolarization and/or the activation of Gq -coupled group 1 metabotropic glutamate receptors. However, the impact of other neurotransmitters and their downstream signaling pathways on synaptic 2-AG signaling has not been intensively studied. Here, we found that DGLα activity in membrane fractions from transfected HEK293T cells was significantly increased by in vitro phosphorylation using cyclic AMP-dependent protein kinase (PKA). Moreover, PKA directly phosphorylated DGLα at Ser798 in vitro. Elevation of cAMP levels in HEK293 cells expressing DGLα increased Ser798 phosphorylation, as detected using a phospho-Ser798-specific antibody, and enhanced DGLα activity; this in situ enhancement of DGLα activity was prevented by mutation of Ser798 to Ala. We investigated the impact of PKA on synaptic 2-AG mobilization in mouse striatal slices by manipulating D1-dopamine receptor (D1R) signaling and assessing depolarization-induced suppression of excitation, a DGLα- and 2-AG-dependent form of short-term synaptic depression. The magnitude of depolarization-enhanced suppression of excitation in direct pathway medium spiny neurons was increased by pre-incubation with a D1R agonist, and this enhancement was blocked by post-synaptic inhibition of PKA. Taken together, these findings provide new molecular insights into the complex mechanisms regulating synaptic endocannabinoid signaling.

Introduction of Gamma-Aminobutyric Acid into the Bloodstream to Negate NMDA Receptor Hypofunction Induced by Delta 9-Tetrahydrocannabinol

Glutamate is the most abundant neurotransmitter found in the brain, controlling fast signalling throughout all sections and being especially involved in memory recollection and learning. Long-Term Potentiation (LTP) is the strengthening of neural connections through receptor synthesis over consistent usage, first triggered by synapse activation by a small amount of glutamate. However, in heavy (prolonged instance of exposure) and habitual users of cannabis, the effects of LTP are exacerbated by N-methyl-D-Aspartic Acid (NMDA) Receptor Hypofunction (NRHypo) which in turn affects memory, learning, reasoning and other aspects of one’s function. Emerging evidence has associated the inhibition of long-term potentiation by Delta 9-Tetrahydrocannabinol (D9-THC) activating presynaptic Cannabinoid Receptor Type 1 (CB1) receptors to the inhibition of the ability to stop production of glutamate (GLU). An excess of glutamate will overstimulate the postsynaptic NMDA and α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid (AMPA) receptors in the neurons commonly in the hippocampus, basal ganglia, and prefrontal cortex, which allow excessive influx of calcium Ca2+ ions, causing neurotoxic conditions. Glutamate Decarboxylase 67 molecule has been shown bind in high concentrations with GLU and lower the harmful effects of D9-THC on the brain by converting GLU to Gamma-Aminobutyric Acid (GABA), an inhibitory neurotransmitter. GAD67 will be distributed to mice in this proposed experiment and the behaviour of the mice will be monitored. D9-THC affected, D9-THC and GAD67 affected, and normal mice will be subjected to behavioral interaction and maze tests which will show differences in their learning, spatial awareness and orientation, and reasoning abilities. Chemical analysis of cerebral fluid and brain slices will determine chemical concentrations of GAD67 and D9-THC in the brain. Using direct injections into the cerebrospinal fluid (CSF) and bloodstream in mouse models, our aim is to determine the selectivity of the blood brain barrier (BBB) to enzymes such as GAD67 via both channels as well as assess the interaction GAD67 has with cascading neurological effects caused by NRHypo and LTP.

Cannabinoid CB1 receptors in the amygdalar cholecystokinin glutamatergic afferents to nucleus accumbens modulate depressive-like behavior.

