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|Neuropsychopharmacology: The Fifth Generation of Progress|
Billy R. Martin
Marijuana continues to be the most frequently abused illicit drug in America. Despite modest declines from the pinnacle of its use in the mid 1970s, an upsurge of use occurred during the 1990s. Marijuana smoking is prevalent regardless of age, ethnicity, and sex. Many health consequences of marijuana still defy simple characterization and remain controversial, whereas others are well established. One objective of this review is to discuss current reports dealing with the adverse actions of marijuana with particular emphasis on the reasons for the continuing ambiguity. The clinical utility of cannabinoids, albeit limited, will also be discussed. The biochemical and pharmacological actions of cannabinoids attracted considerable interest in the 1970s, only to be followed by a quiescent period in the early 1980s. During the past few years, numerous breakthroughs have occurred that have greatly increased our understanding of the cannabinoids. It is now evident that an endogenous cannabinoid system exists. A receptor has been characterized and cloned, second messenger systems identified, and a putative endogenous ligand isolated and synthesized. Our concept now extends beyond the notion that cannabinoids are producing their behavioral effects by directly interfering with neurotransmitter systems. This review will also summarize these findings and discuss their implications for future research.
Approximately 5, 8 and 31% of individuals in the U.S. population aged 12 and over have used marijuana during the past month, during the past year, or at least once in their lifetime, respectively (1). One of the most important statistics is the current weekly use, estimated to be at the 2–3% level. However, juveniles and young adults account for the greatest amount of use. In 1994, 20% of the population aged 12–25 years reported marijuana use during the past month, compared with only 8.5% of those over the age of 25. This finding is consistent with the continued downward trend in the age of first-time users. In the late 1960s, the average age at first use of marijuana was approximately 20 years; today, it is about 16 years. The percentage of eighth, tenth, and twelfth graders who used marijuana during the past month were 8, 16, and 16%, respectively. Currently, approximately one in 25 high school seniors use marijuana on a daily basis (58).
Effects on Performance
Cannabinoids affect sensory, psychomotor, and cognitive function. It is, therefore, not surprising that the ability to perform certain tasks is compromised in some individuals after smoking marijuana. There is little dispute that high doses of marijuana can disrupt performance when the task is difficult. Cannabinoid-induced impairment of flying and driving has been documented. As might be expected, the effects of marijuana on performance become more variable as the complexity of the task is simplified and the dose of marijuana is reduced. In a comprehensive review, Chait and Pierri (14) concluded that marijuana, at doses that produce moderate levels of intoxication, can affect a wide range of learned and unlearned behaviors, including simple motor tasks, and more complex psychomotor and cognitive tasks. Their evaluation of the literature indicated that although marijuana adversely affected gross and simple motor function (body sway and hand tremor), psychomotor behaviors (rotary pursuit, Digit Symbol Substitution Test, reaction time, accuracy in divided attention tasks, and sustained attention), it did not adversely affect simple reaction time and hand-eye coordination. Heishman et al. (47) indicated that marijuana can impair complex human performance in arithmetic and recall tests up to 24 hours after smoking. The results suggested that tolerance may develop in chronic users to some of the acute effects. Thus, the level of intoxication is more difficult to detect in an experienced user, except for those tasks requiring a great deal of skill or manual dexterity and tests for which they have had little previous exposure and training. Therefore, it is not surprising that motor impairment and diminution of cognition could easily lead to accidents and traffic fatalities, and that abuse of marijuana has been linked to accidents (8).
Factors that confound the interpretation of marijuana-induced impairment include co-abuse with other agents, individual variability, development of tolerance, and the normal difficulties associated with a systematic evaluation conducted in the general population. For the above reasons, it has been difficult to assess the consequences of millions of individuals smoking marijuana in terms of personal injury, lost productivity, etc. Although it is indisputable that tolerance develops during chronic exposure to high quantities of D9-THC, it is certainly less definitive following intermittent exposure to marijuana (50). As for motor performance, marijuana intoxication may be difficult to detect in an experienced abuser, except in difficult performance tasks or for those tasks in which they have had little previous training (14). On the other hand, marijuana intoxication in an inexperienced individual can be readily detectable on some performance measures. The other complicating factor in assessing marijuana effects on function is that marijuana is frequently co-abused with other drugs, such as alcohol, that are likely to augment its effects (50). Perez-Reyes et al. (105) reported that ethanol dose-dependent decrements in performance skill necessary for automobile driving were further exacerbated by marijuana. Needless to say, establishing a direct correlation between the degree of impairment and blood concentrations of cannabinoids would be of tremendous benefit in establishing causality in accidents. Given the individual sensitivity to marijuana and the confounding factors discussed above, it not likely that measures of D9-THC or its major metabolites in either blood or urine will become standards for intoxication. However, several theoretical models have been proposed for estimating the time of drug use based upon blood and urinary levels of D9-THC and its metabolites (53).
Memory and Learning
Many of the effects described above on performance involved the performance of a complex combination of cognitive and motor skills. There has always been considerable interest in the effects of chronic marijuana use on cognitive functioning. No consensus exists regarding the effects of D9-THC on memory and learning; results are often inconsistent and test-specific (14,116). Hall et al. (45) concluded from clinical observations and cross-cultural studies that chronic marijuana use does not appear to produce severe gross impairment but rather may produce subtle cognitive deficits. The most frequently mentioned deficits were slower psychomotor performance, poorer perceptual motor coordination, and memory dysfunction. During the past few years, greater attention has been directed toward investigating specific cognitive deficits and relating these effects directly to marijuana use. While THC appears to produce its greatest decrement in free recall or short-term memory, it has been proposed that chronic marijuana use in adolescents may result in long-term memory impairment (116). There are also indications that individuals with learning disabilities may be more susceptible to memory deficits (116). Almost all studies have shown that marijuana has no effect on retrieval of already-learned material. THC reliably alters the perception of time, with subjects overestimating elapsed time or experiencing an increase in the subjective rate of time (14). Evidence has emerged from several studies that chronic marijuana use after many years produces subtle cognitive changes, specifically with regard to attention, and organization and integration of complex information (109,123).
