Coordinated regulation of CB1 cannabinoid receptors and anandamide metabolism stabilize network activity during homeostatic scaling down

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 post-synaptic compartments that regulate synaptic transmission, plasticity, and neuronal excitability throughout much of the brain, by activating pre-synaptic cannabinoid receptor CB1. The eCB system is well situated to regulate neuronal network properties and coordinate pre- and post-synaptic activity. However, the role of the eCB system in homeostatic adaptations to neuronal hyperactivity is unknown. We show that in mature cultured rat cortical neurons, chronic bicuculline treatment, known to induce homeostatic scaling-down, induces a coordinated adaptation to enhance tonic eCB signaling. Hyper-excitation triggers down regulation of fatty acid amide hydrolase (FAAH), the lipase that degrades the eCB anandamide. Subsequently, we measured an accumulation of anandamide and related metabolites, and an upregulation of total and cell surface CB1. We show that bicuculline induced downregulation of surface AMPA-type glutamate receptors and upregulation of CB1 occur through independent mechanisms. Finally, using live-cell microscopy of neurons expressing an extracellular fluorescent glutamate reporter (iGluSnFR), we confirm that cortical neurons in vitro exhibit highly synchronized network activity, reminiscent of cortical up-states in vivo. Up-state like activity in mature cortical cultures requires CB1 signaling under both control conditions and following chronic bicuculline treatment. We propose that during the adaptation to chronic neuronal hyperexcitation, tonic eCB signaling is enhanced through coordinated changes in anandamide metabolism and cell-surface CB1 expression to maintain synchronous network activity.

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Altered network properties in C9ORF72 repeat expansion cortical neurons are due to synaptic dysfunction

Molecular Neurodegeneration ◽

10.1186/s13024-021-00433-8 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Emma M. Perkins ◽

Karen Burr ◽

Poulomi Banerjee ◽

Arpan R. Mehta ◽

Owen Dando ◽

...

Keyword(s):

Synaptic Plasticity ◽

Synaptic Vesicle ◽

Cortical Neurons ◽

Electrode Array ◽

Repeat Expansion ◽

Cortical Network ◽

Synaptic Function ◽

Network Activity ◽

Cortical Function ◽

Network Function

Abstract Background Physiological disturbances in cortical network excitability and plasticity are established and widespread in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those harbouring the C9ORF72 repeat expansion (C9ORF72RE) mutation – the most common genetic impairment causal to ALS and FTD. Noting that perturbations in cortical function are evidenced pre-symptomatically, and that the cortex is associated with widespread pathology, cortical dysfunction is thought to be an early driver of neurodegenerative disease progression. However, our understanding of how altered network function manifests at the cellular and molecular level is not clear. Methods To address this we have generated cortical neurons from patient-derived iPSCs harbouring C9ORF72RE mutations, as well as from their isogenic expansion-corrected controls. We have established a model of network activity in these neurons using multi-electrode array electrophysiology. We have then mechanistically examined the physiological processes underpinning network dysfunction using a combination of patch-clamp electrophysiology, immunocytochemistry, pharmacology and transcriptomic profiling. Results We find that C9ORF72RE causes elevated network burst activity, associated with enhanced synaptic input, yet lower burst duration, attributable to impaired pre-synaptic vesicle dynamics. We also show that the C9ORF72RE is associated with impaired synaptic plasticity. Moreover, RNA-seq analysis revealed dysregulated molecular pathways impacting on synaptic function. All molecular, cellular and network deficits are rescued by CRISPR/Cas9 correction of C9ORF72RE. Our study provides a mechanistic view of the early dysregulated processes that underpin cortical network dysfunction in ALS-FTD. Conclusion These findings suggest synaptic pathophysiology is widespread in ALS-FTD and has an early and fundamental role in driving altered network function that is thought to contribute to neurodegenerative processes in these patients. The overall importance is the identification of previously unidentified defects in pre and postsynaptic compartments affecting synaptic plasticity, synaptic vesicle stores, and network propagation, which directly impact upon cortical function.

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Modulation of Network Oscillatory Activity and GABAergic Synaptic Transmission by CB1 Cannabinoid Receptors in the Rat Medial Entorhinal Cortex

Neural Plasticity ◽

10.1155/2008/808564 ◽

2008 ◽

Vol 2008 ◽

pp. 1-12 ◽

Cited By ~ 11

Author(s):

Nicola H. Morgan ◽

Ian M. Stanford ◽

Gavin L. Woodhall

Keyword(s):