Major depressive disorder is a devastating psychiatric disease that afflicts up to 17% of the world's population. Postmortem brain analyses and imaging studies of patients with depression have implicated basal lateral amygdala (BLA) dysfunction in the pathophysiology of depression. However, the circuit and molecular mechanisms through which BLA neurons modulate depressive behavior are largely uncharacterized. Here, in mice, we identified that BLA cholecystokinin (CCK) glutamatergic neurons mediated negative reinforcement via D2 medium spiny neurons (MSNs) in the nucleus accumbens (NAc) and that chronic social defeat selectively potentiated excitatory transmission of the CCKBLA-D2NAc circuit in susceptible mice via reduction of presynaptic cannabinoid type-1 receptor (CB1R). Knockdown of CB1R in the CCKBLA-D2NAc circuit elevated synaptic activity and promoted stress susceptibility. Notably, selective inhibition of the CCKBLA-D2NAc circuit or administration of synthetic cannabinoids in the NAc was sufficient to produce antidepressant-like effects. Overall, our studies reveal the circuit and molecular mechanisms of depression.

Presynaptic cannabinoid CB2 receptors modulate [3 H]-Glutamate release at subthalamo-nigral terminals of the rat.

Recent studies suggested the expression of CB2 receptors in neurons of the CNS, however, most of these studies have only explored one aspect of the receptors, i.e., expression of protein, messenger RNA, or functional response, and more complete studies appear to be needed to establish adequately their role in the neuronal function. Electron microscopy studies showed the presence of CB2r in asymmetric terminals of the substantia nigra pars reticulata (SNr), and its mRNA appeared is expressed in the subthalamic nucleus. Here, we explore the expression, source, and functional effects of such receptors by different experimental approaches. Through PCR and immunochemistry, we showed mRNA and protein for CB2rs in slices and primary neuronal cultures from subthalamus. GW833972A, GW405833, and JHW 133, three CB2r agonists dose-dependent inhibited K+ -induced [3 H]-Glutamate release in slices of SNr, and the two antagonist/inverse agonists, JTE-907 and AM630, but not AM281, a CB1r antagonist, prevented GW833972A effect. Subthalamus lesions with kainic acid prevented GW833972A inhibition on release and decreased CB2r protein in nigral synaptosomes, thus nigral CB2rs originate in subthalamus. Inhibition of [3 H]-Glutamate release was PTX- and gallein-sensitive, suggesting a Giβγ -mediated effect. P/Q Ca2+ -type channel blocker, ω-Agatoxin-TK, also inhibited the [3 H]-Glutamate release, this effect was occluded with GW833972A inhibition, indicating that the βγ subunit effect is exerted on Ca2+ channel activity. Finally, microinjections of GW833972A in SNr induced contralateral turning. Our data showed that presynaptic CB2rs inhibit [3 H]-Glutamate release in subthalamo-nigral terminals by P/Q-channels modulation through the Giβγ subunit and suggested their participation in motor behavior.

Dopamine D2 receptor activation potently inhibits striatal glutamatergic transmission in a G2019S LRRK2 genetic model of Parkinson's disease

Among genetic abnormalities identified in Parkinson's disease (PD), mutations of the leucine-rich repeat kinase2 (LRRK2) gene, such as the G2019S missense mutation linked to enhanced kinase activity, are the most common. While the complex role of LRRK2 has not been fully elucidated, evidence that mutated kinase activity affects synaptic transmission has been reported. Thus, our aim was to explore possible early alterations of neurotransmission produced by the G2019S LRRK2 mutation in PD. We performed electrophysiological patch-clamp recordings of striatal spiny projection neurons (SPNs) in the G2019S-Lrrk2 knock-in (KI) mouse model of PD, in D1994S kinase-dead (KD), Lrrk2 knock-out (KO) and wild-type (WT) mice. In G2019S Lrrk2 KI mice, basal spontaneous glutamatergic transmission, synaptic facilitation, and NMDA/AMPA ratios were unchanged, whereas the stimulation of dopamine (DA) D2 receptor by quinpirole reduced the spontaneous and evoked excitatory postsynaptic currents (EPSC). Quinpirole reduced the EPSC amplitude of SPNs in KI but not in KD, KO and WT mice, suggesting that the enhanced LRRK2 kinase activity induced by the G2019S mutation is associated with the observed functional alteration of SPNs synaptic transmission. The effect of quinpirole was mediated by a phospholipase C (PLC)-dependent release of endocannabinoid, with subsequent activation of presynaptic cannabinoid receptor 1 and reduced release of glutamate. The key role of DA D2 receptor in reducing glutamatergic output in our LRRK2 genetic model of PD further supports the use of DA agonists in the treatment of early PD patients with LRRK2 mutations to counteract the disease progression.

Fatty-acid–binding protein 5 controls retrograde endocannabinoid signaling at central glutamate synapses

Endocannabinoids (eCBs) are lipid-signaling molecules involved in the regulation of numerous behaviors and physiological functions. Released by postsynaptic neurons, eCBs mediate retrograde modulation of synaptic transmission and plasticity by activating presynaptic cannabinoid receptors. While the cellular mechanisms by which eCBs control synaptic function have been well characterized, the mechanisms controlling their retrograde synaptic transport remain unknown. Here, we demonstrate that fatty-acid-binding protein 5 (FABP5), a canonical intracellular carrier of eCBs, is indispensable for retrograde eCB transport in the dorsal raphe nucleus (DRn). Thus, pharmacological inhibition or genetic deletion of FABP5 abolishes both phasic and tonic eCB-mediated control of excitatory synaptic transmission in the DRn. The blockade of retrograde eCB signaling induced by FABP5 inhibition is not mediated by impaired cannabinoid receptor function or reduced eCB synthesis. These findings indicate that FABP5 is essential for retrograde eCB signaling and may serve as a synaptic carrier of eCBs at central synapses.

Cannabinoid Receptors Modulate Excitation of an Olfactory Bulb Local Circuit by Cortical Feedback.

Recent studies have provided evidence that corticofugal feedback (CFF) from the olfactory cortex to the olfactory bulb (OB) can significantly impact the state of excitation of output mitral cells (MCs) and tufted cells (TCs) and also modulate neural synchrony. Interpreting these effects however has been complicated by the large number of cell targets of CFF axons in the bulb. Within the granule cell layer (GCL) alone, CFF axons target both GABAergic granule cells (GCs) as well as GABAergic deep short-axon cells (dSACs) that inhibit GCs. Because GCs are a major source of inhibition of MCs/TCs, CFF could be inhibitory to MCs (by exciting GCs) or disinhibitory (by exciting dSACs that inhibit GCs). In this study, we used patch-clamp recordings combined with optogenetic and electrical stimulation methods to investigate the role of presynaptic cannabinoid receptors in regulating CFF pathways, which could alter the weights of inhibition and disinhibition. Recording first from dSACs, we found that the cannabinoid receptor (CB-R) agonist WIN-55212.2 (WIN) reduced excitatory post-synaptic currents (CFF-EPSCs) driven by stimulation of CFF axons. The effects were reversed by the Type 1 CB-R (CB1-R)-specific antagonist SR-141716A. Furthermore, prolonged 5-s depolarizations applied to postsynaptic dSACs effectively reduced CFF-EPSCs in a CB1-R-dependent fashion, providing evidence for depolarization-induced suppression of excitation (DSE) at CFF-to-dSAC synapses. Further analysis indicated that CB1-Rs mediate widespread suppressive effects on synaptic transmission, occurring at CFF synapses onto different dSAC subtypes and CFF synapses onto GCs. Feedforward excitation of dSACs, mediated by MCs/TCs, however, was not impacted by CB1-Rs. In recordings from MCs, performed to examine the net effect of CB1-R activation on GC-to-MC transmission, we found that WIN could both increase and decrease disynaptic inhibition evoked by CFF axon stimulation. The exact effect depended on the size of the inhibitory response, reflecting the local balance of dSAC vs. GC activation. Our results taken together indicate that CB1-Rs can bidirectionally alter the weighting of inhibition and disinhibition of MCs through their effects on CFF pathways.

Presynaptic inhibition upon CB1 or mGlu2/3 receptor activation requires ERK/MAPK phosphorylation of Munc18-1.