Great interest has been generated in the effects of marijuana upon adolescent development, educational performance and production of an "amotivational syndrome" (11). The concept of "amotivation" arose from the report of apathy, decreased productivity and lack of ability to carry out long-term goals in adolescents using marijuana (85). Miles (91) reported that marijuana smoking attenuated work activity in male volunteers in a clinical study. On the other hand, several investigators have pointed out the difficulties in establishing that marijuana is the primary cause of amotivation (27,38,50). It has been suggested that the lack of motivation observed in some individuals more likely results from psychosocial problems and polydrug abuse rather than marijuana use alone (127). The controversy that surrounds amotivation and marijuana appears to stem from attempts to establish a primary cause (i.e., marijuana use) for lack of motivation, when it is undoubtedly a multivariate problem. The more relevant question should be the extent to which chronic marijuana use contributes to amotivational states rather than seeking to establish it as a sole causative factor. The difficulty of establishing causality in altered motivational states is an important consideration. On the other hand, sufficient documentation of diminished motivation in chronic marijuana users exists to suggest a link between them.
The relationship between marijuana use and mental illness has always received attention because of the frequency of drug use in individuals with psychological disorders. Despite suggestions that marijuana induces several psychopathological states (98), a "marijuana psychosis" has not been successfully characterized. It is certainly not surprising that marijuana exacerbates pre-existing mental disorders. The detrimental effects of marijuana in schizophrenics are widely recognized, and a significant proportion of these individuals continue to "self-medicate" with marijuana even after recognizing its negative effects (99). Schizophrenics abusing marijuana are more difficult to treat effectively, or their symptoms worsen even when appropriate neuroleptic levels were maintained. The question of the causal relationship between abuse of marijuana and the development of schizophrenia has not been established, although some investigators believe that marijuana use is a causative factor (7,99). It has been pointed out that individuals who abuse marijuana and who also develop psychiatric problems suffer from rapid-onset schizophrenia (7). Since most of these individuals are multiple-drug users, it seem more likely that marijuana or any of the other abused drugs might act as a trigger to precipitate latent schizophrenia (124). A systematic study will be required in order to establish the relative risk of developing psychiatric problems with marijuana abuse. Until proven otherwise, the risk appears to be relatively small given the widespread abuse of marijuana in the general population.
Central Nervous System Physiology
One of the most notable findings in recent years has been the effects of cannabinoids on cerebral blood flow and electroencephalographic (EEG) measures. Mathew and Wilson (83) summarized the recent literature and concluded that marijuana smoking increased cerebral blood flow, with the greatest increases in the frontal region and right hemisphere. Marijuana also increased cerebral arterial blood velocity, thought to be due to increased capillary perfusion. There is continued interest in the effects of marijuana on EEG. Struve et al. (126) reported that THC caused an increase in absolute power of all frequencies over all cortical areas. They also reported that THC produced significant elevations in absolute alpha power, relative alpha power and interhemispheric alpha coherence over frontal and frontal-central areas in chronic users (125). They referred to this phenomenon as alpha hyperfrontality.
The effects of marijuana and cannabinoids on hormonal functions in humans and laboratory animals during pregnancy was reviewed in the last edition and has been updated recently by Wenger (136). Reproduction studies in both animals and humans have produced conflicting results and widely varying conclusions; this may be due in great part to a combination of differences in experimental design and interspecies differences in drug tolerance (136). However, D9-THC has been described in various studies as a reproductive toxicant in both man and animals. In animal studies, THC produced adverse effects on gametogenesis, embryogenesis, and postnatal development (131). Earlier conclusions that marijuana may be linked to infertility were based on reductions in sperm concentrations following administration of four to 16 marijuana joints per week for a four-week period and oligospermia with Leydig and Sertoli cell dysfunction. Wenger (136) concluded that marijuana can produce reversible and irreversible effects on the reproductive system of both sexes. Attenuation of luteinizing hormone by marijuana has been well documented in males (133) and females (90). Yet, despite considerable evidence supporting cannabinoid-induced alterations in reproductive function, there have been no epidemiological studies that have demonstrated a direct link between marijuana use and infertility.
A great deal of effort has been expended on the investigation of the effects of perinatal exposure to_D9-THC. Initial data suggested the existence of various deficits in humans, and it was anticipated that a definable syndrome—equivalent to the Fetal Alcohol Syndrome—would be found. Prenatal marijuana exposure was reported to be related to tremors, increased startle response, and poorer habituation to visual stimuli of human offspring (40). There is no correlation between the number of minor physical anomalies present in an individual and the extent of maternal marijuana use, though two anomalies (true ocular hypertelorism and severe epicanthus) were found only among children of heavy users of cannabis (101). However, a survey of the literature indicates that prenatal exposure to cannabinoids does not produce malformations in humans and only does so in mice following exposure to high intraperitoneal doses (3). Long-term studies on postnatal effects have produced generally inconsistent results, which Abel (3) attributes to methodological flaws in experimental design. Failure to consider the confounding influences of maternal toxicity (prenatal and postnatal) is likely to yield a high rate of false-positive results; this has been observed in studies of marijuana that preceded current concerns for pair-feeding and surrogate fostering (3, 56). Nearly all such studies found neurobehavioral effects that included changes in activity as well as impairments in learning and memory (56). It is now generally concluded that there are few lasting effects that can be demonstrated in offspring exposed to marijuana (56). Transient decrements in rodent body growth (55) and brain protein synthesis (93) have been observed in neonates following perinatal marijuana exposure, but these effects appeared to be due to maternal toxicity (56). When marijuana is inhaled in smoke, a ventilation/perfusion imbalance is created and fetal oxygen availability is reduced (16), but the effect appears to be related to the 30% reduction in maternal respiration. Any fetal effects are the result of indirect toxicity, and this decrement has not been related to any developmental toxicity. Regardless of the causative factors, longitudinal studies have revealed several subtle, cognitive deficits in offspring of mothers who smoked during pregnancy. It will be important to determine whether these offspring exhibit any deficits when they reach adulthood.
