Neuromodulatory signalling is poised to serve as a neural mechanism for gain control, acting as a crucial tuning factor to influence neuronal activity by dynamically shaping excitatory and inhibitory fast neurotransmission. The endocannabinoid (eCB) signalling system, the most widely expressed neuromodulatory system in the mammalian brain, has been demonstrated to filter excitatory and inhibitory inputs through retrograde, presynaptic action in vitro and ex vivo1-9. However, whether eCBs exert retrograde gain control to ultimately facilitate motivated behaviours in freely moving mammals has not been established. Here, using a suite of in vivo physiological, imaging, genetic and machine learning-based approaches, we uncover a fundamental role for the dynamic release of eCBs in controlling behavioural engagement during reward seeking through a genetically and anatomically defined thalamostriatal circuit.

Cannabis-based therapies are widely used for chronic pain, yet their mechanisms and therapeutic windows remain incompletely defined. This review synthesizes preclinical and clinical evidence on how dose, route of administration, treatment duration, and chemical composition shape analgesic efficacy and adverse-effect liability for Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and select cannabis-derived terpenes. Across rodent pain models, acute THC reliably produces antinociception, but its therapeutic window is narrow because analgesic doses overlap with CB1 receptor-mediated side effects such as sedation, hypothermia, hyperphagia, and motor impairment. Repeated THC exposure leads to tolerance and dependence. In contrast, CBD shows limited acute efficacy in naïve and inflammatory models but demonstrates more consistent benefit with repeated dosing in neuropathic and chemotherapy-induced pain, often without cannabimimetic adverse effects. Terpenes such as linalool, β-caryophyllene, myrcene, limonene, α-terpineol, and α-bisabolol exhibit independent antinociceptive and anti-inflammatory properties and are thought to pharmacologically interact with cannabinoids in a dose-, ratio-, and route-dependent "entourage" effect that either enhance or constrain therapeutic benefit. This review also focuses on integrating machine learning-based behavioral phenotyping of rodents to refine cannabinoid analgesia preclinical research. Computer vision pose-estimation and unsupervised clustering approaches enable high-resolution quantification of spontaneous and evoked natural behaviors, allowing the analytical dissociation of true analgesia from sedation, ataxia, or reduced exploration. By coupling these behavioral pipelines with pharmacokinetic and circuit-level analyses, emerging frameworks will define therapeutic windows with greater precision and improve the translational relevance of cannabinoid-based pain therapeutics.

The search for transformative medicines has continuously uncovered select diseases associated with the disruption of the endocannabinoid (eCB) signaling system in the brain and emphasized the therapeutic value of small molecules that rescue this signaling system. In this issue of JCI, Wang et al. report that genetic disruption of PPP2R1A function in mouse forebrain, a preclinical mouse model of neurodevelopmental disorders, resulted in pronounced impairment of eCB signaling. Notably, small-molecule inhibitors of eCB inactivation rescued both eCB signaling and cognitive dysfunction in this model, providing a solid foundation to move such transformative therapeutic approaches based on targeting eCB signaling toward human clinical trial testing.

Endocannabinoids (eCBs) modulate the activity of proteins expressed at the plasma and intracellular membranes. Nothing is known about the dynamic changes in eCB levels in these subcellular compartments. We leveraged the eCB sensor, GRABeCB2.0, to establish the stimulus-induced increases in the eCB, 2-arachidonoyl glycerol (2-AG), at the plasma and intracellular membranes of undifferentiated Neuro2a cells in culture. Activating G protein-coupled B2 receptors with bradykinin increased 2-AG levels at both the plasma and intracellular membranes within ≈5 and ≈15 s, respectively. By contrast, the activation of G proteins by the small peptide mastoparan and the ensuing opening of plasma membrane calcium channels increased 2-AG levels in plasma membrane within ≈1-2 s and in intracellular membranes after ≈30 s. While both these stimuli-induced increases in 2-AG production involved canonical lipases, they required distinct sources of calcium. Thus, distinct stimuli differentially increase 2-AG levels at plasma and intracellular membranes via distinct molecular mechanisms.

While it is known that endocannabinoids (eCB) modulate multiple neuronal functions, the molecular mechanism governing their release and transport remains elusive. Here, we propose an "on-demand release" model, wherein the formation of microvesicles, a specific group of extracellular vesicles (EVs) containing the eCB, 2-arachidonoylglycerol (2-AG), is an important step. A coculture model system that combines a reporter cell line expressing the fluorescent eCB sensor, G protein-coupled receptor-based (GRAB)eCB2.0, and neuronal cells revealed that neurons release EVs containing 2-AG, but not anandamide, in a stimulus-dependent process regulated by protein kinase C, Diacylglycerol lipase, Adenosinediphosphate (ADP) ribosylation factor 6 (Arf6), and which was sensitive to inhibitors of eCB facilitated diffusion. A vesicle contained approximately 2,000 2-AG molecules. Accordingly, hippocampal eCB-mediated synaptic plasticity was modulated by Arf6 and transport inhibitors. The "on-demand release" model, supported by mathematical analysis, offers a cohesive framework for understanding eCB trafficking at the molecular level and suggests that microvesicles carrying signaling lipids in their membrane regulate neuronal functions in parallel to canonical synaptic vesicles.