Synaptic Transmission ◽

Cannabinoid Receptors ◽

Brain Regions ◽

Synaptic Activity ◽

Network Activity ◽

Oscillatory Activity ◽

Gabaergic Neurotransmission ◽

Network Oscillations ◽

Gabaergic Synaptic Transmission

Cannabinoids modulate inhibitory GABAergic neurotransmission in many brain regions. Within the temporal lobe, cannabinoid receptors are highly expressed, and are located presynaptically at inhibitory terminals. Here, we have explored the role of type-1 cannabinoid receptors (CB1Rs) at the level of inhibitory synaptic currents and field-recorded network oscillations. We report that arachidonylcyclopropylamide (ACPA; 10 M), an agonist at CB1R, inhibits GABAergic synaptic transmission onto both superficial and deep medial entorhinal (mEC) neurones, but this has little effect on network oscillations in beta/gamma frequency bands. By contrast, the CB1R antagonist/inverse agonist LY320135 (500 nM), increased GABAergic synaptic activity and beta/gamma oscillatory activity in superficial mEC, was suppressed, whilst that in deep mEC was enhanced. These data indicate that cannabinoid-mediated effects on inhibitory synaptic activity may be constitutively active in vitro, and that modulation of CB1R activation using inverse agonists unmasks complex effects of CBR function on network activity.

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Endocannabinoid System as a Promising Therapeutic Target in Inflammatory Bowel Disease – A Systematic Review

Frontiers in Immunology ◽

10.3389/fimmu.2021.790803 ◽

2021 ◽

Vol 12 ◽

Author(s):

Szymon Hryhorowicz ◽

Marta Kaczmarek-Ryś ◽

Aleksandra Zielińska ◽

Rodney J. Scott ◽

Ryszard Słomski ◽

...

Keyword(s):

Inflammatory Bowel Disease ◽

Bowel Disease ◽

Cannabinoid Receptors ◽

Intestinal Inflammation ◽

Fatty Acid Amide Hydrolase ◽

Acid Amide ◽

Endocannabinoid System ◽

Unknown Etiology ◽

Bioactive Lipids ◽

Inflammatory Bowel

Inflammatory bowel disease (IBD) is a general term used to describe a group of chronic inflammatory conditions of the gastrointestinal tract of unknown etiology, including two primary forms: Crohn’s disease (CD) and ulcerative colitis (UC). The endocannabinoid system (ECS) plays an important role in modulating many physiological processes including intestinal homeostasis, modulation of gastrointestinal motility, visceral sensation, or immunomodulation of inflammation in IBD. It consists of cannabinoid receptors (CB1 and CB2), transporters for cellular uptake of endocannabinoid ligands, endogenous bioactive lipids (Anandamide and 2-arachidonoylglycerol), and the enzymes responsible for their synthesis and degradation (fatty acid amide hydrolase and monoacylglycerol lipase), the manipulation of which through agonists and antagonists of the system, shows a potential therapeutic role for ECS in inflammatory bowel disease. This review summarizes the role of ECS components on intestinal inflammation, suggesting the advantages of cannabinoid-based therapies in inflammatory bowel disease.

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Excitatory synaptic transmission and network activity are depressed following mechanical injury in cortical neurons

Journal of Neurophysiology ◽

10.1152/jn.00467.2010 ◽

2011 ◽

Vol 105 (5) ◽

pp. 2350-2363 ◽

Cited By ~ 42

Author(s):

Paulette B. Goforth ◽

Jianhua Ren ◽

Benjamin S. Schwartz ◽

Leslie S. Satin

Keyword(s):

Synaptic Transmission ◽

Cortical Neurons ◽

Mechanical Injury ◽

Synaptic Function ◽

Transient Increase ◽

Excitatory Synaptic Transmission ◽

Network Activity ◽

Free Calcium ◽

Post Injury

In vitro and in vivo traumatic brain injury (TBI) alter the function and expression of glutamate receptors, yet the combined effect of these alterations on cortical excitatory synaptic transmission is unclear. We examined the effect of in vitro mechanical injury on excitatory synaptic function in cultured cortical neurons by assaying synaptically driven intracellular free calcium ([Ca2+]i) oscillations in small neuronal networks as well as spontaneous and miniature excitatory postsynaptic currents (mEPSCs). We show that injury decreased the incidence and frequency of spontaneous neuronal [Ca2+]i oscillations for at least 2 days post-injury. The amplitude of the oscillations was reduced immediately and 2 days post-injury, although a transient rebound at 4 h post-injury was observed due to increased activity of N-methyl-d-aspartate (NMDARs) and calcium-permeable α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (CP-AMPARs). Increased CP-AMPAR function was abolished by the inhibition of protein synthesis. In parallel, mEPSC amplitude decreased immediately, 4 h, and 2 days post-injury, with a transient increase in the contribution of synaptic CP-AMPARs observed at 4 h post-injury. Decreased mEPSC amplitude was evident after injury, even if NMDARs and CP-AMPARs were blocked pharmacologically, suggesting the decrease reflected alterations in synaptic Glur2-containing, calcium-impermeable AMPARs. Despite the transient increase in CP-AMPAR activity that we observed, the overriding effect of mechanical injury was long-term depression of excitatory neurotransmission that would be expected to contribute to the cognitive deficits of TBI.