Tolerance and Dependence
The degree to which either sensitization or desensitization to marijuana occurs has been debated. The notion that sensitization (or ‘reverse tolerance') occurs with marijuana is based upon reports that some newcomers require several smoking episodes before experiencing the marijuana ‘high'. That tolerance could then develop to marijuana's psychotomimetic effects formed the basis of the proposed "reverse-reverse tolerance" in the early years of marijuana research. There is no doubt that many factors other than the inherent properties of D9-THC are contributors to the development of tolerance, including the variable THC content in marijuana, expectations, environmental influences, individual differences, and the frequency of use, to just name a few. However, there is convincing evidence for the development of tolerance to D9-THC in humans (62). Tolerance developed to a variety of D9-THC's effects following oral administration; these include cannabinoid-induced decreases in cardiovascular and autonomic functions, increases in intraocular pressure, sleep disturbances and mood changes (62). Results are less conclusive for behavioral tolerance. To achieve behavioral tolerance, high doses of D9-THC were administered for a sustained period of time. In one study, tolerance to the subjective effects of D9-THC developed after oral administration (10 mg) for several days; greater tolerance developed with increased amounts of the drug (59). Thus, if the doses of D9-THC are small and infrequent, little behavioral tolerance seems to develop. High doses must be given for long periods of time to produce tolerance.
Chronic marijuana use does not result in severe withdrawal symptoms, but numerous case reports attest to the development of dependence (59). Several early reports came from countries where potent marijuana was used for long periods of time. Upon deprivation of marijuana, users experienced auditory and visual hallucinations and irritability. The development of tolerance and dependence have been studied under rigorous and controlled conditions (59,61,62,63). In one study, a 30-mg dose of marijuana extract or D9-THC was administered orally approximately 6 times/day for up to 21 days. The most prominent subjective symptoms were increased irritability and restlessness. Other symptoms included insomnia, anorexia, increased sweating and mild nausea, although these were variable. Objective symptoms were increased body temperature, weight loss and hand tremor. Re-administration of a marijuana cigarette or oral D9-THC alleviated the objective and subjective effects, suggesting the establishment of a withdrawal symptom. Similar findings were reported by Georgotas and Zeidenberg (43) in abstinent subjects who had smoked high quantities of marijuana over a long period of time.
Epidemiological data support marijuana dependence (as reviewed by Hall et al. ). Numerous reports of individuals seeking treatment for dependence in which marijuana is the primary cause have emerged over the past several years (32,60,92,114). These patients typically complained of being unable to stop or decrease their use despite experiencing sleepiness, depression, inability to concentrate, memorization difficulties, etc. that they directly attributed to marijuana exposure. Kandel and Davis (64) found similar problems in daily marijuana users. Several groups of investigators have used DSM-III-R and DSM-IV criteria to diagnose marijuana dependence (67,100,115). With regard to prevalence of marijuana abuse and dependence, the strongest evidence was provided by the Epidemiological Catchment Area study involving 20,000 individuals in five geographical areas of the U.S. (112). Approximately 4.4% of the population were considered marijuana abusers and/or marijuana-dependent; three-fifths of these individuals met the criteria for dependence. After an extensive review of the literature, Hall et al. (45) concluded that the risk of developing marijuana dependence was probably similar to that of alcohol, and that daily use over a period of weeks to months resulted in the greatest risk of dependence. Kandel and Davis (64) estimated that the risk of dependence in near daily marijuana users was one in three. Hall et al. (45) estimated that the risk of developing dependence was 10% for those who have ever used marijuana, with the risk rising to 20–30% for those who had used the drug more than five times. Factors that have been associated with marijuana dependence include poor academic achievement, deviant behavior, rebelliousness, maladjustment, difficult parental relations, early initiation of drug use, and family history of drug use (45). The major complaints of marijuana-dependent individuals are loss of control over drug use, cognitive and motivational impairments, lowered self-esteem, depression, and spousal discord.
Our understanding of some of the potential adverse consequences of marijuana use have been alluded to above. There are always reasons to be concerned about drug abuse during pregnancy and in adolescents. The dangers of inhalation of foreign substances have been well documented, particularly substances formed under pyrolytic conditions and that may be highly reactive. Several investigators have expressed concern regarding the pathophysiologic effects of marijuana on various organ systems (98). While relatively little new information regarding specific detrimental effects of marijuana has appeared in the past few years, there has been renewed interest in assessing the health consequences of both acute intoxication and long-term exposure. These concerns have arisen because of a reversal in the formerly downward trend in marijuana use during the past few years. Thus, increased attention has been directed toward the risks and benefits of using of marijuana for medicinal purposes.
Acute Pharmacological Effects
Relatively little is known about the effects of marijuana on animals, due to the constraints of replicating the conditions of smoking the drug in non-human subjects. Most of our knowledge is based upon studies of D9-THC administration to numerous animal species, the results of which reveal a striking resemblance to the pharmacological effects reported above with marijuana administration to humans. Motor incoordination has been reported in dogs, monkeys, and rodents; catalepsy has been reported in mice and rats; antinociception in mice and rats; hypothermia in rats, squirrel monkeys, and mice; and drug discrimination in rats, pigeons, and monkeys (82,110). In addition, memory impairment has been described in rats (49,72). Prominent cardiovascular effects have been reported in both dogs (81) and rats (28), the major effects are bradycardia and prolonged hypotension.
