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1.
Mol Pharmacol ; 102(6): 259-268, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36153039

RESUMEN

The two main constituents of cannabis are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). While Δ9-THC pharmacology has been studied extensively, CBD-long considered inactive-is now the subject of vigorous research related to epilepsy, pain, and inflammation and is popularly embraced as a virtual cure-all. However, our understanding of CBD pharmacology remains limited, although CBD inhibits cannabinoid CB1 receptor signaling, likely as a negative allosteric modulator. Cannabis synthesizes (-)-CBD, but CBD can also exist as an enantiomer, (+)-CBD. We enantioselectively synthesized both CBD enantiomers using established conditions and describe here a new, practical, and reliable, NMR-based method for confirming the enantiomeric purity of two CBD enantiomers. We also investigated the pharmacology of (+)-CBD in autaptic hippocampal neurons, a well-characterized neuronal model of endogenous cannabinoid signaling, and in CHO-K1 cells. We report the inhibition constant for displacing CP55,940 at CB1 by (+)-CBD, is 5-fold lower than (-)-CBD. We find that (+)-CBD is ∼10 times more potent at inhibiting depolarization-induced suppression of excitation (DSE), a form of endogenous cannabinoid-mediated retrograde synaptic plasticity. (+)-CBD also inhibits CB1 suppression of cAMP accumulation but with less potency, indicating that the signaling profiles of the enantiomers differ in a pathway-specific manner. In addition, we report that (+)-CBD stereoselectively and potently activates the sphingosine-1 phosphate (S1P) receptors, S1P1 and S1P3 These results provide an attractive method for synthesizing and distinguishing enantiomers of CBD and related phytocannabinoids and provide further evidence that these enantiomers have their own unique and interesting signaling properties. SIGNIFICANCE STATEMENT: Cannabidiol (CBD) is the subject of considerable scientific and popular interest, but we know little of the enantiomers of CBD. We find that the enantiomer (+)-CBD is substantially more potent inhibitor of cannabinoid CB1 receptors and that it activates sphingosine-1-phosphate receptors in an enantiomer-specific manner; we have additionally developed an improved method for the synthesis of enantiomers of CBD and related compounds.


Asunto(s)
Cannabidiol , Cannabidiol/farmacología , Dronabinol/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Endocannabinoides , Transducción de Señal , Receptor Cannabinoide CB1 , Receptor Cannabinoide CB2
2.
Molecules ; 26(17)2021 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-34500785

RESUMEN

Cannabis contains more than 100 phytocannabinoids. Most of these remain poorly characterized, particularly in neurons. We tested a panel of five phytocannabinoids-cannabichromene (CBC), cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), and Δ9-tetrahydrocannabivarin (THCV) in two neuronal models, autaptic hippocampal neurons and dorsal root ganglion (DRG) neurons. Autaptic neurons expressed a form of CB1-dependent retrograde plasticity while DRGs expressed a variety of transient receptor potential (TRP) channels. CBC, CBDA, and CBDVA had little or no effect on neuronal cannabinoid signaling. CBDV and THCV differentially inhibited cannabinoid signaling. THCV inhibited CB1 receptors presynaptically while CBDV acted post-synaptically, perhaps by inhibiting 2-AG production. None of the compounds elicited a consistent DRG response. In summary, we find that two of five 'minor' phytocannabinoids tested antagonized CB1-based signaling in a neuronal model, but with very different mechanisms. Our findings highlight the diversity of potential actions of phytocannabinoids and the importance of fully evaluating these compounds in neuronal models.


Asunto(s)
Cannabinoides/farmacología , Modelos Biológicos , Neuronas/efectos de los fármacos , Fitoquímicos/farmacología , Animales , Cannabinoides/química , Células Cultivadas , Humanos , Ratones , Neuronas/metabolismo , Fitoquímicos/química
3.
J Neurosci ; 34(15): 5152-63, 2014 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-24719095

RESUMEN

For many G-protein-coupled receptors (GPCRs), including cannabinoid receptor 1 (CB1R), desensitization has been proposed as a principal mechanism driving initial tolerance to agonists. GPCR desensitization typically requires phosphorylation by a G-protein-coupled receptor kinase (GRK) and interaction of the phosphorylated receptor with an arrestin. In simple model systems, CB1R is desensitized by GRK phosphorylation at two serine residues (S426 and S430). However, the role of these serine residues in tolerance and dependence for cannabinoids in vivo was unclear. Therefore, we generated mice where S426 and S430 were mutated to nonphosphorylatable alanines (S426A/S430A). S426A/S430A mutant mice were more sensitive to acutely administered delta-9-tetrahydrocannabinol (Δ(9)-THC), have delayed tolerance to Δ(9)-THC, and showed increased dependence for Δ(9)-THC. S426A/S430A mutants also showed increased responses to elevated levels of endogenous cannabinoids. CB1R desensitization in the periaqueductal gray and spinal cord following 7 d of treatment with Δ(9)-THC was absent in S426A/S430A mutants. Δ(9)-THC-induced downregulation of CB1R in the spinal cord was also absent in S426A/S430A mutants. Cultured autaptic hippocampal neurons from S426A/S430A mice showed enhanced endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and reduced agonist-mediated desensitization of DSE. These results indicate that S426 and S430 play major roles in the acute response to, tolerance to, and dependence on cannabinoids. Additionally, S426A/S430A mice are a novel model for studying pathophysiological processes thought to involve excessive endocannabinoid signaling such as drug addiction and metabolic disease. These mice also validate the approach of mutating GRK phosphorylation sites involved in desensitization as a general means to confer exaggerated signaling to GPCRs in vivo.


Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Dronabinol/farmacología , Tolerancia a Medicamentos , Mutación Missense , Receptor Cannabinoide CB1/metabolismo , Secuencias de Aminoácidos , Animales , Sensibilización del Sistema Nervioso Central , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiología , Potenciales de la Membrana , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Sustancia Gris Periacueductal/efectos de los fármacos , Sustancia Gris Periacueductal/metabolismo , Sustancia Gris Periacueductal/fisiología , Fosforilación , Unión Proteica , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB1/genética , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/fisiología
4.
Mol Pain ; 10: 56, 2014 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-25189223

RESUMEN

BACKGROUND: Cisplatin, a platinum-derived chemotherapeutic agent, produces antineoplastic effects coupled with toxic neuropathic pain and impaired general health status. These side-effects complicate long term studies of neuropathy or analgesic interventions in animals. We recently demonstrated that pretreatment with sodium bicarbonate (4% NaHCO3) prior to cisplatin (3 mg/kg i.p. weekly up to 5 weeks) was associated with improved health status (i.e. normal weight gain, body temperature, creatinine and ketone levels, and kidney weight ratio) in rats (Neurosci Lett 544:41-46, 2013). To reduce the nephrotoxic effects of cisplatin treatment in mice, we compared effects of sodium bicarbonate (4% NaHCO3 s.c.), vitamin C (25 mg/kg s.c.), resveratrol (25 mg/kg s.c.) and saline (0.9% NaCl) pretreatment on cisplatin-induced changes in animal health status, neuropathic pain and proinflammatory cytokine levels in spinal cord and kidney. RESULTS: Cisplatin-treated mice receiving saline pretreatment exhibited elevated ketone, creatinine and kidney weight ratios, representative of nephrotoxicity. Vitamin C and sodium bicarbonate lowered creatinine/ketone levels and kidney weight ratio whereas resveratrol normalized creatinine levels and kidney weight ratios similar to saline pretreatment. All pretreatments were associated with decreased ketone levels compared to saline pretreatment. Cisplatin-induced neuropathy (i.e. mechanical and cold allodynia) developed equivalently in all pretreatment groups and was similarly reversed by either morphine (6 mg/kg i.p.) or ibuprofen (6 mg/kg i.p.) treatment. RT-PCR showed that mRNA levels for IL-1ß were increased in lumbar spinal cord of cisplatin-treated groups pretreated with either saline, NaHCO3 or resveratrol/cisplatin-treated groups. However, IL-6 and TNF-alpha were elevated in the kidneys in all cisplatin-treated groups. Our studies also demonstrate that 60 days after the last cisplatin treatment, body weight, body temperature, kidney functions and mRNA levels have returned to baseline although the neuropathic pain (mechanical and cold) is maintained. CONCLUSIONS: Studies employing cisplatin should include NaHCO3 or vitamin C pretreatment to improve animal health status and reduce nephrotoxicity (lower creatinine and kidney weight ratio) without affecting the development of chemotherapy-induced neuropathy or analgesic efficacy.