Microtubule targeting agents (MTAs) disrupt the mitotic process and kill cancer cells by triggering apoptosis and autophagy. ST-401 is a mild inhibitor of microtubule (MT) assembly that does not disrupt mitosis and kills glioblastoma (GBM) cells in interphase by disrupting the molecular machinery involved in mitochondrial energy supply. ST-401 passes the blood brain barrier and exhibits significant in vivo antitumor activity in GBM mouse models. Here we sought to identify the small molecule chemical characteristics required to mildly inhibit MT assembly and kill GBM cells in interphase by disrupting energy supply as a foundation for drug development. The solved Cryo-EM structure of ST-403, the active enantiomer of ST-401, bound to the colchicine site of a,b-tubulin and ensuing computational analysis of its docking revealed its unique structural stabilization mode of a,b-tubulin. Molecular dynamic analysis of related compounds that bind the colchicine site with comparable mild inhibitory activities provided a model depicting the structural stabilization induced by mild inhibitors of MT. Accordingly, ST-403 and these mild inhibitors of MT assembly similarly kill GBM cells in culture and reduce their mitochondrial energy supply. These results outline the fundamental chemical determinants required for brain penetrant mild inhibitors of MT assembly to kill GBM cells by reducing mitochondrial energy supply. We then used an unbiased phospho-proteomics approach to identify signaling networks that are modulated by mild inhibitors of MT assembly and identified mitochondrial fission induced by Drp1 as a key mediator validated by its genetic knockout and overexpression. Accordingly, low Drp1 expression represents a potential prognostic biomarker of increased survival in GBM patients. Our study unravels a previously unappreciated molecular mechanism that links brain-penetrant mild inhibitors of MT assembly to the killing GBM cells in interphase by disrupting mitochondria energy supply.

How Δ9-tetrahydrocannabinol (THC) impairs natural behaviors in mice remains unknown. We developed a video-monitored behavioral platform with machine learning classifiers to unravel discrete changes in natural mouse behaviors. THC infusion into the medial prefrontal cortex (mPFC) disrupted walking kinematic features characteristic of impairment responses. THC predominantly increased mPFC GABAergic activity preceding walk initiation shifting the mPFC excitatory/inhibitory (E/I) balance. Pose-defined closed loop photo-stimulation of mPFC GABAergic neurons demonstrated that THC exacerbates selected parameters of motor impairment. Surprisingly, THC also induced a time locked, movement-induced, transient potentiation of mPFC endocannabinoid (eCB) release and ensuing CB1R-mediated synaptic inhibition. Here we establish that THC-modifies mPFC E/I balance to excitation via dynamic changes in eCB release which acts to induce behavioral impairment.

Cannabidiol (CBD) is increasingly used as a health supplement, though few clinical studies have demonstrated benefits. The primary objective of this study was to evaluate the effects of an oral CBD-terpene formulation on sleep physiology in individuals with insomnia. In this double-blind, placebo-controlled, randomized clinical trial, 125 individuals with insomnia received an oral administration of CBD (300 mg) and terpenes (1 mg each of linalool, myrcene, phytol, limonene, α-terpinene, α-terpineol, α-pinene, and β-caryophyllene) for ≥ 4 days/wk over 4 weeks using a crossover design. The study medication was devoid of Δ9-tetrahydrocannabinol. The primary outcome measure was the percentage of time participants spent in the combination of slow-wave sleep (SWS) and rapid eye movement (REM) sleep stages, as measured by a wrist-worn sleep-tracking device. This CBD-terpene regimen marginally increased the mean nightly percentage of time participants spent in SWS + REM sleep compared to the placebo (mean [standard error], 1.3 https://clinicaltrials.gov/study/NCT05233761 ; Identifier: NCT05233761. Wang M, Faust M, Abbott S, et al. Effects of a cannabidiol/terpene formulation on sleep in individuals with insomnia: a double-blind, placebo-controlled, randomized, crossover study. J Clin Sleep Med. 2025;21(1):69–80.