Presynaptic cannabinoid (CB1R) and metabotropic glutamate receptors (mGluR2/3) regulate synaptic strength by inhibiting secretion. Here, we reveal a presynaptic inhibitory pathway activated by extracellular signal-regulated kinase (ERK) that mediates CB1R- and mGluR2/3-induced secretion inhibition. This pathway is triggered by a variety of events, from foot shock-induced stress to intense neuronal activity, and induces phosphorylation of the presynaptic protein Munc18-1. Mimicking constitutive phosphorylation of Munc18-1 results in a drastic decrease in synaptic transmission. ERK-mediated phosphorylation of Munc18-1 ultimately leads to degradation by the ubiquitin-proteasome system. Conversely, preventing ERK-dependent Munc18-1 phosphorylation increases synaptic strength. CB1R- and mGluR2/3-induced synaptic inhibition and depolarization-induced suppression of excitation (DSE) are reduced upon ERK/MEK pathway inhibition and further reduced when ERK-dependent Munc18-1 phosphorylation is blocked. Thus, ERK-dependent Munc18-1 phosphorylation provides a major negative feedback loop to control synaptic strength upon activation of presynaptic receptors and during intense neuronal activity.

Impaired 2-AG Signaling in Hippocampal Glutamatergic Neurons: Aggravation of Anxiety-Like Behavior and Unaltered Seizure Susceptibility.

Background:Postsynaptically generated 2-arachidonoylglycerol activates the presynaptic cannabinoid type-1 receptor, which is involved in synaptic plasticity at both glutamatergic and GABAergic synapses. However, the differential function of 2-arachidonoylglycerol signaling at glutamatergic vs GABAergic synapses in the context of animal behavior has not been investigated yet.Methods:Here, we analyzed the role of 2-arachidonoylglycerol signaling selectively in hippocampal glutamatergic neurons. Monoacylglycerol lipase, the primary degrading enzyme of 2-arachidonoylglycerol, is expressed at presynaptic sites of excitatory and inhibitory neurons. By adeno-associated virus-mediated overexpression of monoacylglycerol lipase in glutamatergic neurons of the mouse hippocampus, we selectively interfered with 2-arachidonoylglycerol signaling at glutamatergic synapses of these neurons.Results:Genetic modification of monoacylglycerol lipase resulted in a 50% decrease in 2-arachidonoylglycerol tissue levels without affecting the content of the second major endocannabinoid anandamide. A typical electrophysiological read-out for 2-arachidonoylglycerol signaling is the depolarization-induced suppression of excitation and of inhibition. Elevated monoacylglycerol lipase levels at glutamatergic terminals selectively impaired depolarization-induced suppression of excitation, while depolarization-induced suppression of inhibition was not significantly changed. At the behavioral level, mice with impaired hippocampal glutamatergic 2-arachidonoylglycerol signaling exhibited increased anxiety-like behavior but showed no alterations in aversive memory formation and seizure susceptibility.Conclusion:Our data indicate that 2-arachidonoylglycerol signaling selectively in hippocampal glutamatergic neurons is essential for the animal’s adaptation to aversive situations.

Weeding out bad waves: towards selective cannabinoid circuit control in epilepsy.

Endocannabinoids are lipid-derived messengers, and both their synthesis and breakdown are under tight spatiotemporal regulation. As retrograde signalling molecules, endocannabinoids are synthesized postsynaptically but activate presynaptic cannabinoid receptor 1 (CB1) receptors to inhibit neurotransmitter release. In turn, CB1-expressing inhibitory and excitatory synapses act as strategically placed control points for activity-dependent regulation of dynamically changing normal and pathological oscillatory network activity. Here, we highlight emerging principles of cannabinoid circuit control and plasticity, and discuss their relevance for epilepsy and related comorbidities. New insights into cannabinoid signalling may facilitate the translation of the recent interest in cannabis-related substances as antiseizure medications to evidence-based treatment strategies.

Cannabinoid-mediated short-term plasticity in hippocampus.