Asunto(s)
Ácido Ascórbico/administración & dosificación , Estado de Salud , Enfermedades del Sistema Nervioso Periférico/prevención & control , Bicarbonato de Sodio/administración & dosificación , Vitaminas/administración & dosificación , Animales , Antineoplásicos/toxicidad , Glucemia/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Cisplatino/toxicidad , Creatinina/sangre , Modelos Animales de Enfermedad , Esquema de Medicación , Hiperalgesia/inducido químicamente , Hiperalgesia/prevención & control , Cetonas/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Umbral del Dolor/efectos de los fármacos , Enfermedades del Sistema Nervioso Periférico/sangre , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico
5.
Mol Pharmacol ; 84(2): 296-302, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23748223

RESUMEN

Cannabinoids are part of an endogenous signaling system consisting of cannabinoid receptors and endogenous cannabinoids as well as the enzymatic machinery for their synthesis and degradation. Depolarization-induced suppression of excitation (DSE) is a form of cannabinoid CB(1) receptor-mediated inhibition of synaptic transmission that involves the production of the endogenous cannabinoid 2-arachidonoyl glycerol (2-AG). Both diacylglycerol lipase α (DAGLα) and DAGLß can produce 2-AG in vitro, but evidence from knockout animals argues strongly for a predominant, even exclusive, role for DAGLα in regulation of 2-AG-mediated synaptic plasticity. What role, if any, might be played by DAGLß remains largely unknown. Cultured autaptic hippocampal neurons exhibit robust DSE. With the ability to rapidly modulate expression of DAGLα and DAGLß in these neurons with short hairpin RNA, they are well suited for a comparative study of the roles of each isoform in mediating DSE. We find that RNA interference knockdown of DAGLα substantially reduces autaptic DSE, shifting the "depolarization-response curve" from an ED(50) value of 1.7 seconds to 3.0 seconds. Surprisingly, DAGLß knockdown diminishes DSE as much or more (ED(50) 6.4 seconds), suggesting that DAGLß is also responsible for a portion of 2-AG production in autaptic neurons. Similarly, the two DAGLs both contribute to the production of 2-AG via group I metabotropic glutamate receptors. Our results provide the first explicit evidence for a role of DAGLß in modulating neurotransmission.


Asunto(s)
Ácidos Araquidónicos/biosíntesis , Endocannabinoides/biosíntesis , Glicéridos/biosíntesis , Hipocampo/metabolismo , Lipoproteína Lipasa/metabolismo , Neuronas/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Células Cultivadas , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Células HEK293 , Humanos , Ratones , Receptores de Glutamato Metabotrópico/metabolismo , Transmisión Sináptica/fisiología
6.
Methods Mol Biol ; 2576: 385-393, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36152204

RESUMEN

Measuring protein levels of receptors and enzymes involved in endocannabinoid metabolism is an important step for understanding the distribution, function, and regulation of these components of the endocannabinoid system. A common approach for detecting proteins from complex biological systems is western blotting. In this chapter, we describe a general approach to western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, with due attention paid to the validation of the primary antibodies used, it can provide quantitative information on protein expression levels. Additional information can also be inferred from western blotting such as potential pre- and post-translational modifications (e.g., alternative splicing, phosphorylation, or glycosylation) that can be further evaluated by specific analytical techniques.


Asunto(s)
Cannabinoides , Endocannabinoides , Western Blotting , Colodión , Endocannabinoides/metabolismo , Dodecil Sulfato de Sodio
7.
Methods Mol Biol ; 2576: 395-406, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36152205

RESUMEN

Measuring the functional behavior of G protein-coupled receptors (GPCRs) has been a major focus of academic and pharmaceutical research for many decades. These efforts have led to the development of many assays to measure the downstream effects of ligand binding on receptor activity. In this chapter, we describe an internalization/recycling assay that can be used to track changes in receptor number at the plasma membrane. Used in concert with other assays, this antibody-based technique can provide dynamic information on GPCR activation by receptor-specific ligands.


Asunto(s)
Proteínas de Unión al GTP , Receptores Acoplados a Proteínas G , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Unión al GTP/metabolismo , Ligandos , Receptores Acoplados a Proteínas G/metabolismo
8.
Cannabis Cannabinoid Res ; 8(4): 612-622, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-35639364

RESUMEN

Introduction: Adolescence is an important phase in brain maturation, specifically it is a time during which weak synapses are pruned and neural pathways are strengthened. Adolescence is also a time of experimentation with drugs, including cannabis, which may have detrimental effects on the developing nervous system. The cannabinoid type 1 receptor (CB1) is an important modulator of neurotransmitter release and plays a central role in neural development. Neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB), are also critical during development for axon guidance and synapse specification. Objective: The objective of this study was to examine the effects of the phytocannabinoids, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on the expression of BDNF, its receptor TrkB, and other synaptic markers in the adolescent mouse hippocampus. Materials and Methods: Mice of both sexes were injected daily from P28 to P49 with 3 mg/kg THC, CBD, or a combination of THC/CBD. Brains were harvested on P50, and the dorsal and ventral hippocampi were analyzed for levels of BDNF, TrkB, and several synaptic markers using quantitative polymerase chain reaction, western blotting, and image analyses. Results: THC treatment statistically significantly reduced transcript levels of BDNF in adolescent female (BDNF I) and male (BDNF I, II, IV, VI, and IX) hippocampi. These changes were prevented when CBD was co-administered with THC. CBD by itself statistically significantly increased expression of some transcripts (BDNF II, VI, and IX for females, BDNF VI for males). No statistically significant changes were observed in protein expression for BDNF, TrkB, phospho-TrkB, phospho-CREB (cAMP response element-binding protein), and the synaptic markers, vesicular GABA transporter, vesicular glutamate transporter, synaptobrevin, and postsynaptic density protein 95. However, CB1 receptors were statistically significantly reduced in the ventral hippocampus with THC treatment. Conclusions: This study found changes in BDNF mRNA expression within the hippocampus of adolescent mice exposed to THC and CBD. THC represses transcript expression for some BDNF variants, and this effect is rescued when CBD is co-administered. These effects were seen in both males and females, but sex differences were observed in specific BDNF isoforms. While a statistically significant reduction in CB1 receptor protein in the ventral dentate gyrus was seen, no other changes in protein levels were observed.


Asunto(s)
Cannabidiol , Femenino , Masculino , Ratones , Animales , Cannabidiol/farmacología , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Dronabinol/farmacología , Tropomiosina/metabolismo , Tropomiosina/farmacología , Hipocampo
9.
iScience ; 26(6): 106948, 2023 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-37332596

RESUMEN

Cannabis use has become popular among athletes, many of whom are exposed to repetitive subconcussive head impacts. We aimed to test whether chronic cannabis use would be neuroprotective or exacerbating against acute subconcussive head impacts. This trial included 43 adult soccer players (Cannabis group using cannabis at least once a week for the past 6 months, n = 24; non-cannabis control group, n = 19). Twenty soccer headings, induced by our controlled heading model, significantly impaired ocular-motor function, but the degrees of impairments were less in the cannabis group compared to controls. The control group significantly increased its serum S100B level after heading, whereas no change was observed in the cannabis group. There was no group difference in serum neurofilament light levels at any time point. Our data suggest that chronic cannabis use may be associated with an enhancement of oculomotor functional resiliency and suppression of the neuroinflammatory response following 20 soccer headings.

10.
Cannabis Cannabinoid Res ; 8(5): 749-767, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37015060

RESUMEN

Introduction: How sex influences prefrontal cortexes (PFCs) synaptic development through adolescence remains unclear. Materials and Methods: In this study we describe sex-specific cellular and synaptic trajectories in the rat PFC from adolescence to adulthood. Results: The excitability of PFC layer 5 pyramidal neurons was lower in adult females compared with other developmental stages. The developmental course of endocannabinoid-mediated long-term depression (eCB-LTD) was sexually dimorphic, unlike long-term potentiation or mGluR3-LTD. eCB-LTD was expressed in juvenile females but appeared only at puberty in males. Endovanilloid TRPV1R or eCB receptors were engaged during LTD in a sequential and sexually dimorphic manner. Gene expression of the eCB/vanilloid systems was sequential and sex specific. LTD-incompetent juvenile males had elevated expression levels of the CB1R-interacting inhibitory protein cannabinoid receptor interacting protein 1a and of the 2-arachidonoylglycerol-degrading enzyme ABHD6. Pharmacological inhibition of ABHD6 or MAGL enabled LTD in young males, whereas inhibition of anandamide degradation was ineffective. Conclusions: These results reveal sex differences in the maturational trajectories of the rat PFC.


Asunto(s)
Endocannabinoides , Maduración Sexual , Ratas , Femenino , Animales , Masculino , Endocannabinoides/metabolismo , Plasticidad Neuronal/genética , Potenciación a Largo Plazo , Expresión Génica
11.
Eur J Med Chem ; 249: 115123, 2023 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-36708677