Chronic administration of D9-THC and related analogs results in development of tolerance to almost all behavioral effects in rats, mice, pigeons, and monkeys (19). Some reports actually indicated lack of activity following chronic treatment with doses that were 300- or 6000-fold higher than those initially effective in producing an effect (86). Tolerance has also been shown to the lethal effect of THC in pigeons; a dose of 180 mg/kg, though inactive in tolerant animals, proved to be lethal in drug-naive pigeons. Recently, tolerance to the discriminative cue of D9-THC was shown in rats. Studies conducted with the potent cannabinoid analog CP 55,940 demonstrated that the magnitude of tolerance can exceed 50-fold (33). It has also been established that tolerance is associated with cellular changes in the central nervous system (discussed later in this chapter) rather than alterations in pharmacodynamics (29).
The two major approaches for demonstrating drug dependence are though self-administration studies and the occurrence of physical withdrawal symptoms upon either cessation of chronic treatment or administration of an antagonist. Efforts to train animals to self-administer cannabinoids have proven unsuccessful. Recent attempts to maintain intravenous self-administration of D9-tetrahydrocannabinol and CP 55,940, a potent analog, under a fixed-interval schedule in rhesus monkeys also failed (77). Termination of chronic administration of cannabinoids in an attempt to produce abrupt withdrawal has produced conflicting results. McMillan et al. (86) failed to detect withdrawal symptoms upon termination of chronic administration of cannabinoids to pigeons. A few reports noted that abrupt cessation of cannabinoids produced certain behavioral changes, including increased grooming, motor activity (66), aggression (9), and susceptibility to electroshock-induced convulsions (65). However, re-administration of a cannabinoid did not reverse these effects. Failure of other laboratories to observe abrupt withdrawal has made it difficult to draw definitive conclusions regarding dependence. Since cannabinoids and opioids both produce antinociception, several investigators challenged rats treated chronically with D9-THC with naloxone in an attempt to precipitate physical withdrawal. Although the symptomatology differed somewhat from that described for opioid dependence, naloxone administration produced some physical signs (66). Fortunately, a selective and highly potent cannabinoid antagonist (SR 141716A, Figure 1) was developed, the pharmacology of which will be discussed later (111). When rats that had been treated chronically with D9-THC were challenged with this antagonist, a prominent physical withdrawal syndrome ensued (4,5,130). A marked change in the D9-THC-infused animals was evident approximately 10 minutes after the injection of SR 141716A, and these effects subsided within an hour. The behavioral signs included head shakes, facial tremors, tongue rolling, biting, wet-dog shakes, eyelid ptosis, facial rubbing, paw treading, retropulsion, immobility, ear twitch, chewing, licking, stretching and arched back. The signs of facial rubbing and wet-dog shakes were quantified and found to be statistically greater than those observed in vehicle-infused rats. These studies provided convincing evidence that cannabinoids can produce dependence consistent with the observations made in chronic marijuana users. The challenge is to understand the relationship between these animal models and the abuse pattern observed with cannabinoid use in humans and to devise means for treating individuals who seek assistance in terminating their marijuana use.
The chemistry of marijuana has been well characterized for many years. In addition, there is continuing interest in the levels of D9-THC found in different varieties of marijuana. Evaluation of confiscated material indicates that the D9-THC content in marijuana rose markedly in the 1970s and peaked at approximately 3% in the early 1980s. Occasional samples containing more than 15% D9-THC have been seized. Although the average THC content today is considerably higher than that reported 25 years ago, levels have remained relatively constant during the past 10–15 years. It would not appear that the amount of THC in marijuana influences use patterns, given the fall and rebound in marijuana use during the period that THC levels were relatively stable.
The development of highly potent cannabinoid agonists lagged behind the progress made for many other centrally acting agents. Fortunately, these agonists have now been developed and indeed have played key roles in recent advances in cannabinoid pharmacology. The research group at Pfizer embarked on a synthetic strategy that resulted in novel bicyclic structures that proved to be 4–25 times more potent than D9-THC, depending upon the pharmacological measure (20). The success of Melvin and Johnson (57) not only helped redefine many of the structural determinants of cannabinoid action, it also resulted in novel bi- and tricyclic analogs that are as much as 700 times more potent than D9-THC (74). A second group of compounds with potent agonists properties has also emerged. Mechoulam et al. (87) prepared 11-OH-D8-THC-dimethylheptyl (DMH) [Figure 1] that, as they had predicted, proved to be several hundred times more potent than D8-THC in several behavioral tests (75). The corresponding 11-OH-D9-THC-DMH also exhibited similar high potency (80). Equally important was the preparation of highly pure enantiomers of 11-OH-D8-THC-DMH. These enantiomers were used to finally establish that cannabinoids indeed exhibit high enantioselectivity (87). Findings of high potency and high enantioselectivity reinforced the notion that cannabinoids act through receptor mechanisms. A future challenge will be to develop therapeutically useful cannabinoids that are devoid of unwanted side effects.