Endocannabinoids (eCBs) modulate both excitatory and inhibitory neurotransmission in hippocampus via activation of pre-synaptic cannabinoid receptors. Here, we present a model for cannabinoid mediated short-term depression of excitation (DSE) based on our recently developed model for the equivalent phenomenon of suppressing inhibition (DSI). Furthermore, we derive a simplified formulation of the calcium-mediated endocannabinoid synthesis that underlies short-term modulation of neurotransmission in hippocampus. The simplified model describes cannabinoid-mediated short-term modulation of both hippocampal inhibition and excitation and is ideally suited for large network studies. Moreover, the implementation of the simplified DSI/DSE model provides predictions on how both phenomena are modulated by the magnitude of the pre-synaptic cell's activity. In addition we demonstrate the role of DSE in shaping the post-synaptic cell's firing behaviour qualitatively and quantitatively in dependence on eCB availability and the pre-synaptic cell's activity. Finally, we explore under which conditions the combination of DSI and DSE can temporarily shift the fine balance between excitation and inhibition. This highlights a mechanism by which eCBs might act in a neuro-protective manner during high neural activity.

A biophysical model of endocannabinoid-mediated short term depression of excitation in hippocampus

Cannabinoids are a group of chemical substances that activate cannabinoid receptors. The general types of cannabinoids are (1) phyto-cannabinoids, naturally occurring in the cannabis plant, (2) synthetic cannabinoids, produced in the laboratory, and (3) endocannabinoids which naturally form endogenously in the body. The endocannabinoid signalling system is known to have a neuromodulatory role in many physiological processes including neurotransmission, synaptic plasticity, learning and memory [1]. Endocannabinoids are synthesised in response to neuronal activity in a calcium-dependent manner and/or due to G protein-coupled receptor activation. Once released into the synaptic cleft, they act in a retrograde fashion on pre-synaptic cannabinoid receptors resulting to inhibition of neurotransmitter release. Endocannabinoids are known to underlie certain forms of short- and long-term forms of synaptic plasticity at both excitatory and inhibitory synapses in various brain areas [1]. In hippocampus, a form of endocannabinoid-mediated short-term depression (eCB-STD), which is calcium-dependent, is known as depolarization-induced suppression of inhibition (DSI) or excitation (DSE) [3]. Both hippocampal DSI and DSE can be induced by application of cannabinoid receptor agonists and are blocked by cannabinoid receptor antagonists [2,4]. Although DSI and DSE in hippocampus share many common biochemical signalling pathways, DSE is less prominent than DSI and requires longer depolarisations for induction supposedly due to the lower expression and sensitivity of cannabinoid receptors on excitatory cells [3]. However, the expression of cannabinoid receptors on excitatory terminals is more homogeneous than in inhibitory terminals (only about 50% of inhibitory terminals are sensitive to eCB-STD) [3]. Here, we present the development of a mathematical model of the endocannabinoid signalling underlying DSE at hippocampal excitatory synapses on excitatory cells which is (a) biophysically plausible, describing the most essential pathways underlying this phenomenon (b) detailed enough to describe the key DSE experimental characteristics, and, (c) is simple enough to use in network studies. The main components of the DSE model are (i) a post-synaptic single compartment model for the excitatory cell with endocannabinoid synthesis mediated by intracellular calcium dynamics, and, (ii) a pre-synaptic single compartment model for the excitatory cell with cannabinoid receptor dynamics modulating excitatory synaptic transmission. As a modelling framework for the cannabinoid signalling we consider a recently developed biophysical model for endocannabinoid-mediated short-term depression of inhibition in hippocampus [5]. We extend and adapt this model in order to account for the reduced magnitude and higher induction threshold for DSE versus DSI. The model successfully reproduces the mediation of DSE due to endocannabinoid-mobilising stimulation (depolarising voltage pulses), as observed experimentally [3]. The model also successfully captures the mediation of endocannabinoid-mediated short-term depression of excitation due to cannabinoid agonist administration (WIN55,212) [3]. The development of the DSE model opens the way for comprehensive networks studies including both DSI and DSE, in order to decipher the role of cannabinoids in synaptic transmission in network emergent behaviour related to memory and learning.