RESUMEN

The alkylindole (AI), WIN55212-2, modulates the activity of several proteins, including cannabinoid receptors 1 and 2 (CB1R, CB2R), and at least additional G protein-coupled receptor (GPCR) that remains uncharacterized with respect to its molecular identity and pharmacological profile. Evidence suggests that such AI-sensitive GPCRs are expressed by the human kidney cell line HEK293. We synthesized fourteen novel AI analogues and evaluated their activities at AI-sensitive GPCRs using [35S]GTPγS and [3H]WIN55212-2 binding in HEK293 cell membranes, and performed in silico pharmacophore modeling to identify characteristics that favor binding to AI-sensitive GPCRs versus CB1R/CB2R. Compounds 10 and 12 stimulated [35S]GTPγS binding (EC50s = 3.5 and 1.1 nM, respectively), and this response was pertussis toxin-sensitive, indicating that AI-sensitive GPCRs couple to Gi/o proteins. Five AI analogues reliably distinguished two binding sites that correspond to the high and low affinity state of AI-sensitive GPCRs coupled or not to G proteins. In silico pharmacophore modeling suggest 3 characteristics that favor binding to AI-sensitive GPCRs versus CB1R/CB2R: 1) an s-cis orientation of the two aromatic rings in AI analogues, 2) a narrow dihedral angle between the carbonyl group and the indole ring plane [i.e., O-C(carbonyl)-C3-C2] and 3) the presence of a carbonyl oxygen. The substituted alkylindoles reported here represent novel chemical tools to study AI-sensitive GPCRs.


Asunto(s)
Cannabinoides , Humanos , Cannabinoides/farmacología , Guanosina 5'-O-(3-Tiotrifosfato) , Células HEK293 , Receptores Acoplados a Proteínas G/metabolismo , Receptor Cannabinoide CB2 , Receptor Cannabinoide CB1 , Receptores de Cannabinoides/metabolismo
12.
Sci Rep ; 12(1): 14182, 2022 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-35986066

RESUMEN

Saliva serves multiple important functions within the body that we typically take for granted, such as helping prepare food for swallowing and defense against oral pathogens. Dry mouth is a primary symptom of SjÓ§gren's syndrome and is a side effect of many drug treatments. Cannabis users frequently report dry mouth, but the basis for this is still unknown. If the effects occur via the endogenous cannabinoid signaling system, then this may represent a novel mechanism for the regulation of salivation. We examined expression of cannabinoid CB1 receptors in submandibular salivary gland using immunohistochemistry and tested regulation of salivation by THC and cannabinoid-related ligands. We now report that CB1 receptors are expressed in the axons of cholinergic neurons innervating the submandibular gland. No staining is seen in submandibular gland epithelial cells (acinar and ductal), or myoepithelial cells (MECs). Treatment with THC (4 mg/kg, IP) or the cannabinoid receptor agonist CP55940 (0.5 mg/kg) reduced salivation in both male and female mice 1 h after treatment. CBD had no effect on its own but reversed the effect of THC in a concentration-dependent manner. Neither the CB1 receptor antagonist SR141716 (4 mg/kg) nor the CB2-selective agonist JWH133 (4 mg/kg) had an effect on salivation. We also found that fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide and related lipids, regulates salivation. Salivation was reduced in FAAH knockout mice as well as mice treated with the FAAH blocker URB597 (4 mg/kg). URB597 had no effect in CB1 knockout mice. FAAH protein is detected intracellularly in acinar but not ductal epithelial cells. In lipidomics experiments, we found that FAAH knockout mice chiefly had elevated levels of acylethanolamines, including anandamide, and reduced levels of acyglycines. Our results are consistent with a model wherein endocannabinoids activate CB1 receptors on cholinergic axons innervating the submandibular gland. THC likely acts by plugging into this system, activating CB1 receptors to reduce salivation, thus offering a mechanism underlying the dry mouth reported by cannabis users.


Asunto(s)
Cannabinoides , Xerostomía , Amidohidrolasas/metabolismo , Animales , Agonistas de Receptores de Cannabinoides/farmacología , Dronabinol/farmacología , Endocannabinoides/metabolismo , Femenino , Masculino , Ratones , Ratones Noqueados , Alcamidas Poliinsaturadas/metabolismo , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/genética , Receptores de Cannabinoides , Salivación
13.
Eur J Neurosci ; 32(5): 693-706, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21050275