The development of potent cannabinoids, such as CP 55,940, provided new opportunities to explore cannabinoid receptors (89). Indeed, it was not until the potent analgesic CP-55,940 was radiolabeled and used as a ligand that a specific and saturable cannabinoid binding site was shown to exist (25). Behaviorally active cannabinoids, including D9-THC, exhibit high affinity for this site, lending credence to the hypothesis that it was the cannabinoid receptor. Subsequently, this receptor was characterized using other radiolabeled ligands such as 3H-11-OH-D9-THC-DMH, 3H-WIN 55212-2, and 3H-11-OH-hexahydro-THC-DMH. D9-THC competes with these ligands with KIs in the range of 1–40 nM. There are subtle differences in the binding characteristics of these various ligands, but they all appear to bind to the same site in a similar fashion. There is an excellent correlation between the pharmacological potency of cannabinoids and their affinity for this binding site. The most extensive study has been conducted by Compton et al. (21), who demonstrated that the binding affinities of 60 cannabinoids were consistent with the pharmacological potencies of these agents in numerous pharmacological assays in mice and rats. Their findings suggested that this single receptor type could account for the behavioral and pharmacological effects of the cannabinoids. Similarly, computer modeling studies indicated that a single pharmacophore could accommodate all of the agents that interact at this cannabinoid site (129). Although it is reasonable to speculate that multiple cannabinoid receptors might exist, evidence has not yet been forthcoming.
A key determinant for any receptor is its selectivity for a given class of compounds. This point is particularly important for the cannabinoids, because they have been shown to share numerous pharmacological properties with neurotransmitters and other centrally acting agents (27,78). Howlett (52) has examined an impressive array of centrally acting compounds and has found that the following agents do not compete for cannabinoid binding: adrenergic, dopaminergic, opioid, sigma, cholinergic, neuroleptics, GABAergic, or serotonergic agents; hormones, steroids, amino acids, peptides, nonsteroidal anti-inflammatory agents, arachidonic acid and metabolites, prostaglandins, and leukotrienes, as well as numerous other agents.
The anatomical distribution of the receptor throughout the brain, as well as its localization within the neuron, has been determined autoradiographically (48). The binding sites are most dense in the substantia nigra pars reticulata, globus pallidus, interpeduncular nucleus, caudate-putamen and the molecular layer of the cerebellum. Low levels of binding occur in the brain stem (medulla and pons), thalamic nuclei, hypothalamus, corpus callosum, and the deep nuclear layer of the cerebellum. Intermediate levels of binding are seen in layers I and VI of the cortex, and the dentate gyrus and CA pyramidal cell regions of the hippocampus.
This discrete distribution of receptors throughout the brain provides some insight into their functional significance. High receptor densities in the extrapyramidal motor system and the cerebellum are consistent with the effects of cannabinoids on many forms of movement. The hippocampal formation is also a brain region demonstrating relatively dense binding of cannabinoids. The hippocampus is involved in coding sensory information and storing memory. Importantly, D9-THC disrupts short-term memory in man, as discussed earlier. Therefore, the relatively dense localization of receptors in the hippocampus and cortex may be the source of the effects of cannabinoids on cognition and memory. The presence of cannabinoid receptors in the ventromedial striatum and nucleus accumbens suggests an association with dopamine neurons that may mediate brain reward (41). D9-THC augmented self-administered electrical stimulation in the rat medial forebrain bundle and enhanced both potassium-stimulated presynaptic dopamine efflux in rat neostriatum and presynaptic basal dopamine efflux in the nucleus accumbens of freely moving rats. Despite the rather low density of receptors in the hypothalamus, cannabinoids produced hypothermia in mice when given intravenously (74), intracerebroventricularly, or directly into the preoptic area (37). Lastly, the low density of cannabinoid receptors in medullary nuclei would be expected, given that there are almost no reports of marijuana producing profound respiratory depression in humans.
Convincing evidence for a cannabinoid receptor was provided by the cloning experiments of Matsuda et al. (84). These investigators isolated a clone from a rat brain library that had a high degree of homology with other G-protein coupled receptors. This receptor gained "orphan" status when all of the traditional agonists of G-protein coupled receptors failed to interact with it. However, discovery that the distribution of mRNA of the clone paralleled that of the cannabinoid receptor, as reported by Herkenham et al. (48), led them to speculate that they had cloned the cannabinoid receptor. When cells were transfected with this clone, CP-55,940, D9-THC, and other psychoactive cannabinoids were able to inhibit adenylyl cyclase, whereas untransfected cells were non-responsive. The human cannabinoid receptor was subsequently cloned (44). This cannabinoid receptor appears to be part of a G protein-coupled receptor subfamily (94). Knowledge of the structure of the cannabinoid receptor offers us the opportunity to study its interactions with both ligands and second messenger systems. More importantly, it demonstrates that the cannabinoid receptor undoubtedly plays an important functional role.
As for multiple receptors, a second cannabinoid receptor clone (CB2) that has a different sequence (44% amino acid identity with the brain clone) but a similar binding profile to the CB1 clone was discovered in differentiated myeloid cells (95). The CB2 receptor has been found primarily in the spleen and cells of the immune system. Munro et al. (95) reported that the receptor's affinities for several cannabinoids are comparable to those of the brain receptor. The exception is cannabinol, which has some selectivity for the CB2 receptor. Synthetic ligands have now been developed that have approximately 40-fold selectivity for the CB2 receptor (118). The role of the cannabinoid receptor in immune function has not been established, but it is reasonable to speculate on a modulatory role given the immunosuppressive effects of cannabinoids.
All evidence points to the brain cannabinoid receptor as being coupled to G-proteins. Ligand binding at the cannabinoid receptor was reduced by the non-hydrolyzable guanine nucleotide analog Gpp(NH)p . Studies with [35S]GTPgS also confirmed that receptor activation resulted in increased binding of this non-hydrolyzable analog (117,120), thereby lending further support to the premise of receptor-G-protein coupling. The most likely candidate for a second messenger system is adenylyl cyclase (51). Numerous laboratories have demonstrated that cannabinoids inhibit adenylyl cyclase both in vivo and in vitro, probably by interaction with Gi. However, the effects of cannabinoids are not confined to adenylyl cyclase. Electrophysiological studies in neuroblastoma cells indicated that cannabinoids inhibited an omega conotoxin-sensitive, high voltage-activated calcium channel. This effect is blocked by the administration of pertussis toxin and is independent of the formation of cAMP (76). It was hypothesized that N-type calcium channels were affected, because the L-type calcium channel blocker nitrendipine failed to alter the effect of the cannabinoids.
