Jean Lud Cadet Karen Bolla and Ronald I Herning
This chapter summarizes the neurological approaches used to assess the potential long-term effects of drugs on the nervous system of drug abusers. These include the use of neuropsycho-logical assessments, transcranial Doppler TCD sonography, and electroencephalographic EEG recordings. Neuropsychological procedures are used in an effort to provide an unbiased estimate of the individual's cognitive capacity, and included tests of language skills, attention, memory, and motor skills. TCD allows...
Splenocyte and TCell Proliferation Assay
1. BrdU cell proliferation ELISA kit from Roche Molecular Biochemicals Indianapolis, IN a. BrdU labeling reagent 1000X 10 mM 5'bromo-2'-deoxyuridine in PBS, pH 7.4 provided in kit . b. FixDenaturant ready to use provided in kit . c. Anti-BrdU-POD lyophilisate, stabilized. Monoclonal antibody from mouse-mouse hybrid cells clone BMG 6H8, Fab fragments conjugated with peroxi-dase POD provided in kit . d. Antibody dilution solution ready to use provided in kit . e. Washing buffer 10X in PBS...
Patricia Tagliaferro Alberto J Ramos Emmanuel S Onaivi Sergio G Evrard Maite
One of the major goals for the use of digital image analysis systems in neuroanatomy is to visualize structures, cells, or other tissue components in order to compare various populations. In addition, digital image analysis allows semi-quantification of cell labeling because it is capable of measuring simultaneously the staining intensity, location, size, and shape of labeled profiles. In the present work, the morphological changes in the CB1 hippocampal area and corpus striatum induced by...
In Vitro Studies on Isolated Gastrointestinal Muscle Preparations
Contractions of the intestine are affected by the activities of two layers of smooth muscle cells an outer layer with the long axis of the cells arranged longitudinally and an inner layer with the long axis of the cells arranged circularly. The study of inhibitory effects of cannabinoids on the contraction of isolated gastrointestinal tissue began over three decades ago in the laboratory of Paton 31 . These experiments involved the utilization of guinea pig ileum in which contractions were...
Acknowledgments Ybv
Supported by grants from National Institutes on Drug Abuse DA9158, DA03801, and DA07215 . Scheme 17. Synthesis of tail-modified analog 73 95 . Reagents and conditions a I2, PPh3, imidazole, Et2O CH3CN, 0 to 23 C, 1 h, 98 b PPh3, CH3CN, reflux, 18 h, 90 c NaHMDS, THF HMPA, 80, -78 to 23 C, 2 h, 61 d LiOH, MeOH H2O, 23 C, 18 h, 94 e oxalyl chloride, CH2Cl2, 0 C, 2 h, 100 f 2-ethanolamine, CH2Cl2, 0 to 23 C. Methyl iodide 79 . To a stirred solution of triphenylphosphine 456 mg, 1.74 mmol and...
Drug Discrimination
1. Rodents are trained to discriminate cannabinoids from drugs of other classes, and they will generalize responses to other cannabinoids. 2. In the drug-discrimination paradigm see ref. 19 and Note 3 , laboratory animals learn to recognize the presence of a certain drug such as nicotine, morphine, LSD, A9-THC and express the discrimination between the drug under investigation and a control substance placebo in a two-choice situation, where the correct choice is rewarded.
Aminoalkylindoles
The fourth chemical class of cannabinergic compounds, the aminoalkylindoles AAIs , were initially developed at Sterling Winthrop as potential Fig. 5. Representative aminoalkylindoles. Fig. 5. Representative aminoalkylindoles. nonulcerogenic analogs of nonsteroidal anti-inflammatory drugs NSAIDs 64 and bear no structural relationship to the cannabinoids e.g., 48,49, Fig. 5 . These analogs also exhibited antinociceptive properties that eventually were attributed to their interactions with the...
References
1. Matsuda, L. A., Lolait, T. I., Brownstein, M. J., Young, A. C., and Bonner, T. I. 1990 Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346, 561-564. 2. Chakrabarti, A., Onaivi, E. S., and Chaudhuri, G. 1995 Cloning and sequencing of a cDNA encoding the mouse brain-type cannabinoid receptor protein. DNA Sequence 5, 385-388. 3. Munro, S., Thomas, K. L., and Abu-Shaar, M. 1993 Molecular characterization of a peripheral cannabinoid receptor. Nature 365,...
CB2R Knockout Mouse
To investigate the role of the CB2R in immunomodulation, we created a mouse deficient for the CB2R using homologous recombination CB2R knockout or CB2R- - mouse 31 . The CB2R- - gene is encoded by a single exon. Upon homologous recombination, 341 bp from the coding region's 3' end was replaced by the neomycin gene. This mouse model as well as cell lines derived from these mice have been successfully used to answer questions regarding the role of the CB2R- - in the immune system 32 . In...
Reward and Reinforcement
3.3.1. Intracranial Self-Stimulation Self-Delivered Electrical Stimulation of CNS Reward Circuits 1. Electrodes are placed in the reward pathway at the level of the medial forebrain bundle of the animal's most frequently rat brain see Note 4 . 2. Thus, when microelectrodes were implanted in the medial forebrain bundle, a low dose of A9-THC 1.5 mg kg ip decreased the electrophysiological threshold for the delivery of intracranial self-stimulation ICSS 40,41 . 3. The rat is placed in an operant...
In Vitro Peristalsis Studies in Isolated Intestine
Peristalsis is a coordinated motor process that allows the intestine to propel its content toward the anus. It consists of a preparatory phase in which longitudinal muscles contract in response to effluent infusion and a subsequent emptying phase in which coordinated contractions of circular muscles expel the effluent from the stomach to the anal end. Peristalsis is induced in 5- to 10-cm isolated segments of guinea pig ileum by either a constant fluid perfusion 41,42 or electrical field...
Methods for Double and SingleLabel In Situ Hybridization Andrea G Hohmann
This chapter presents a reliable, detailed method for performing double-label in situ hybridization ISH that has been validated for use in studies identifying the co-localization of cannabinoid CB1 receptor mRNA with other distinct species of mRNAs.This method permits simultaneous detection of two different species of mRNA within the same tissue section. Doublelabel ISH may be accomplished by hybridizing tissue sections with a combination of radiolabeled and digoxigenin-labeled RNA probes that...
Appetite and Food Intake
3.6.1. Human Studies see Note 10 1. In open pilot studies, dronabinol A9-THC treatment caused weight gain in the majority of subjects 58 . 2. A relatively low dose of dronabinol, 2.5 mg twice daily, enhanced appetite and stabilized body weight in acquired immunodeficiency syndrome AIDS patients suffering from anorexia 59 for at least 7 mo, although in another study, dronabinol was not effective 60 . 3. When dronabinol was administered to healthy volunteers, an increase in caloric intake was...
Immunoblotting of Cannabinoid Receptors
3.5.1. Protein Extraction From Blood or Tissue Samples 1. Blood or tissue samples are obtained for CBr protein determinations from the subjects. Fig. 4. 3H Anandamide uptake by CHO cells expressing hDAT black bar and two unidentified transporters P16, slashed bar and G25, hatched bar . Uptake was carried out for 5 min at 37 C in KRH buffer see Subheading 3. . These CHO cells are stable cell lines. Data are mean SEM from three experiments. Fig. 4. 3H Anandamide uptake by CHO cells expressing...
Cytokine Assays
2.3.1. Splenocyte Cytokine Assay 1. Bio-Plex cytokine assay kit Bio-Rad Laboratories, Hercules, CA . a. Anti-cytokine conjugated beads 25X , 2.5 x 106 beads mL per cytokine provided in kit . b. Cytokine detection antibody 8-plex 50X 0.05 mg mL per cytokine provided in kit . c. Cytokine standard lyophilized , 25 ng per cytokine provided in kit . d. Bio-Plex assay buffer A provided in kit . e. Bio-Plex wash buffer A provided in kit . f. Bio-Plex detection antibody diluent A provided in kit . g....
Nonclassical Cannabinoids
A second class of cannabinergic ligands possessing close similarity with CCs was developed at Pfizer in an effort to simplify the CC structure, while maintaining or improving biological activity 52-56 . This group of compounds is generally designated as nonclassical cannabinoids and includes AC-bicyclic e.g., 26 and 27, Fig. 2 and ACD-tricyclic e.g., 28, Fig. 2 ligands lacking the pyran Bring of CCs. NCCs share some of the key pharmacophores of the CCs, namely, the phenolic hydroxyl, the side...
Index
2-AG, see 2-Arachidonoylglycerol AM251, synthesis, 136 AM281, synthesis, 135-137 AM708, synthesis, 121 AM1241, synthesis, 134, 135 Aminoalkylindoles, cannabinoid analog synthesis, 138-142 cannabinoid receptor subtype affinity, 138 COS cell uptake assay, 12, 16 gastrointestinal effects, 174, 180 metabolism, see Fatty acid amide hydrolase structure-activity relationship studies, 138 synaptosome transporter, assay, materials, 164, 165, 167 synaptosome preparation, 166 uptake assay, 166, 167 brain...
Assessing Cannabis Craving in a Treatment Setting
In a treatment or clinic setting, patients are typically assessed with numerous forms, questionnaires, and interviews. To reduce burden on patients, we suggest using the 12-item MCQ rather than the full version. 3.2.1. Training on Completion of MCQ As discussed above Subheading 3.1.3. , patients should be given instructions before completing the MCQ see Note 2 . The use of the MCQ in a treatment setting may be more flexible than in a research setting. A patient's level of craving may be...
Phagocytosis Assay
1. Red-dyed polystyrene latex particles 0.8 im Bangs Laboratories, Fishers, IN 2.5 solids in 5 heat-inactivated FCS and incubated at 37 C for 30 min immediately prior to use. Incubation with FCS allows optimal opsonization. 2. Fluorescent microspheres Yellow-green Fluoresbrite polystyrene latex particles 1 im Polyscience, Inc., Warrington, PA 1 solids in 5 heat-inactivated FCS and incubated at 37 C for 30 min immediately prior to use. Incubation with FCS allows optimal opsonization. 1. Bacteria...
Stephen J Heishman and Edward G Singleton
Cannabis is the most widely used illicit drug in the United States, with 14.6 million current users. Cannabis-dependent individuals presenting for treatment typically report cannabis craving however, the phenomenon has received little research attention. In the absence of a valid, multidimensional questionnaire to assess cannabis craving, we developed the Marijuana Craving Questionnaire MCQ . The MCQ consists of four constructs or factors that characterize cannabis craving compulsivity,...
References Uex
1. Fride, E. and Shohami, E. 2002 The endocannabinoid system function in survival of the embryo, the newborn and the neuron Neuroreport 13, 1833-1841. 2. Mechoulam, R., Hanus, L, and. Fride, E. 1998 Towards cannabinoid drugs revisited. Prog. Med. Chem. 35, 199-243. 3. Treit, D. and Menard, J. 1998 Animal models of anxiety and depression, in In Vivo Neuromethods Boulton, E., Baker, G., and Bateson, A., eds. , Humana Press, Totowa, NJ. 4. Martin, B. R., Compton, D. R., Thomas, B. F., et al. 1991...
Info Jbh
Fig. 1. Dose-response curves of anandamide in the mouse tetrad comparison with A8-THC. Performance of Sabra mice in the tetrad test for cannabinoid activity. Female Sabra mice were injected ip with anandamide 0.01-100 mg kg or with A8-THC 50 mg kg and were tested in the tetrad for cannabinoid-induced effects after 15 or 30 min, respectively. Mice were tested for ambulation A and rearing not shown in an open field catalepsy on a ring B rectal temperature C and analgesia on a hot plate D . n 5-8....
Riboprobe Hybridization
For double-label ISH, slide-mounted sections are hybridized with a cocktail of 35S -labeled and digoxigenin-labeled riboprobes. We have found the greatest sensitivity without additional steps for amplification when the 35S -labeled probe is used to label CB1 mRNA and the digoxigenin-labeled probe is used to label typically more abundant peptide mRNAs of interest e.g., prepro-tachykinin A, prodynorphin, and proenkephalin mRNA . In adjacent sections, the correspondence of the 35S -labeled...
Radiochromatographic Assay of FAAH Activity
This radiochromatographic method, joining the resolution and reproducibility of HPLC with the sensitivity of scintillation counting, allows the detection of FAAH at the femtomolar level in specimens available not only in large amounts, but also in small quantities, such as brain areas or primary cells in culture. The method involves the procedure for both extraction of the proteins from samples and enzyme assay, performed by measuring the release of 3H arachi-donic acid from 3H anandamide....
References Ycl
1. Block, R. I. and Wittenborn, J. R. 1984 Marijuana effects on semantic memory Verification of common and uncommon category members. Psychol. Rep. 55, 503-512. 2. Block, R. I. and Wittenborn, J. R. 1984 Marijuana effects on visual imagery in a paired-associate task. Percept. Mot. Skills 58, 759-766. 3. Block, R. I. and Wittenborn, J. R. 1985 Marijuana effects on associative processes. Psychopharmacology 85, 426-430. 4. Block, R. I. and Wittenborn, J. R. 1986 Marijuana effects on the speed of...
Cannabinoid CB1 Receptor Radioligand Binding
Cannabinoid receptor ligands are generally highly lipophilic, and the concentrations that can be used are limited by this fact see also Notes 2 and 3 . A sample saturation assay of the commonly used CB1-selective antagonist SR141716A is illustrated in Fig. 1. The figure shows how concentrations of this ligand above 5 nM result in more nonspecific binding than specific binding, and thus rarely yield usable results. This is true even in rat cortex membranes where CB1 is expressed at very high...
Splenocyte and TCell Proliferation Assays
1. The protocol described in the BrdU cell proliferation ELISA kit is used to assess cell proliferation. 2. Forty-eight hours after the cells were stimulated with ConA or plate-bound anti-CD3 antibody, treat each well in the 96-well microtiter plates with 10 L of bro-modeoxyuridine BrdU -labeled stock solution. Return the plates to the same 5 CO2 incubator as described in the kit. After incubating for 24 h more, remove the 96-well microtiter plates containing the BrdU-labeled cells from the...
Diarylpyrazoles
The most widely studied compound of the diarylpyrazole class is SR141716A Rimonabant 59, Fig. 7 , developed by Rinaldi-Carmona and coworkers at Sanofi, which has passed phase III clinical trials for the treatment of obesity and smoking cessation 69 . This highly potent and selective CBi receptor ligand has served as a unique pharmacological and biochemical tool for further characterization of the CB1 receptor 70,71 . Other diarylpyrazole ligands that have contributed to our understanding of CB1...
Acknowledgment
This work was supported by National Institute on Drug Abuse intramural funds. References 1. United Nations Office on Drugs and Crime. 2003 Global Illicit Drug Trends, 2003, United Nations Office on Drugs and Crime,New York. 2. Substance Abuse amp Mental Health Services Administration. 2002 National Survey on Drug Use and Health, 2002, Substance Abuse amp Mental Health Services Administration, Rockville MD. 3. McPartland J. M. and Pruitt P. L. 1999 Side effects of pharmaceuticals not elicited by...
TCD Sonography
The routine TCD evaluation of cerebral blood flow velocity began in the 1980s 41 . TCD provides clinically useful information in assessing abnormal hemodynamics in cerebral arteries. Current clinical applications of TCD include screening children 2-16 yr of age with sickle cell disease for stroke risk, the detection and monitoring vasospasm after subarachnoid hemorrhage, detection of intracranial steno-occlusive disease, monitoring vasomotor reactivity, detection cerebral circulatory arrest,...
Cell Cultures Splenocyte Cell Cultures
1. First, sacrifice the mice by CO2 asphyxiation. Working in a sterile hood, cut into the animal's skin below the front legs,and pull away the skin to expose the peritoneum and the underlying internal organs. Make a sterile incision into the peritoneal membrane on the animal's left side. Then remove the spleen with forceps. Pool together three spleens from CB2R and CB2R- - mice groups and process them for primary splenocyte culture preparations. 2. Transfer the three spleens to a sterile 70- im...
Contributors
B. Emmanuel Akinshola Department of Pharmacology, Howard University College of Medicine, Washington DC Natalia Battista Department of Biomedical Sciences, University of Teramo, Teramo, Italy Monica Bari Department of Experimental Medicine and Biochemical Sciences, University of Rome, Tor Vergata, Rome, Italy Robert I. Block Department of Anesthesia, University of Iowa, Iowa City, IA Karen Bolla Molecular Neuropsychiatry Branch, Intramural Research Program, National Institute on Drug...
Methods for the Synthesis of Cannabinergic Ligands
Ganesh A. Thakur, Spyros P. Nikas, Richard I. Duclos, Jr., and Alexandros Makriyannis During the last decade, numerous cannabinergic ligands with high affinity and selectivity profiles for cannabinoid receptors CB1 and CB2 emerged from rigorously pursued structure-activity relationship studies. This chapter focuses on the synthetic aspects of key cannabi-noid receptor probes representing the different classes of cannabinergic ligands that encompasses classical cannabinoids CCs including some...
Nissar A Darmani
This chapter briefly describes the physiological neural mechanisms by which diverse neuro-transmitter receptor systems control several aspects of gastrointestinal functions such as motility, secretion, feeding, and emesis. The current techniques used to study the effects of cannabinoids on these gastrointestinal functions are then sequentially described, starting with isolated gastrointestinal muscle preparations and ultimately evolving to whole animal models. Both A9-tetrahydro-cannibinol...
Acute Effects Xvz
As indicated in Table 3, smoking a marijuana cigarette containing 10 mg of THC did not affect recall in a paired-associate learning test in which subjects were presented with a list of pairs of words 2 . Memory was assessed by pre- Note For abbreviations, see Table 1. Note For abbreviations, see Table 1. Reference studied mean age characteristics Min 2 mo in last 6 mo, median 2.3 wk in last 6 mo 88 current, 12 experienced but not current users median for current users 1-4 wk 88 current, 12...