RESUMEN

A role for endocannabinoid signaling in neuronal morphogenesis as the brain develops has recently been suggested. Here we used the developing somatosensory circuit as a model system to examine the role of endocannabinoid signaling in neural circuit formation. We first show that a deficiency in cannabinoid receptor type 1 (CB(1)R), but not G-protein-coupled receptor 55 (GPR55), leads to aberrant fasciculation and pathfinding in both corticothalamic and thalamocortical axons despite normal target recognition. Next, we localized CB(1)R expression to developing corticothalamic projections and found little if any expression in thalamocortical axons, using a newly established reporter mouse expressing GFP in thalamocortical projections. A similar thalamocortical projection phenotype was observed following removal of CB(1)R from cortical principal neurons, clearly demonstrating that CB(1)R in corticothalamic axons was required to instruct their complimentary connections, thalamocortical axons. When reciprocal thalamic and cortical connections meet, CB(1)R-containing corticothalamic axons are intimately associated with elongating thalamocortical projections containing DGLß, a 2-arachidonoyl glycerol (2-AG) synthesizing enzyme. Thus, 2-AG produced in thalamocortical axons and acting at CB(1)Rs on corticothalamic axons is likely to modulate axonal patterning. The presence of monoglyceride lipase, a 2-AG degrading enzyme, in both thalamocortical and corticothalamic tracts probably serves to restrict 2-AG availability. In summary, our study provides strong evidence that endocannabinoids are a modulator for the proposed 'handshake' interactions between corticothalamic and thalamocortical axons, especially for fasciculation. These findings are important in understanding the long-term consequences of alterations in CB(1)R activity during development, a potential etiology for the mental health disorders linked to prenatal cannabis use.


Asunto(s)
Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Morfogénesis/fisiología , Vías Nerviosas/crecimiento & desarrollo , Células Piramidales/crecimiento & desarrollo , Receptor Cannabinoide CB1/fisiología , Tálamo/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Moduladores de Receptores de Cannabinoides/metabolismo , Moduladores de Receptores de Cannabinoides/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis/genética , Vías Nerviosas/embriología , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Células Piramidales/embriología , Receptor Cannabinoide CB1/genética , Receptores de Cannabinoides/genética , Receptores de Cannabinoides/fisiología , Tálamo/embriología
14.
Anal Chem ; 82(1): 359-67, 2010 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-19968249

RESUMEN

The ability to analyze complex (macro) molecules is of fundamental importance for understanding chemical, physical, and biological processes. Complexity may arise from small differences in structure, large dynamic range, as well as a vast range in solubility or ionization, imposing daunting tasks in areas as different as lipidomics and proteomics. Here, we describe a rapid matrix application that permits the deposition of matrix-assisted laser desorption/ionization (MALDI) matrix solvent-free. This solvent-free one-step automatic matrix deposition is achieved through vigorous movements of beads pressing the matrix material through a metal mesh. The mesh (20 mum) produces homogeneous coverage of <12 microm crystals (DHB, CHCA matrixes) in 1 min, as determined by optical microscopy, permitting fast uniform coverage of analyte and possible high-spatial resolution surface analysis. Homogenous tissue coverage of <5 microm sized crystals is achieved using a 3 microm mesh. Solvent-free MALDI analysis on a time-of-flight (TOF) mass analyzer of mouse brain tissue homogenously covered with CHCA matrix subsequently provides a homogeneous response in ion signal intensity. Total solvent-free analysis (TSA) by mass spectrometry (MS) of tissue sections is carried out by applying the MALDI matrix solvent-free for subsequent ionization and gas phase separation for decongestion of complexity in the absence of any solvent using ion mobility spectrometry (IMS) followed by MS detection. Isobaric compositions were well-delineated using TSA by MS.


Asunto(s)
Encéfalo/anatomía & histología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Ratones , Ratones Endogámicos C57BL , Solventes
15.
J Vis Exp ; (158)2020 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-32391813

RESUMEN

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5-1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 °C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.


Asunto(s)
Encéfalo/citología , Criopreservación/métodos , Animales , Costos y Análisis de Costo , Criopreservación/economía , Masculino , Ratones , Ratas
16.
Invest Ophthalmol Vis Sci ; 61(10): 48, 2020 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-32852544

RESUMEN

Purpose: Aqueous deficiency dry eye (ADDE) is a chronic condition affecting millions, with symptoms ranging from a dry itchiness to blurred vision and accompanied by an increased risk of eye infections. ADDE typically arises from disorders of the lacrimal gland that produces tears necessary for eye lubrication. Cannabis users frequently report dry eye, but the basis for this is unknown. If the effects occur via the endogenous cannabinoid signaling system, then this may represent a novel mechanism for the regulation of tearing. Methods: We examined expression of cannabinoid CB1 receptors in the lacrimal gland using immunohistochemistry, Western blotting, and PCR and tested tetrahydrocannabinol (THC) regulation of tearing in wild-type and CB1-null mice. Results: We now report that CB1 receptors are expressed in the axons of cholinergic neurons innervating the lacrimal gland. Little if any staining is seen in lacrimal gland epithelial cells (acinar and ductal) or myoepithelial cells (MECs). Activation of CB1 receptors by THC or the cannabinoid agonist CP55940 reduces tearing in male mice. In female mice, THC has no effect, but CP55940 increases tearing. In both sexes, the effect of CP55940 is absent in CB1 knockout mice. CB1 mRNA and protein levels are approximately four- to fivefold higher in males than females. In male knockouts, THC increases tearing, suggesting that THC also acts through different receptors. Conclusions: Our results suggest a novel, albeit sex-dependent, physiologic basis for the dry eye symptoms experienced by cannabis users: activation of neuronal CB1 receptors in the lacrimal gland reduces tearing.