As I have alluded to above, there is considerable interest in establishing the pharmacological effects that result from activation of cannabinoid receptors. Traditionally, specific receptor antagonists have played major roles in discriminating between receptor and non-receptor mechanisms. The development of an antagonist that is selective for the brain CB1 receptor has provided such an opportunity (111). Researchers demonstrated that this antagonist blocks cannabinoid-induced inhibition of adenylyl cyclase and smooth muscle contractions. Others found that this compound antagonized a wide range of cannabinoid pharmacological effects in mice (18), THC drug discrimination in rats (137), cannabinoid-induced decrements in performance of rats in an eight-arm radial maze (72), antinociception in rats (73) and cannabinoid-induced blockade of long-term potentiation in rat hippocampus (17).
The development of pharmacological tolerance was accompanied by down-regulation of cannabinoid receptors in selected brain areas, as measured by radiolabeled ligand binding (33,103,113). It is interesting to note that in one study, the decrease in receptor number was not paralleled by changes in adenylyl cyclase functionality (33). On the other hand, others have shown that cannabinoid-induced G-protein activation is diminished in brains from cannabinoid-tolerant rats (119). While the cellular mechanisms responsible for these adaptive processes remain to be established, an increase in mRNA for the CB1 receptor has been detected in cerebellum of rats (33). The detection of concomitant pharmacological tolerance and adaptation in the biochemical events associated with the CB1 receptor provides a framework for elucidating the neurochemical actions of the endogenous cannabinoid system.
PUTATIVE ENDOGENOUS LIGANDS
Devane et al. (26) postulated that an endogenous cannabinoid ligand is likely to be a highly lipophilic agent and, therefore, undertook to isolate endogenous THC-like substances from lipid extracts of porcine brain. An ethanolamide derivative of arachidonic acid was isolated and found to bind to the cannabinoid receptor. This compound (anandamide; Figure 1) inhibited electrically stimulated contractions of smooth muscle in much the same fashion as D9-THC. Anandamide shares some of the pharmacological effects of D9-THC (39), although it is considerably less potent (4–20 fold, depending upon the pharmacological action) and shorter acting (121). Additionally, it inhibits adenylyl cyclase and N-type calcium channels (34). Since fatty acids are so plentiful, it is not unreasonable to predict that an entire family of anandamide-like compounds may exist. Indeed, Hanus et al. (46) have identified homo-g-linolenylethanolamide and docosatetraenylethanolamide as constituents in porcine brain that also compete for cannabinoid receptor binding.
Anandamide binds to both the CB1 and CB2 cannabinoid receptors. This has been demonstrated in membrane preparations from brain and in transfected cells (6,26,95,118). Anandamide's affinity for the CB2 receptor was considerably less than that for the CB1 receptor (95); however, subsequent studies demonstrated that anandamide's affinity for CB2 receptors was approximately four-fold less than that for CB1 receptors in stably transfected cells (35,118).
Anandamide is rapidly degraded by amidases (15,22). This has hindered its detection and quantitation in tissue until recently, when the compound was detected in human and rat brain areas (36). There have been two major proposals for the synthesis of anandamide. The first involves the simple condensation of arachidonic acid with ethanolamine, as evidenced by the formation of anandamide when these precursors are added to brain tissue (22,23,24). However, others have shown that anandamide formation occurs through phosphodiesterase-mediated cleavage of a novel phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine (30), in a process that is controlled by calcium and c-AMP (13). This latter proposal suggests that anandamide is stored and then released upon appropriate stimulation, the manner of which is as yet undetermined.
As for characterization of the full physiological role and pharmacological profile of anandamide, several laboratories have conducted structure-activity relationship studies (see review by Mechoulam et al. ). Adams et al. (6) developed several analogs that were considerably more potent than anandamide and thereby provide unique tools for exploring the neurochemistry of this class of compounds. The prediction that non-hydrolyzable analogs would have higher potency was borne out with the synthesis of methanandamide (2).
The future challenge will be to establish the physiological role for the endogenous cannabinoids. Is anandamide a neurotransmitter or a neuromodulator? Does an entire cannabinoid family of amide derivatives of fatty acids exist, each of which has a distinct neurochemical role? If cannabinoids serve a normal physiological role, then what are the consequences of an imbalance in this system? Based upon the pharmacological effects of cannabinoids, motor imbalance, altered pain sensitivity and cognitive impairment are all possible outcomes. Answers to these and related questions may provide an entirely new perspective on the way we view cannabinoids.
The interactions of cannabinoids with central neurotransmitters and neuromodulators has been reviewed recently by Pertwee (106), who pointed out that although there is a well established cannabinoid interaction with these systems, the nature of these interactions is less well defined. The cholinergic nervous system appears to be involved in the production of cannabinoid-induced catalepsy, in that cholinergic agonists potentiate this effect, whereas antagonists attenuate it. The cholinergic system is not involved in all of the actions of the cannabinoids. because cholinergic antagonists fail to alter the discriminative stimulus of D9-THC. The cholinergic system also does not appear to be involved in the antinociceptive effects of D9-THC. Efforts to define a role for the cholinergic system in the production of cannabinoid-induced hypothermia have produced somewhat equivocal results.
The interactions among the cannabinoids, dopamine, and norepinephrine are of particular interest. One measure of motor function which has received considerable attention has been cannabinoid-induced catalepsy. Numerous laboratories demonstrated that agents that stimulate the dopaminergic system also attenuate cannabinoid-induced catalepsy, whereas dopamine antagonists enhance catalepsy. The adrenergic system has been implicated in the antinociceptive effects of cannabinoids. For example, the a2 antagonist yohimbine, administered intrathecally (i.t.), will block cannabinoid antinociception (70). Gardner (41) summarized the findings concerning cannabinoids and brain reward systems. He and his colleagues have previously shown that cannabinoids do enhance brain-stimulating reward in the Lewis rat. An argument was made that D9-THC enhances both basal and potassium-stimulated extracellular dopamine efflux in brain loci involved in reward mechanisms.
There are relatively few studies implicating the serotonergic system in the actions of the cannabinoids. As Pertwee noted (106), consistent results have not emerged from studies characterizing the influence of serotonergic agonists and antagonists on cannabinoid-induced hypothermia. On the other hand, numerous results suggest that activation of the serotonergic system enhances the cataleptic effects of the cannabinoids, whereas serotonergic antagonists attenuate these effects. Spinally administered methysergide had no effect on D9-THC-induced antinociception (70).
Several lines of research suggest a link between GABAergic compounds and cannabinoids. First, D9-THC acts synergistically with both GABAA (muscimol) and GABAB [(-)-baclofen] agonists in producing catalepsy (107). Additionally, benzodiazepines that facilitate GABA interaction with GABAA receptors act synergistically with D9-THC in producing catalepsy, and this state can be blocked by flumazenil. Studies from our own laboratory have shown that cannabinoids that exhibit anxiogenic properties (increased aversion to open arms of the elevated plus maze) can be blocked by either diazepam or flumazenil (102). Furthermore, there are reports of partial generalization by diazepam to the THC discriminative cue, and a report of cannabinoids influencing benzodiazepine receptor binding. Childer's laboratory has found that cannabinoids and GABAB receptor agonists decrease cAMP levels in cerebellar granule cells in a non-additive fashion, whereas they act in an additive fashion in stimulating GTPase in cerebellar membranes (79). They interpreted these findings as a case of receptor convergence, in which both classes of drugs share common adenylyl cyclase catalytic units without sharing common G-proteins.
The observation that cannabinoids and opioids exhibit several similar pharmacological properties prompted speculation that they share a common mechanism of action. There are indications that opioids and cannabinoids may produce some cross tolerance in selected tests, although the data are far from conclusive. The cannabinoids, like the opioids, produce antinociception and analgesia. Human subjects receiving oral doses of 10 and 20 mg/kg D9-THC indicated analgesia comparable to that of codeine; however, a significant number of undesirable side effects were also present. Intravenous administration of D9-THC to human dental patients produced analgesia accompanied by dysphoria and anxiety. Animal studies have also revealed spinal and supraspinal antinociception. Several potent cannabinoid analogs have been shown to produce antinociceptive effects upon intrathecal (i.t.) administration to rats and dogs. Studies conducted in mice and rats also indicated that the cannabinoid-induced antinociceptive effects are mediated at both spinal and supraspinal sites (71,135).
The localization of cannabinoid receptors allows for an interaction with the opioid system. The binding of CP-55,940 is dense in the striatum (48), an area also associated with dense opioids binding. Although cannabinoid receptors are relatively sparse in the medulla, there are moderate concentrations in the periaqueductal gray, a structure that contains high concentrations of opioid receptors and plays an important role in analgesia. Binding of CP-55,940 in the substantia gelatinosa of the spinal cord has been shown, although it is rather low (48). Nonetheless, it is much higher than the binding in the dorsal horn of the spinal cord of Substance P, a major transmitter involved with pain processing in the spinal cord. The substantia gelatinosa is also the principle binding site of the opioids in the dorsal horn and the major site of the processing of pain signals for transmission to the spinothalamic tract.
There have been a few suggestions that opioid antagonists such as naloxone can block the antinociceptive effects of cannabinoids. By and large, however, most studies have failed to support such a contention. Indeed, no study has demonstrated that cannabinoids and opioids have high affinity for each other's receptors. The antinociceptive effects of D9-THC and morphine are additive following intravenous administration, thus implying distinct mechanisms of action (42). Recently, Welch (134) published the intriguing observation that cannabinoid antinociception was blocked by the k opioid antagonist, nor-binaltorphimine (nor-BNI). The d antagonist ICI 174,864 (10 mg/mouse, administered i.t.) failed to block the effects of any of the cannabinoids administered i.v. Moreover, the nor-BNI blockade of D9-THC (i.t.) antinociception was specific for antinociception and did not block catalepsy, hypothermia, or hypoactivity (122). The lack of naloxone blockade of the cannabinoid-induced antinociception leads to the question of opioid involvement in the effects of nor-BNI. To date all k opioid antinociceptive effects are blocked by naloxone, albeit at high doses of naloxone. In addition, k opioid binding remained unaltered by cannabinoids (132), and cannabinoid binding in the brain was not displaced by nor-BNI or the k antagonist U50,488H (134). Thus, the exact nature of the nor-BNI blockade is not known.
Enkephalinergic neurons are known to synapse on dopaminergic neurons in the nucleus accumbens, the site proposed to modulate the reward system for all addicting drugs (41). The cannabinoids appear to interact with opioids allosterically, either presynaptically on the enkephalinergic neuron or with the opioid receptor on the dopaminergic neuron to enhance reinforcing effects. Although there are numerous distinct differences in their mechanisms of action, opioid/cannabinoid interactions cannot be ruled out.
A great deal of information has been generated regarding the actions of cannabinoids on prostaglandin synthesis, a topic which has been reviewed by Burstein (12). Interest initially arose because these two classes of compounds share some similar pharmacological properties, namely the production of analgesia and hypothermia. Additionally, discovery of abundant quantities of arachidonic acid and prostaglandins in the central nervous system led to speculation that these compounds may play a role in normal brain function. Several investigators have shown that blockers of prostaglandin formation, such as aspirin and indomethacin, attenuate the antinociceptive, cataleptic, and hypotensive effects of D9-THC (12). Similar findings have been reported in humans (104), where selected behavioral effects of the cannabinoids were blocked by indomethacin. Immunization of mice against PGE2 led to reduced responsiveness to cannabinoids (54), and administration of D9-THC produced a rise in levels of PGE2 and PGF2a (10). Despite these indications that eicosanoids are involved in the actions of the cannabinoids, defining a precise role for them has not been possible. Needless to say, the discovery of an arachidonic acid metabolite as a putative endogenous cannabinoid intensified interest in this area.