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells escape death in mitosis, exit mitosis and become malignant polyploid giant cancer cells (PGCC). Considering the low number of cancer cells undergoing mitosis in tumor tissues, killing them in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule (MT) assembly, preferentially kills cancer cells in interphase as opposed to mitosis, a cell death mechanism that avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces a transient integrated stress response, reduces energy metabolism, and promotes mitochondria fission. This cell response may underly death in interphase and avoid the development of PGCC. Considering that ST-401 is a brain-penetrant MTA, we validated these results in glioblastoma cell lines and found that ST-401 also reduces energy metabolism and promotes mitochondria fission in GBM sensitive lines. Thus, brain-penetrant mild inhibitors of MT assembly, such as ST-401, that induce death in interphase through a previously unanticipated antitumor mechanism represent a potentially transformative new class of therapeutics for the treatment of GBM.

Background and Purpose Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2‐arachidonoyl glycerol (2‐AG). While it is known that extracellular ATP stimulates 2‐AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real‐time changes in eCB levels with the genetically encoded sensor, GRAB eCB2.0 , can address this shortfall. Experimental Approach 2‐AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC‐MS, and GRAB eCB2.0 fluorescence changes were detected using live‐cell confocal microscopy and a 96‐well fluorescence plate reader. Key Results 2‐AG and AEA increased GRAB eCB2.0 fluorescence in N2a cells with EC 50 values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB 1 R) antagonist SR141617 and absent in cells expressing the mutant‐GRAB eCB2.0 . ATP increased only 2‐AG levels in N2a cells, as measured by LC‐MS, and induced a transient increase in the GRAB eCB2.0 signal within minutes primarily via activation of P2X 7 receptors (P2X 7 R). This response was dependent on diacylglycerol lipase β activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2‐AG hydrolysing enzyme, α/β‐hydrolase domain containing 6 (ABHD6). Conclusions and Implications Considering that P2X 7 R activation increases 2‐AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation.

Targeting the endocannabinoid (eCB) signaling system for pain relief is an important treatment option that is only now beginning to be mechanistically explored. In this review, we focus on two recently appreciated cannabinoid-based targeting strategies, treatments with cannabidiol (CBD) and α/β-hydrolase domain containing 6 (ABHD6) inhibitors, which have the exciting potential to produce pain relief through distinct mechanisms of action and without intoxication. We review evidence on plant-derived cannabinoids for pain, with an emphasis on CBD and its multiple molecular targets expressed in pain pathways. We also discuss the function of eCB signaling in regulating pain responses and the therapeutic promises of inhibitors targeting ABHD6, a 2-arachidonoylglycerol (2-AG)-hydrolyzing enzyme. Finally, we discuss how the novel cannabinoid biosensor GRABeCB2.0 may be leveraged to enable the discovery of targets modulated by cannabinoids at a circuit-specific level. SIGNIFICANCE STATEMENT Cannabis has been used by humans as an effective medicine for millennia, including for pain management. Recent evidence emphasizes the therapeutic potential of compounds that modulate endocannabinoid signaling. Specifically, cannabidiol and inhibitors of the enzyme ABHD6 represent promising strategies to achieve pain relief by modulating endocannabinoid signaling in pain pathways via distinct, nonintoxicating mechanisms of action.

No preclinical experimental approach enables the study of voluntary oral consumption of high-concentration Δ9-tetrahydrocannabinol (THC) and its intoxicating effects, mainly owing to the aversive response of rodents to THC that limits intake. Here, we developed a palatable THC formulation and an optimized access paradigm in mice to drive voluntary consumption. THC was formulated in chocolate gelatin (THC-E-gel). Adult male and female mice were allowed ad libitum access for 1 and 2 hr. Cannabimimetic responses (hypolocomotion, analgesia, and hypothermia) were measured following access. Levels of THC and its metabolites were measured in blood and brain tissue. Acute acoustic startle responses were measured to investigate THC-induced psychotomimetic behavior. When allowed access for 2 hr to THC-E-gel on the second day of a 3-day exposure paradigm, adult mice consumed up to ≈30 mg/kg over 2 hr, which resulted in robust cannabimimetic behavioral responses (hypolocomotion, analgesia, and hypothermia). Consumption of the same gelatin decreased on the following third day of exposure. Pharmacokinetic analysis shows that THC-E-gel consumption led to parallel accumulation of THC and its psychoactive metabolite, 11-OH-THC, in the brain, a profile that contrasts with the known rapid decline in brain 11-OH-THC levels following THC intraperitoneal (i.p.) injections. THC-E-gel consumption increased the acoustic startle response in males but not in females, demonstrating a sex-dependent effect of consumption. Thus, while voluntary consumption of THC-E-gel triggered equivalent cannabimimetic responses in male and female mice, it potentiated acoustic startle responses preferentially in males. We built a dose-prediction model that included cannabimimetic behavioral responses elicited by i.p. versus THC-E-gel to test the accuracy and generalizability of this experimental approach and found that it closely predicted the measured acoustic startle results in males and females. In summary, THC-E-gel offers a robust preclinical experimental approach to study cannabimimetic responses triggered by voluntary consumption in mice, including sex-dependent psychotomimetic responses.

