an-vitro-preparation-isolated-enteric-neurons-glia-from-myenteric

An <em>In-vitro</em> Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Virginia Commonwealth University

Repeated administration of Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa, induces profound tolerance that correlates with desensitization and downregulation of CB(1) cannabinoid receptors in the CNS. However, the consequences of repeated administration of the endocannabinoid N-arachidonoyl ethanolamine (anandamide, AEA) on cannabinoid receptor regulation are unclear because of its rapid metabolism by fatty acid amide hydrolase (FAAH). FAAH(-/-) mice dosed subchronically with equi-active maximally effective doses of AEA or THC displayed greater rightward shifts in THC dose-effect curves for antinociception, catalepsy, and hypothermia than in AEA dose-effect curves. Subchronic THC significantly attenuated agonist-stimulated [(35)S]GTP gamma S binding in brain and spinal cord, and reduced [(3)H]WIN55,212-2 binding in brain. Interestingly, AEA-treated FAAH(-/-) mice showed less CB(1) receptor downregulation and desensitization than THC-treated mice. Experiments examining tolerance and cross-tolerance indicated that the behavioral effects of THC, a low efficacy CB(1) receptor agonist, were more sensitive to receptor loss than those of AEA, a higher efficacy agonist, suggesting that the expression of tolerance was more affected by the intrinsic activity of the ligand at testing than during subchronic treatment. In addition, the CB(1) receptor antagonist, rimonabant, precipitated a markedly reduced magnitude of withdrawal in FAAH(-/-) mice treated subchronically with AEA compared with mice treated repeatedly with THC. The findings that repeated AEA administration produces lesser adaptive changes at the CB(1) receptor and has reduced dependence liability compared with THC suggest that pharmacotherapies targeting endocannabinoid catabolic enzymes are less likely to promote tolerance and dependence than direct acting CB(1) receptor agonists.

FAAH-/- mice display differential tolerance, dependence, and cannabinoid receptor adaptation after delta 9-tetrahydrocannabinol and anandamide administration.

The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G-protein-coupled receptors (GPCRs), including CB(1) and CB(2) receptors. The CB(1) receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB(1) receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G-proteins. These proteins, which include the GPCR kinases, beta-arrestins, GPCR-associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor-interacting proteins: CRIP(1a/b), are thought to play important roles in the regulation of intracellular trafficking, desensitization, down-regulation, signal transduction and constitutive activity of CB(1) receptors. This review examines CB(1) receptor-interacting proteins, including heterotrimeric G-proteins, but with particular emphasis on non-G-protein entities, that might comprise the CB(1) receptosomal complex. The evidence for direct interaction with CB(1) receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery.

Cannabinoid CB1 receptor-interacting proteins: novel targets for central nervous system drug discovery?

Î²-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of Î²-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 Î¼M) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC(50) values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 Â± 0.08 (n = 9) to 5.45 Â± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from Î²-arrestin2 knockout mice. Î²-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of Î²-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 Î¼M) exposure. Naloxone prevented the decrease in Î²-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in Î²-arrestin2 occurred. We conclude that a decrease in Î²-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

The role of Î²-arrestin2 in the mechanism of morphine tolerance in the mouse and guinea pig gastrointestinal tract.

Gastrointestinal peristalsis is significantly dependent on the enteric nervous system. Constipation due to reduced peristalsis is a major side-effect of morphine, which limits the chronic usefulness of this excellent pain reliever in man. The ionic basis for the inhibition of enteric neuron excitability by morphine is not well characterized as previous studies have mainly utilized microelectrode recordings from whole mount myenteric plexus preparations in guinea pigs. Here we have developed a Swiss-Webster mouse myenteric neuron culture and examined their electrophysiological properties by patch-clamp techniques and determined the mechanism for morphine-induced decrease in neuronal excitability. Isolated neurons in culture were confirmed by immunostaining with pan-neuronal marker, Î²-III tubulin and two populations were identified by calbindin and calretinin staining. Distinct neuronal populations were further identified based on the presence and absence of an afterhyperpolarization (AHP). Cells with AHP expressed greater density of sodium currents. Morphine (3 ÂµM) significantly reduced the amplitude of the action potential, increased the threshold for spike generation but did not alter the resting membrane potential. The decrease in excitability resulted from inhibition of sodium currents. In the presence of morphine, the steady-state voltage dependence of Na channels was shifted to the left with almost 50% of channels unavailable for activation from hyperpolarized potentials. During prolonged exposure to morphine (two hours), action potentials recovered, indicative of the development of tolerance in single enteric neurons. These results demonstrate the feasibility of isolating mouse myenteric neurons and establish sodium channel inhibition as a mechanism for morphine-induced decrease in neuronal excitability.

Article	Year
FAAH-/- mice display differential tolerance, dependence, and cannabinoid receptor adaptation after delta 9-tetrahydrocannabinol and anandamide administration. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology\| PubMed ID: 20357755	2010
Cannabinoid CB1 receptor-interacting proteins: novel targets for central nervous system drug discovery? British journal of pharmacology\| PubMed ID: 20590557	2010
The role of Î²-arrestin2 in the mechanism of morphine tolerance in the mouse and guinea pig gastrointestinal tract. The Journal of pharmacology and experimental therapeutics\| PubMed ID: 22129596	2012
Morphine decreases enteric neuron excitability via inhibition of sodium channels. PloS one\| PubMed ID: 23028881	2012

Tricia H. Smith

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