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DNA Methyltransferase 1 (DNMT1) Shapes Neuronal Activity of Human iPSC-Derived Glutamatergic Cortical Neurons

International Journal of Molecular Sciences ◽

10.3390/ijms22042034 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2034

Author(s):

Sarah Bachmann ◽

Jenice Linde ◽

Michael Bell ◽

Marc Spehr ◽

Hans Zempel ◽

...

Keyword(s):

Dna Methylation ◽

Neuronal Activity ◽

Cortical Neurons ◽

Dna Methyltransferase ◽

Synaptic Function ◽

Synaptic Activity ◽

Epigenetic Mechanism ◽

Human Neurons ◽

Excitatory Neurons ◽

Methyltransferase 1

Epigenetic mechanisms are emerging key players for the regulation of brain function, synaptic activity, and the formation of neuronal engrams in health and disease. As one important epigenetic mechanism of transcriptional control, DNA methylation was reported to distinctively modulate synaptic activity in excitatory and inhibitory cortical neurons in mice. Since DNA methylation signatures are responsive to neuronal activity, DNA methylation seems to contribute to the neuron’s capacity to adapt to and integrate changing activity patterns, being crucial for the plasticity and functionality of neuronal circuits. Since most studies addressing the role of DNA methylation in the regulation of synaptic function were conducted in mice or murine neurons, we here asked whether this functional implication applies to human neurons as well. To this end, we performed calcium imaging in human induced pluripotent stem cell (iPSC)-derived excitatory cortical neurons forming synaptic contacts and neuronal networks in vitro. Treatment with DNMT1 siRNA that diminishs the expression of the DNA (cytosine-5)-methyltransferase 1 (DNMT1) was conducted to investigate the functional relevance of DNMT1 as one of the main enzymes executing DNA methylations in the context of neuronal activity modulation. We observed a lowered proportion of actively firing neurons upon DNMT1-knockdown in these iPSC-derived excitatory neurons, pointing to a correlation of DNMT1-activity and synaptic transmission. Thus, our experiments suggest that DNMT1 decreases synaptic activity of human glutamatergic neurons and underline the relevance of epigenetic regulation of synaptic function also in human excitatory neurons.

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N-Docosahexaenoylethanolamide promotes development of hippocampal neurons

Biochemical Journal ◽

10.1042/bj20102118 ◽

2011 ◽

Vol 435 (2) ◽

pp. 327-336 ◽

Cited By ~ 74

Author(s):

Hee-Yong Kim ◽

Hyun-Seuk Moon ◽

Dehua Cao ◽

Jeongrim Lee ◽

Karl Kevala ◽

...

Keyword(s):

Protein Expression ◽

Hippocampal Neurons ◽

Memory Function ◽

Acid Amide ◽

Neurite Growth ◽

Synaptic Function ◽

Synaptic Activity ◽

Neuronal Cultures ◽

Synaptic Protein

DHA (docosahexaenoic acid, C22:6,n−3) has been shown to promote neurite growth and synaptogenesis in embryonic hippocampal neurons, supporting the importance of DHA known for hippocampus-related learning and memory function. In the present study, we demonstrate that DHA metabolism to DEA (N-docosahexaenoylethanolamide) is a significant mechanism for hippocampal neuronal development, contributing to synaptic function. We found that a fatty acid amide hydrolase inhibitor URB597 potentiates DHA-induced neurite growth, synaptogenesis and synaptic protein expression. Active metabolism of DHA to DEA was observed in embryonic day 18 hippocampal neuronal cultures, which was increased further by URB597. Synthetic DEA promoted hippocampal neurite growth and synaptogenesis at substantially lower concentrations in comparison with DHA. DEA-treated neurons increased the expression of synapsins and glutamate receptor subunits and exhibited enhanced glutamatergic synaptic activity, as was the case for DHA. The DEA level in mouse fetal hippocampi was altered according to the maternal dietary supply of n–3 fatty acids, suggesting that DEA formation is a relevant in vivo process responding to the DHA status. In conclusion, DHA metabolism to DEA is a significant biochemical mechanism for neurite growth, synaptogenesis and synaptic protein expression, leading to enhanced glutamatergic synaptic function. The novel DEA-dependent mechanism offers a new molecular insight into hippocampal neurodevelopment and function.

