Cells and Molecules Underpinning Cannabis-Related Variations in Cortical Thickness during Adolescence.

During adolescence, cannabis experimentation is common, and its association with interindividual variations in brain maturation well studied. Cellular and molecular underpinnings of these system-level relationships are, however, unclear. We thus conducted a three-step study.

A type II cannabis extract and a 1:1 blend of Δ(9)-tetrahydrocannabinol and cannabidiol display distinct antinociceptive profiles and engage different endocannabinoid targets when administered into the subarachnoid space.

Cannabis extracts are being increasingly used to mitigate chronic pain. Current guidelines for their prescription rely on Δ-tetrahydrocannabinol (THC) and cannabidiol (CBD) content as well as the ratio of these major cannabinoids present in the blend. Here we assessed whether these descriptors were representative of product effectiveness to produce a desired outcome such as analgesia.

Acan downregulation in parvalbumin GABAergic cells reduces spontaneous recovery of fear memories.

While persistence of fear memories is essential for survival, a failure to inhibit fear in response to harmless stimuli is a feature of anxiety disorders. Extinction training only temporarily suppresses fear memory recovery in adults, but it is highly effective in juvenile rodents. Maturation of GABAergic circuits, in particular of parvalbumin-positive (PV) cells, restricts plasticity in the adult brain, thus reducing PV cell maturation could promote the suppression of fear memories following extinction training in adults.

Neuronal GPR81 regulates developmental brain angiogenesis and promotes brain recovery after a hypoxic ischemic insult.

Perinatal hypoxic/ischemic (HI) brain injury is a major clinical problem with devastating neurodevelopmental outcomes in neonates. During HI brain injury, dysregulated factor production contributes to microvascular impairment. Glycolysis-derived lactate accumulated during ischemia has been proposed to protect against ischemic injury, but its mechanism of action is poorly understood.

Comprehensive Signaling Profiles Reveal Unsuspected Functional Selectivity of δ-Opioid Receptor Agonists and Allow the Identification of Ligands with the Greatest Potential for Inducing Cyclase Superactivation.

Prolonged exposure to opioid receptor agonists triggers adaptations in the adenylyl cyclase (AC) pathway that lead to enhanced production of cyclic adenosine monophosphate (cAMP) upon withdrawal. This cellular phenomenon contributes to withdrawal symptoms, hyperalgesia and analgesic tolerance that interfere with clinical management of chronic pain syndromes. Since δ-opioid receptors (DOPrs) are a promising target for chronic pain management, we were interested in finding out if cell-based signaling profiles as generated for drug discovery purposes could inform us of the ligand potential to induce sensitization of the cyclase path.

Signaling diversity of mu- and delta- opioid receptor ligands: Re-evaluating the benefits of β-arrestin/G protein signaling bias.

Opioid analgesics are elective for treating moderate to severe pain but their use is restricted by severe side effects. Signaling bias has been proposed as a viable means for improving this situation. To exploit this opportunity, continuous efforts are devoted to understand how ligand-specific modulations of receptor functions could mediate the different in vivo effects of opioids.

Signal profiling of the βAR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by βAR and βAR.

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β-adrenergic receptor (βAR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR.

Author Correction: Exploring use of unsupervised clustering to associate signaling profiles of GPCR ligands to clinical response.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ligand-specific recycling profiles determine distinct potential for chronic analgesic tolerance of delta-opioid receptor (DOPr) agonists.

δ-opioid receptor (DOPr) agonists have analgesic efficacy in chronic pain models but development of tolerance limits their use for long-term pain management. Although agonist potential for inducing acute analgesic tolerance has been associated with distinct patterns of DOPr internalization, the association between trafficking and chronic tolerance remains ill-defined. In a rat model of streptozotocin (STZ)-induced diabetic neuropathy, deltorphin II and TIPP produced sustained analgesia  following daily (intrathecal) i.

Exploring use of unsupervised clustering to associate signaling profiles of GPCR ligands to clinical response.

Signaling diversity of G protein-coupled (GPCR) ligands provides novel opportunities to develop more effective, better-tolerated therapeutics. Taking advantage of these opportunities requires identifying which effectors should be specifically activated or avoided so as to promote desired clinical responses and avoid side effects. However, identifying signaling profiles that support desired clinical outcomes remains challenging.

Molecular aspects of delta opioid receptors.

The delta opioid receptor (DOP) belongs to the Class A, rhodopsin-like family of G protein-coupled receptors. Although this receptor has a high level of similarity with the other opioid receptors, it displays unique aspects and functions. Indeed, as opposed to most membrane receptors, DOP is poorly addressed to the plasma membrane.

N-Guanidyl and C-Tetrazole Leu-Enkephalin Derivatives: Efficient Mu and Delta Opioid Receptor Agonists with Improved Pharmacological Properties.

Leu-enkephalin and d-Ala-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells.

Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors.

G protein-coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho.

Delta opioid receptors recycle to the membrane after sorting to the degradation path.

Soon after internalization delta opioid receptors (DOPrs) are committed to the degradation path by G protein-coupled receptor (GPCR)-associated binding protein. Here we provide evidence that this classical post-endocytic itinerary may be rectified by downstream sorting decisions which allow DOPrs to regain to the membrane after having reached late endosomes (LE). The LE sorting mechanism involved ESCRT accessory protein Alix and the TIP47/Rab9 retrieval complex which supported translocation of the receptor to the TGN, from where it subsequently regained the cell membrane.

Analyzing biased responses of GPCR ligands.

G protein-coupled receptors (GPCRs) are valuable targets for drug discovery. They exist in interconverting states differentially stabilized by diverse signaling partners. A ligand's capacity to distinguish among receptors associated with different partners is the basis of bias.

Molecular Pharmacology of δ-Opioid Receptors.

Opioids are among the most effective analgesics available and are the first choice in the treatment of acute severe pain. However, partial efficacy, a tendency to produce tolerance, and a host of ill-tolerated side effects make clinically available opioids less effective in the management of chronic pain syndromes. Given that most therapeutic opioids produce their actions via µ-opioid receptors (MOPrs), other targets are constantly being explored, among which δ-opioid receptors (DOPrs) are being increasingly considered as promising alternatives.

Practical guide for calculating and representing biased signaling by GPCR ligands: A stepwise approach.

Signaling bias makes reference to the capacity of G-protein coupled receptor (GPCR) ligands to direct pharmacological stimuli to a subset of effectors among all of those controlled by the receptor. This new signaling modality has added texture to the classical notion of efficacy. In doing so, it has opened new avenues for the development of therapeutic GPCR ligands that specifically modulate signals underlying desired effects while sparing those that support undesired drug actions.

Design and construction of conformational biosensors to monitor ion channel activation: A prototype FlAsH/BRET-approach to Kir3 channels.

Ion channels play a vital role in numerous physiological functions and drugs that target them are actively pursued for development of novel therapeutic agents. Here we report a means for monitoring in real time the conformational changes undergone by channel proteins upon exposure to pharmacological stimuli. The approach relies on tracking structural rearrangements by monitoring changes in bioluminescence energy transfer (BRET).

Kir3 channels undergo arrestin-dependant internalization following delta opioid receptor activation.

Kir3 channels control excitability in the nervous system and the heart. Their surface expression is strictly regulated, but mechanisms responsible for channel removal from the membrane remain incompletely understood. Using transfected cells, we show that Kir3.

Kir3 channel signaling complexes: focus on opioid receptor signaling.

Opioids are among the most effective drugs to treat severe pain. They produce their analgesic actions by specifically activating opioid receptors located along the pain perception pathway where they inhibit the flow of nociceptive information. This inhibition is partly accomplished by activation of hyperpolarizing G protein-coupled inwardly-rectifying potassium (GIRK or Kir3) channels.

Label-free monitoring of μ-opioid receptor-mediated signaling.

In this study, we used a combination of traditional signaling investigation approaches, bioluminescence resonance energy transfer (BRET) biosensors, and the label-free approach surface plasmon resonance (SPR) spectroscopy to monitor the signaling cascades of the μ-opioid receptor (MOP). In human embryonic kidney cells stably expressing a Flag-tagged version of human MOP, we compared the signals triggered by the noninternalizing and internalizing MOP agonists morphine and DAMGO (Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol), respectively. We studied three major and well described components of MOP signaling: receptor internalization, G protein coupling, and activation of extracellular signal-regulated kinase ERK1/ERK2.

Identifying ligand-specific signalling within biased responses: focus on δ opioid receptor ligands.

Unlabelled: Opioids activate GPCRs to produce powerful analgesic actions but at the same time induce side effects and generate tolerance, which restrict their clinical use. Reducing this undesired response profile has remained a major goal of opioid research and the notion of 'biased agonism' is raising increasing interest as a means of separating therapeutic responses from unwanted side effects. However, to fully exploit this opportunity, it is necessary to confidently identify biased signals and evaluate which type of bias may support analgesia and which may lead to undesired effects.

Endocytic profiles of δ-opioid receptor ligands determine the duration of rapid but not sustained cAMP responses.

Traditional assays that monitor cAMP inhibition by opioid receptor ligands require second-messenger accumulation over periods of 10-20 minutes. Since receptor regulation occurs within a similar time frame, such assays do not discriminate the actual signal from its modulation. Here we used bioluminescence resonance energy transfer to monitor inhibition of cAMP production by δ-opioid receptor (DOR) agonists in real time.

Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists.

Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells.

Conformational dynamics of Kir3.1/Kir3.2 channel activation via δ-opioid receptors.

This study assessed how conformational information encoded by ligand binding to δ-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels.