Emmanuel S. Onaivi

In-vitro and in-vivo action of cannabinoids.

The discovery of endocannabinoids such as anandamide and the wide spread localization of cannabinoid receptors in the brain and peripheral tissues, suggests that the cannabinoid system represents a previously unrecognized ubiquitous net work in the nervous system, whose physiology and function is unfolding. In this study, we tested the hypothesis that some of the actions of anandamide are independent of a cannabinoid receptor mechanism. This was accomplished by the use of cannabinoid agonist and antagonist interaction in an in-vitro and in-vivo test systems. In-vitro, we used Xenopus laevis oocytes expression system and two-voltage clamp technique in combination with differential display polymerase chain reaction to determine whether the differential display of genes following treatment with anandamide may be linked to AMPA glutamate receptor. The differential expression of genes in vivo after the sub-acute administration of anandamide could not be directly linked with the AMPA glutamate receptor. In the voltage clamp studies we investigated the effects of anandamide on recombinant AMPA GluR3 sub-unit currents generated by kainic acid in oocytes expressing the AMPA glutamate receptor. In the in-vitro studies, we present evidence that anandamide inhibited the kainate activated currents in oocytes expressing AMPA glutamate receptor involves cAMP transduction via a cannabinoid receptor independent mechanism. In the in-vivo studies, SR141716A, the CB1 antagonist, induced anxiolysis, that was dependent on the mouse strain used in the anxiety model and blocked the anxiogenic effects of anandamide or methanandamide whereas SR141716A had no effect on the anandamide inhibition of kainate activated currents in-vitro.

Neurobehavioral Effects of Anandamide and Cannabinoid Receptor Gene Expression in Mice

The objective of the present study was to determine the neurobehavioral effects of the putative endogenous cannabinoid ligand, anandamide, and its influence on cannabinoid (CB1) receptor gene expression. The effect of acute administration of anandamide to C57BL/6, DBA/2, and ICR mice were evaluated in motor function and emotionality tests. The C57BL/6 and ICR mouse strains were more sensitive than the DBA/2 strain to the depression of locomotor activity and stereotyped behavior caused by anandamide. Although anandamide produced catalepsy in all three strains, anandamide induced ataxia in the minus-maze test only in the C57BL/6 animals and only at the lowest dose used. In the plus-maze test system, anandamide produced a mild aversive response, and by the third day of treatment the mouse strains developed an intense aversion to the open arms of the plus-maze. Northern analysis data using the recently cloned mouse cannabinoid receptor cDNA as a probe indicated that there was abundant expression of CB1 gene in the whole brain of the ICR mouse than in the brains of the C57BL/6 and DBA/2 strains with or without pretreatment with anandamide. The anandamide induced neurobehavioral profile does not seem to correspond to the CB1 gene expression in the mouse strains. It is, therefore, unlikely that the CB1 receptor mediates all the cannabinomimetic effects of anandamide in the brain.

Expression of cannabinoid receptors and their gene transcripts in human blood cells

1. This study shows that the human cannabinoid receptors and their gene transcripts can be analyzed in blood samples when combined with polymerase chain reaction. The results also demonstrate that the expression of the cannabinoid receptors is dependent on gender and ethnic background. 2. Normal human volunteers who do not use marijuana have genes that encode for the marijuana (cannabinoid) receptor proteins. 3. Primer pairs from CB1 and CB2 cDNA coding region sequences showed identical amplified DNA band sizes in both DNA-PCR and reverse PCR, with human templates. This suggests that the CB1 and CB2 genes are intronless at least in their coding regions. 4. An advantage of the coding region being intronless may be that the expression of these genes will have one major RNA processing event to skip, thus making the conditions of their expression relatively quick and simple. This advantage may have implications related to the biological functions of these proteins. 5. We therefore concluded that the existence of human cannabinoid receptors and genes along with the discovery of endogenous cannabinoids (endocannabinoids) may be useful markers in elucidating the role(s) and mechanism(s) of action of cannabinoids.

Anxiolytic-like effects of DAIZAC, a selective high-affinity 5-HT3 receptor antagonist, in the mouse elevated plus-maze

Abstract Behavioral effects of desamino-3-iodozacopride (DAIZAC) [( S )-5-chloro-3-iodo-2-methoxy- N -(1-azabicyclo[2.2.2]oct-3-yl)benzamide], a selective high-affinity 5-HT 3 receptor antagonist ( K D 0.14 nM), were evaluated in the mouse elevated plus-maze using the anxiolytic benzodiazepine, diazepam, as a positive control. DAIZAC treatment produced a significant dose-related increase in the time spent in the open arm. The increased total time in the open arm resulted from a significant dose-dependent increase in the number of entries into that arm. The minimum dose of DAIZAC associated with a statistically significant increase in entries and time spent in the open arm was 0.05 mg/kg ip, consistent with its high affinity for the 5-HT 3 receptor. DAIZAC did not affect the amount of time spent in the open arm after each entry. Thus, DAIZAC reduced apparent avoidance of the open arm when the animal was in the central compartment, without affecting active avoidance of that arm when the animal was in the exposed condition. The increase in the open-arm entries was accompanied by a corresponding reduction in the number of entries into the closed arm with a consequent reduction in the time spent in the closed arm. The time spent in the closed arm after each entry was not altered by DAIZAC administration. As such, the sole apparent effect of DAIZAC was to alter the choice of arm to enter when the animal was in the central compartment. Diazepam also significantly increased total time in the open arm; however, the increase was not attributable to a single behavioral factor. The anxiolytic-like effects of DAIZAC reached maximum by 20–30 min and returned to baseline levels by 90 min. Ex vivo binding studies found that levels of DAIZAC-like activity assayed in brains of mice 25 min after DAIZAC injection were significantly correlated with the behavioral parameters associated with anxiolysis. These results indicate that DAIZAC produces dose-dependent anxiolytic-like behavioral changes in the mouse elevated plus-maze that are correlated with brain DAIZAC-like activity.

In Vivo Ibogaine Blockade and In Vitro PKC Action of Cocainea

ABSTRACT: Ibogaine may have antiaddiction potential against alcohol, psychostimulant and opiate abuse, but its mechanism of action is unclear. Ibogaine, however, has been demonstrated in numerous studies to have effects in multiple central nervous system (CNS) neurotransmitters systems. We are using in vitro and in vivo systems to study the effects of cocaine and whether these effects can be blocked by ibogaine. For the In Vivo studies, we first determined the acute and subacute effects of ibogaine (1–5.0 mg/kg) in mice using the plus‐maze test. Acutely increasing doses of ibogaine produced a reduced aversion to the open arms. The subacute administration provoked a variable response which was characterized by fluctuations in aversive and antiaversive behavior of the animals to the open arms of the plus‐maze during the 14‐day treatment period. A separate group of mice received 1.0 mg/kg cocaine for 14 days, and upon abrupt cessation from cocaine treatment, ibogaine 2.5 mg/kg was administered to a subgroup of these mice. Ibogaine reversed the withdrawal aversions produced by the abrupt cessation from cocaine administration. For the in vitro studies, the expression and activity of protein kinase C (PKC) isoforms and Ca2+ levels were examined following the incubation of PC 12 cells with cocaine. This is because PKC plays a key role in a number of cellular and neuronal functions. We report that cocaine disrupts signal transduction in PC 12 cells by altering the expression and activity of PKC isoforms and Ca2+ levels. The data obtained suggest (1) that the PC 12 cells may be useful in studying the neurobiology of abused drugs, like cocaine in vitro, (2) that if anxiety is a factor in drug dependency, then the antiaddictive property of ibogaine In Vivo may be associated with modifying the CNS neurotransmission that may be involved in anxiety. It remains to be determined whether the signaling involving PKC is important in the antiaddictive properties of ibogaine.

Chapter 7 Changes in gene expression and signal transduction following ibogaine treatment

Publisher Summary The chapter presents a study on the changes in gene expression and signal transduction following ibogaine treatment. Drug addiction may not be mediated by one neurochemical pathway and brain structure, but by a complex interaction of programs of gene expression with specific signal transduction pathways and environmental factors. The putative antiaddictive effect of ibogaine may result from the restoration of altered or disrupted programs of central and peripheral neuroadaptative processes involving programmed genes and their associated signaling mechanisms. One new hypothesis being explored is that both the peripheral and central actions of abused substances contribute to drug addiction. One new hypothesis being explored is that both the peripheral and central actions of abused substances contribute to drug addiction. Treatment with cocaine influences the regulation of certain genes in the brain, as indicated by the activation and inhibition of the expression sequence tags (ESTs) that have been isolated. The cAMP-responsive element binding protein (CREB), the first CRE-binding factor to be characterized ( 5 ), is a transcription factor of general importance in both neuronal and other cells. CREB phosphorylation on Ser-133 promotes the activation of genes with an upstream CRE element. If anxiety or stress is a factor in drug dependency, then the anti-addictive property of ibogaine in vivo may be associated with modifying the CNS neurotransmission that may be involved in anxiety.