Dissection
The animals used in this laboratory are albino MF1 mice that are at least 4 wk old 30-50 g . Each mouse is stunned by a blow to the head and killed by cervical dislocation. A vertical cut is made through the lower abdominal wall. Removal of the fatty tissue reveals the testes. These can be used to identify the vasa deferentia, each of which is attached to a testis via the epididymis. Gripping the epididymis with forceps, each vas deferens is cut free first from the testis and then from the...
Assays Using Electrically Unstimulated Tissue
As stated in Subheading 3.2., even when bioassays are to be performed without electrical stimulation, all tissues are first subjected to an equilibration procedure that involves electrical stimulation Subheading 3.2.3. . Bioassays without electrical stimulation are used, for example, to measure the ability of cannabinoids to modulate responses to ai-adrenoceptor and P2X receptor agonists that act directly on the smooth muscle to induce contractions. Care must be taken in such experiments to...
AgonistStimulated [35SGTPyS Binding to Cell Membrane Homogenates
When performing these assays in a new tissue for the first time, it is helpful to first perform an assay to optimize assay conditions by determining nonspecific, basal, and agonist-stimulated using a maximally effective concentration of agonist with different amounts of membrane protein see Fig. 2 . Typically, using brain homogenates, 3-20 g of membrane protein per test tube yields good results. With cultured cells, the amount of protein required depends on the receptor expression level and,...
The Mouse Tetrad Test
The mouse tetrad consists of four simple evaluations, which may be measured in sequence in the same animal. 1. Motor activity in an open field is measured for various lengths of time, but typically for 10 min, by digitized or manual observation. 2. The amount of time in which the mouse is immobile after placement on a metal ring of 5.5 cm diameter, fixed at approx 16 cm above a table top, is recorded for 4-5 min. This method was developed by Pertwee 13 and is taken as a measure of catalepsy....
Vomiting Studies
In the past two decades there have been important basic and clinical advances in the understanding of the mechanisms by which chemo- and radiotherapy produce emesis. These findings led to the introduction of clinically useful antiemetics such as serotonin 5-HT3 receptor antagonists in the early 1990s, which revolutionized the treatment of both radiotherapy- and the initial phase of chemotherapy-induced nausea and vomiting. However, 5-HT3 receptor antagonists by themselves are ineffective in the...
Filomena Fezza Natalia Battista Monica Bari and Mauro Maccarrone
Anandamide AEA is the most studied member of a new class of lipid mediators, collectively called endocannabinoids. The biological activity of AEA at cannabinoid and noncannabinoid receptors depends on its life span in the extracellular space, which is regulated by a rapid cellular uptake, followed by intracellular degradation by the enzyme AEA hydrolase fatty acid amide hydrolase . Here, we present the methodological details of the procedures that we have developed to assay fatty acid amide...
Robert I Block
This chapter summarizes the methods and results of studies in which the author examined the acute or chronic effects of marijuana on human associative processes and memory. Eleven tests used to assess marijuana's effects on associative processes, semantic memory, and episodic memory are described. Key Words A9-tetrahydrocannabinol acute effects associative processes chronic effects constrained associations episodic memory free associations free recall human learning marijuana memory...
Introduction Tnv
The use of marijuana has continued unabated among the US population and worldwide. Its use is often associated with acute neuropsychiatric signs and symptoms, which include euphoria, altered levels of consciousness, and drug intoxication 1 . It has also been shown that the drug can affect an individual's equilibrium and psychomotor performance to such an extent as to impair his or her ability to function in a test of simulated driving 2 . Moreover, abstinent marijuana abusers have been reported...
Preparation of Cell Membrane Homogenates
The membrane homogenate preparation procedure isolates cell membranes and membrane-associated macromolecules and assures their homogeneity for assaying small fractions of the preparation in each of many test tubes. It removes the majority of soluble macromolecules, small molecules including endogenous receptor ligands , and minerals via multiple centrifugation steps each followed by resuspension in fresh buffer containing the divalent ion chelator ethylenediamine tetraacetic acid EDTA . Some...
Analysis of CB1r Gene and Genetic Variation in Polysubstance Abuse
1. Cannabinoid receptor gene variation in polysubstance abusers can be studied using bioinformatics, resequencing, and other genotyping methods, such as restriction fragment length polymorphism RFLP , primer extension methods,and or 5' nucle-ase assay. 2. Genotyping information is used for further analysis to evaluate haplotype blocks and associations linkages with the phenotypes. 3. cDNA analysis demonstrates the isoforms, RNA editings, and genetic expression patterns in tissue samples. 4. In...