Nitric oxide‐dependent long‐term depression but not endocannabinoid‐mediated long‐term potentiation is crucial for visual recognition memory

Synaptic plasticity in perirhinal cortex is essential for recognition memory. Nitric oxide and endocannabinoids (eCBs), which are produced in the postsynaptic cell and act on the presynaptic terminal, are implicated in mechanisms of long-term potentiation (LTP) and long-term depression (LTD) in other brain regions. In this study, we examine these two retrograde signalling cascades in perirhinal cortex synaptic plasticity and in visual recognition memory in the rat. We show that inhibition of NO-dependent signalling prevented both carbachol- and activity (5 Hz)-dependent LTD but not activity (100 Hz theta burst)-dependent LTP in the rat perirhinal cortex in vitro. In contrast, inhibition of the eCB-dependent signalling prevented LTP but not the two forms of LTD in vitro. Local administration into perirhinal cortex of the nitric oxide synthase inhibitor NPA (2 μm) disrupted acquisition of long-term visual recognition memory. In contrast, AM251 (10 μm), a cannabinoid receptor 1 antagonist, did not impair visual recognition memory. The results of this study demonstrate dissociation between putative retrograde signalling mechanisms in LTD and LTP in perirhinal cortex. Thus, LTP relies on cannabinoid but not NO signalling, whilst LTD relies on NO- but not eCB-dependent signalling. Critically, these results also establish, for the first time, that NO- but not eCB-dependent signalling is important in perirhinal cortex-dependent visual recognition memory.

Abstract 117: Contribution of Endocannabinoids via CB1 Receptors to the Impaired Baroreflex Sensitivity in (mRen2)27 Transgenic Hypertensive Rats

There is growing evidence for a role of the endocannabinoid system in the pathogenesis of hypertension and associated metabolic syndrome. Presynaptic CB1 cannabinoid receptors, the neuronal target of endocannabinoids, are densely expressed in the solitary tract nucleus (NTS), the primary site for termination of baroreceptor afferent nerve fibers, and in vagal afferent neurons of the nodose ganglion. Our studies in anesthetized, normotensive Sprague-Dawley rats suggest that activation of cannabinoid receptors within the NTS via bilateral microinjection of the CB1 agonist CP55,940 modulates baroreflex sensitivity (BRS) for control of heart rate (HR) in a dose-related biphasic manner. Administration of the CB1-selective antagonist SR141716A (36 pmol) fully prevented or reversed these changes in BRS, and there was no significant effect of the antagonist alone in these normotensive animals (1.04 ± 0.05 ms/mmHg baseline vs. 1.12 ± 0.11 ms/mmHg after antagonist treatment, n = 5). We have now determined the effects of blockade of CB1 receptors in a model of angiotensin (Ang) II-dependent hypertension and insulin resistance with impaired BRS. In contrast to observations in normotensive rats, SR141716A dose-dependently improved BRS for control of HR when microinjected into the NTS of hypertensive, transgenic (mRen2)27 rats with impaired baseline BRS, without changing resting MAP or HR. Microinjection of 0.36 pmol SR141716A (n = 5) increased BRS from a baseline of 0.43 ± 0.03 ms/mmHg to 0.68 ± 0.02 (p < 0.001), and 36 pmol SR141716A (n = 5) increased BRS from 0.47 ± 0.02 to 1.05 ± 0.07 ms/mmHg (p < 0.001), a level that was comparable with baseline BRS of normotensive Sprague-Dawley rats. The demonstration of dose-dependent actions of the CB1 agonist on BRS in normotensive animals coupled with new findings that blockade of CB1 receptors improves BRS, an index of vagal function, in (mRen2)27 hypertensive animals is consistent with the interpretation that elevated endocannabinoids within the NTS contribute to impaired BRS. Thus, our findings suggest that blockade of CB1 receptors will influence cardiovagal reflexes in a positive manner in the context of hypertension and metabolic syndrome.