Asunto(s)
Dronabinol/metabolismo , Receptor Cannabinoide CB1/metabolismo , Lágrimas/fisiología , Animales , Western Blotting , Ciclohexanoles/farmacología , Dronabinol/antagonistas & inhibidores , Síndromes de Ojo Seco/metabolismo , Femenino , Aparato Lagrimal/metabolismo , Aparato Lagrimal/fisiología , Masculino , Ratones , Ratones Noqueados , Receptor Cannabinoide CB1/antagonistas & inhibidores , Factores Sexuales , Lágrimas/efectos de los fármacos , Lágrimas/metabolismo
17.
Biol Psychiatry ; 87(7): 666-677, 2020 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-31653479

RESUMEN

BACKGROUND: Cannabis usage is increasing with its widespread legalization. Cannabis use by mothers during lactation transfers active cannabinoids to the developing offspring during this critical period and alters postnatal neurodevelopment. A key neurodevelopmental landmark is the excitatory to inhibitory gamma-aminobutyric acid (GABA) switch caused by reciprocal changes in expression ratios of the K+/Cl- transporters potassium-chloride cotransporter 2 (KCC2) and sodium-potassium-chloride transporter (NKCC1). METHODS: Rat dams were treated with Δ9-tetrahydrocannabinol or a synthetic cannabinoid during the first 10 days of postnatal development, and experiments were then conducted in the offspring exposed to these drugs via lactation. The network influence of GABA transmission was analyzed using cell-attached recordings. KCC2 and NKCC1 levels were determined using Western blot and quantitative polymerase chain reaction analyses. Ultrasonic vocalization and homing behavioral experiments were carried out at relevant time points. RESULTS: Treating rat dams with cannabinoids during early lactation retards transcriptional upregulation and expression of KCC2, thereby delaying the GABA switch in pups of both sexes. This perturbed trajectory was corrected by the NKCC1 antagonist bumetanide and accompanied by alterations in ultrasonic vocalization without changes in homing behavior. Neurobehavioral deficits were prevented by CB1 receptor antagonism during maternal exposure, showing that the CB1 receptor underlies the cannabinoid-induced alterations. CONCLUSIONS: These results reveal how perinatal cannabinoid exposure retards an early milestone of development, delaying the trajectory of GABA's polarity transition and altering early-life communication.


Asunto(s)
Cannabinoides , Alucinógenos , Animales , Dronabinol , Femenino , Lactancia , Masculino , Embarazo , Ratas , Ácido gamma-Aminobutírico
18.
Mol Pharmacol ; 76(6): 1220-7, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19767452

RESUMEN

Depolarization-induced suppression of excitation (DSE) is a major form of cannabinoid-mediated short-term retrograde neuronal plasticity and is found in numerous brain regions. Autaptically cultured murine hippocampal neurons are an architecturally simple model for the study of cannabinoid signaling, including DSE. The transient nature of DSE--tens of seconds--is probably determined by the regulated hydrolysis of the endocannabinoid 2-arachidonoyl glycerol (2-AG). No less than five candidate enzymes have been considered to serve this role: fatty acid amide hydrolase (FAAH), cyclooxygenase-2 (COX-2), monoacylglycerol lipase (MGL), and alpha/beta-hydrolase domain (ABHD) 6 and 12. We previously found that FAAH and COX-2 do not have a role in determining the duration of autaptic DSE. In the current study, we found that two structurally distinct inhibitors of MGL [N-arachidonoyl maleimide and 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184)] prolong DSE in autaptic hippocampal neurons, whereas inhibition of ABHD6 by N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4'-(aminocarbonyl)[1,1'-biphenyl]-4-yl ester, carbamic acid (WWL70) had no effect. In addition, we developed antibodies against MGL and ABHD6 and determined their expression in autaptic cultures. MGL is chiefly expressed at presynaptic terminals, optimally positioned to break down 2-AG that has engaged presynaptic CB(1) receptors. ABHD6 is expressed in two distinct locations on autaptic islands, including a prominent localization in some dendrites. In summary, we provide strong pharmacological and anatomical evidence that MGL regulates DSE in autaptic hippocampal neurons and, taken together with other studies, emphasizes that endocannabinoid signaling is terminated in temporally diverse ways.