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Bidirectional neuron-glioma interactions: effects of glioma cells on synaptic activity and its impact on tumor growth

Neuro-Oncology ◽

10.1093/neuonc/noz167.000 ◽

2019 ◽

Vol 21 (Supplement_4) ◽

pp. iv1-iv1

Author(s):

Elena Tantillo ◽

Eleonora Vannini ◽

Chiara Cerri ◽

Chiara Maria Mazzanti ◽

Mario Costa ◽

...

Keyword(s):

Tumor Growth ◽

Neural Activity ◽

Visual Stimulation ◽

Glioma Cells ◽

Cortical Activity ◽

Synaptic Function ◽

Synaptic Activity ◽

Network Activity ◽

Functional Changes ◽

Glioma Progression

Abstract Gliomas grow in a neuronal environment, but little is known on the functional changes in peritumoral neurons during tumor development. Moreover, investigations on the role of neural activity in glioma progression have yielded contradictory results. Here, we monitored longitudinal changes in network activity by recording visual evoked potentials (VEP) and local field potentials (LFP) after transplant of GL261 glioma cells in mouse visual cortex. We detected a progressive deterioration of VEP amplitudes in glioma-bearing mice, accompanied by an increase in slow network oscillations. At the cellular level, the analysis of microdissected peritumoral neurons showed alterations in both pre- and post-synaptic markers. To investigate whether glioma-driven alterations in synaptic function may impact on tumor growth, we manipulated levels of afferent cortical activity in glioma-bearing mice. Specifically, we tested blockade of synaptic activity via botulinum neurotoxin A (BoNT/A), visual deprivation (dark rearing, DR) and visual stimulation (VS). Replicating cells were quantified via immunostaining for BrdU and Ki67. We found that manipulation of cortical activity bidirectionally regulated glioma proliferation. Silencing of cortical synapses with BoNT/A and DR increased tumor proliferation while daily VS had the opposite effect. These findings demonstrate reduced responsiveness of peritumoral neurons which may in turn stimulate tumor cell proliferation, thus triggering a vicious loop that exacerbates glioma progression. Physiological stimulation of neural activity appears to restrain glioma proliferation so it could be implemented in the clinical setting as an adjuvant therapy.

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Low Basal CB2R in Dopamine Neurons and Microglia Influences Cannabinoid Tetrad Effects

International Journal of Molecular Sciences ◽

10.3390/ijms21249763 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9763

Author(s):

Qing-Rong Liu ◽

Ana Canseco-Alba ◽

Ying Liang ◽

Hiroki Ishiguro ◽

Emmanuel S. Onaivi

Keyword(s):

Cortical Neurons ◽

Cannabinoid Receptors ◽

Dynamic Range ◽

Conditional Knockout ◽

Dopamine Neurons ◽

In Vivo Studies ◽

Mouse Strains ◽

Genotypic Differences ◽

Mrna Levels ◽

Selective Agonist

There are two well-characterized cannabinoid receptors (CB1R and CB2R and other candidates): the central nervous system (CNS) enriched CB1R and peripheral tissue enriched CB2R with a wide dynamic range of expression levels in different cell types of human tissues. Hepatocytes and neurons express low baseline CB1R and CB2R, respectively, and their cell-type-specific functions are not well defined. Here we report inducible expression of CB1R in the liver by high-fat and high sugar diet and CB2R in cortical neurons by methamphetamine. While there is less controversy about hepatocyte CB1R, the presence of functional neuronal CB2R is still debated to date. We found that neuron CB2R basal expression was higher than that of hepatocyte CB1R by measuring mRNA levels of specific isoform CB2A in neurons isolated by fluorescence-activated cell sorting (FACS) and CB1A in hepatocytes isolated by collagenase perfusion of liver. For in vivo studies, we generated hepatocyte, dopaminergic neuron, and microglia-specific conditional knockout mice (Abl-Cnr1Δ, Dat-Cnr2Δ, and Cx3cr1-Cnr2Δ) of CB1R and CB2R by crossing Cnr1f/f and Cnr2f/f strains to Abl-Cre, Dat-Cre, and Cx3cr1-Cre deleter mouse strains, respectively. Our data reveals that neuron and microglia CB2Rs are involved in the “tetrad” effects of the mixed agonist WIN 55212-2, CB1R selective agonist arachidonyl-2′-chloroethylamide (ACEA), and CB2R selective agonist JWH133. Dat-Cnr2Δ and Cx3cr1-Cnr2Δ mice showed genotypic differences in hypomobility, hypothermia, analgesia, and catalepsy induced by the synthetic cannabinoids. Alcohol conditioned place preference was abolished in DAT-Cnr2Δ mice and remained intact in Cx3cr1-Cnr2Δ mice in comparison to WT mice. These Cre-loxP recombinant mouse lines provide unique approaches in cannabinoid research for dissecting the complex endocannabinoid system that is implicated in many chronic disorders.

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