Glioblastoma is universally fatal and characterized by frequent chromosomal copy number alterations harboring oncogenes and tumor suppressors. In this study, we analyzed exome-wide human glioblastoma copy number data and found that cytoband 6q27 is an independent poor prognostic marker in multiple data sets. We then combined CRISPR-Cas9 data, human spatial transcriptomic data, and human and mouse RNA sequencing data to nominate PDE10A as a potential haploinsufficient tumor suppressor in the 6q27 region. Mouse glioblastoma modeling using the RCAS/tv-a system confirmed that Pde10a suppression induced an aggressive glioma phenotype in vivo and resistance to temozolomide and radiation therapy in vitro. Cell culture analysis showed that decreased Pde10a expression led to increased PI3K/AKT signaling in a Pten-independent manner, a response blocked by selective PI3K inhibitors. Single-nucleus RNA sequencing from our mouse gliomas in vivo, in combination with cell culture validation, further showed that Pde10a suppression was associated with a proneural-to-mesenchymal transition that exhibited increased cell adhesion and decreased cell migration. Our results indicate that glioblastoma patients harboring PDE10A loss have worse outcomes and potentially increased sensitivity to PI3K inhibition.

Evidence suggests that inhibition of & alpha;/& beta; hydrolase-domain containing 6 (ABHD6) reduces seizures; however, the molecular mechanism of this therapeutic response remains unknown. We discovered that heterozygous expression of Abhd6 (Abhd6+/- ) significantly reduced the premature lethality of Scn1a+/- mouse pups, a genetic mouse model of Dravet Syndrome (DS). Both Abhd6+/- mutation and pharmacological inhibition of ABHD6 reduced the duration and incidence of thermally induced seizures in Scn1a+/- pups. Mechanistically, the in vivo anti-seizure response resulting from ABHD6 inhibition is mediated by potentiation of gamma-aminobutyric acid receptors Type-A (GABAAR). Brain slice electrophysiology showed that blocking ABHD6 potentiates extrasynaptic (tonic) GABAAR currents that reduce dentate granule cell excitatory output without affecting synaptic (phasic) GABAAR currents. Our results unravel an unexpected mechanistic link between ABHD6 activity and extrasynaptic GABAAR currents that controls hippocampal hyperexcitability in a genetic mouse model of DS. Brief summary: This study provides the first evidence for a mechanistic link between ABHD6 activity and the control of extrasynaptic GABAAR currents that controls hippocampal hyperexcitability in a genetic mouse model of Dravet Syndrome and can be targeted to dampened seizures.

The same T98G cell that is shown in Movie S1 imaged 10 min after the addition of 0.5 µM ST-11. Speed = 10Ã-.

Introduction The endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamine (AEA), are produced by separate enzymatic pathways, activate cannabinoid receptors with distinct pharmacology, and differentially regulate pathophysiological processes. The genetically encoded sensor, GRABeCB2.0, detects real-time changes in eCB levels in cells in culture and preclinical model systems; however, its activation by eCB analogues produced by cells and by phyto-cannabinoids remains uncharacterized, a current limitation when interpreting changes in its response. This information could provide additional utility for the tool in in vivo pharmacology studies of phyto-cannabinoid action. Methods GRABeCB2.0 was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements. Results 2-AG increased GRABeCB2.0 fluorescent signal (EC50 = 85 nM), and the cannabinoid 1 receptor (CB1R) antagonist, SR141617, decreased GRABeCB2.0 signal (SR1, IC50 = 3.3 nM), responses that mirror their known potencies at cannabinoid 1 receptors (CB1R). GRABeCB2.0 fluorescent signal also increased in response to AEA (EC50 = 815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (2-LG and 2-OG, EC50s = 1.5 and 1.0 μM, respectively), Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-THC (EC50s = 1.6 and 2.0 μM, respectively), and the artificial CB1R agonist, CP55,940 (CP, EC50 = 82 nM); however their potencies were less than what has been described at CB1R. Cannabidiol (CBD) did not affect basal GRABeCB2.0 fluorescent signal and yet reduced the 2-AG stimulated GRABeCB2.0 responses (IC50 = 8.8 nM). Conclusions 2-AG and SR1 modulate the GRABeCB2.0 fluorescent signal with EC50s that mirror their potencies at CB1R whereas AEA, eCB analogues, THC and CP increase GRABeCB2.0 fluorescent signal with EC50s significantly lower than their potencies at CB1R. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRABeCB2.0 retains the negative allosteric modulator (NAM) property of CBD at CB1R. This study describes the pharmacological profile of GRABeCB2.0 to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CB1R ligands.