Asunto(s)
Moduladores de Receptores de Cannabinoides/farmacología , Endocannabinoides , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/efectos de los fármacos , Monoacilglicerol Lipasas/fisiología , Animales , Ácidos Araquidónicos/farmacología , Benzodioxoles/farmacología , Compuestos de Bifenilo/farmacología , Línea Celular , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Glicéridos/farmacología , Hipocampo/enzimología , Humanos , Ratones , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Piperidinas/farmacología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Pirazoles/farmacología , Rimonabant
19.
Elife ; 72018 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-30201092

RESUMEN

Cannabinoids can cross the placenta, thus may interfere with fetal endocannabinoid signaling during neurodevelopment, causing long-lasting deficits. Despite increasing reports of cannabis consumption during pregnancy, the protracted consequences of prenatal cannabinoid exposure (PCE) remain incompletely understood. Here, we report sex-specific differences in behavioral and neuronal deficits in the adult progeny of rat dams exposed to low doses of cannabinoids during gestation. In males, PCE reduced social interaction, ablated endocannabinoid long-term depression (LTD) and heightened excitability of prefrontal cortex pyramidal neurons, while females were spared. Group 1 mGluR and endocannabinoid signaling regulate emotional behavior and synaptic plasticity. Notably, sex-differences following PCE included levels of mGluR1/5 and TRPV1R mRNA. Finally, positive allosteric modulation of mGlu5 and enhancement of anandamide levels restored LTD and social interaction in PCE adult males. Together, these results highlight marked sexual differences in the effects of PCE and introduce strategies for reversing detrimental effects of PCE.


Asunto(s)
Cannabinoides/farmacología , Corteza Prefrontal/fisiopatología , Efectos Tardíos de la Exposición Prenatal/patología , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Caracteres Sexuales , Regulación Alostérica/efectos de los fármacos , Animales , Ansiedad/patología , Ácidos Araquidónicos/metabolismo , Conducta Animal , Endocannabinoides/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Plasticidad Neuronal/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/patología , Alcamidas Poliinsaturadas/metabolismo , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/patología , Embarazo , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Receptor Cannabinoide CB1/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Conducta Social , Aislamiento Social , Canales Catiónicos TRPV/metabolismo
20.
Cannabis Cannabinoid Res ; 2(1): 235-246, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29098186

RESUMEN

Introduction: The high prevalence of adolescent cannabis use, the association between this use and later psychiatric disease, and increased access to high-potency cannabis highlight the need for a better understanding of the long-term effects of adolescent cannabis use on cognitive and behavioral outcomes. Furthermore, increasing Δ9-tetrahydrocannabinol (THC) in high-potency cannabis is accompanied by a decrease in cannabidiol (CBD), thus an understanding of the interactions between CBD and THC in the neurodevelopmental effects of THC is also important. The current study examined the immediate and long-term behavioral consequences of THC, CBD, and their combination in a mouse model of adolescent cannabis use. Materials and Methods: Male CD1 mice received daily injections of THC (3 mg/kg), CBD (3 mg/kg), CBD+THC (3 mg/kg each), vehicle, or remained undisturbed in their home cage (no handling/injections), either during adolescence (postnatal day [PND] 28-48) or during early adulthood (PND 69-89). Animals were then evaluated with a battery of behavioral tests 1 day after drug treatment, and again after 42 drug-free days. The tests included the following: open field (day 1), novel object recognition (NOR; day 2), marble burying (day 3), elevated plus maze (EPM; day 4), and Nestlet shredding (day 5). Results: Chronic administration of THC during adolescence led to immediate and long-term impairments in object recognition/working memory, as measured by the NOR task. In contrast, adult administration of THC caused immediate, but not long term, impairment of object/working memory. Adolescent chronic exposure to THC increased repetitive and compulsive-like behaviors, as measured by the Nestlet shredding task. Chronic administration of THC, either during adolescence or during adulthood, led to a delayed increase in anxiety as measured by the EPM. All THC-induced behavioral abnormalities were prevented by the coadministration of CBD+THC, whereas CBD alone did not influence behavioral outcomes. Conclusion: These data suggest that chronic exposure to THC during adolescence leads to some of the behavioral abnormalities common in schizophrenia. Interestingly, CBD appeared to antagonize all THC-induced behavioral abnormalities. These findings support the hypothesis that adolescent THC use can impart long-term behavioral deficits; however, cotreatment with CBD prevents these deficits.

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