The influence of cannabinoids on the adrenal-pituitary has been reviewed by Eldridge and Landfield (31), who pointed out that the D9-THC-induced release of glucocorticoids is most likely regulated by central mechanisms. Although marijuana is typically perceived as anxiolytic, it also has anxiogenic properties that are probably dependent upon the environmental situation and individual reaction to marijuana. Interest has been revived in the possible interaction between glucocorticoids and cannabinoids because of the observation that chronic administration of D9-THC to rats resulted in aging-like degenerative changes in the hippocampus that strongly resembled those produced by either stress or elevated glucocorticoid secretion. These authors suggested that cannabinoids either have glucocorticoid agonist effects or inhibit the negative feedback control that produces enhanced output of adrenocortical steroids.
POTENTIAL THERAPEUTIC USES
Although the above mentioned analogs have proven to be extremely valuable as receptor probes in laboratory studies, none have emerged as clinically useful agents. The lack of pharmacological specificity is a major impediment to the therapeutic use of cannabinoids. The primary cannabinoid that is used clinically is D9-THC itself, which has been given the generic name dronabinol.
Considerable interest exists in the therapeutic potential of cannabinoids for several reasons. First, there have always been folklore and anecdotal reports of marijuana being used to treat a wide range of disorders. Some of the more plausible therapeutic uses include treatment of pain, convulsions, glaucoma, muscle spasticity, bronchial asthma, loss of appetite, nausea, and vomiting (50). Second, cannabinoids represent a novel therapeutic option because they differ from the agents traditionally used to treat these disorders. This latter point is important because new strategies are crucial for treating patients who are unresponsive to current therapy or who suffer severe side effects from traditional medications.
Impassioned pleas on both sides have been raised as to the merits of using D9-THC and marijuana as therapeutic agents. There are those who argue that marijuana is a highly efficacious agent without serious side effects; others contend that marijuana and D9-THC lack sufficient efficacy to warrant therapeutic use and that they are, in addition, highly dangerous substances. It is a well known fact that it is much more difficult to develop substances that are subject to abuse for medical uses. Until the mid-1980s, D9-THC was a Schedule I drug—in other words, an abused substance without any medical use. The compound was subsequently reclassified as a Schedule II drug. D9-THC was formulated in sesame oil, given the name dronabinol, and marketed as Marinolâ (Roxane Laboratories, Columbus, OH).
Nausea and Vomiting
The indication studied most extensively has been nausea and vomiting. It is generally thought that marijuana, D9-THC, and some analogs (nabilone and levonantradol) are effective in managing chemotherapy-induced vomiting (68). In 1987, D9-THC (dronabinol) was introduced in the United States for use as an anti-emetic in patients suffering from nausea and vomiting induced by cancer chemotherapy and who were refractory to the usual antiemetic drugs. It has proven to be a useful anti-emetic, although it is less certain that D9-THC is effective in patients refractory to other treatments. More recently, marijuana has been used by AIDS victims to block the nausea of chemotherapy and to stimulate their appetite. The FDA has granted orphan status to D9-THC for stimulating appetite and preventing weight loss in AIDS patients. Clinical trials with dronabinol in AIDS patients have suggested improved appetite at a dose that was tolerated during chronic administration (108). However, it would appear that sanctioning the use of marijuana and D9-THC for appetite stimulation was based more on political expediency than on sound scientific principles. Even though there is a lack of conclusive evidence that D9-THC adversely affects the immune system in healthy adults, there is a wealth of information demonstrating cannabinoid-induced alterations in the immune system in laboratory animals (96). It should be obvious that AIDS patients are at a higher risk than the normal population for averse effects related to immunosuppression. With the development of any therapeutic agent, the next required step is evaluation of the efficacy and safety of the drug in the appropriate population.
Cannabinoids are potent antinociceptive agents in laboratory animal models, as indicated in the above description of cannabinoid-opioid interactions. However, cannabinoid analgesia can only be elicited at doses that also produce other behavioral side effects, and these agents are no more efficacious than the more commonly used opioid analgesics. One goal has been to develop therapeutically useful cannabinoid derivatives with significantly fewer undesirable side effects (110). There is ample evidence thus far that cannabinoids can be highly potent antinociceptive agents. Most studies also demonstrate that the production of antinociception is usually accompanied by catalepsy and hypothermia, as well as sedation. The pharmacological profile of these drugs and their diversity of effects may indicate that multiple mechanisms of action are involved. In addition, the pharmacological profile and the known mechanisms of action of the cannabinoids appear to be distinguishable from other antinociceptive agents. Thus, these drugs may work via a unique mechanism for controlling pain. The results with nor-BNI blockade of cannabinoid-induced antinociception are important because the other effects of cannabinoids were not influenced. The use of nor-BNI may prove a useful tool in the elucidation of the mechanism underlying the antinociceptive effects of the cannabinoids.
Memory and Cognition
There is abundance evidence to show that cannabinoids interfere with working memory in laboratory animals (49,69). Moreover, the cannabinoid antagonist SR 141716A is able to reverse the effects of D9-THC in the eight-arm radial maze (72). These latter findings suggest that these disruptive effects of cannabinoids on memory are mediated specifically through the cannabinoid CB1 receptor. This observation provides insight into the possible physiological role of the endogenous cannabinoid system. If activation of the endogenous system by an agonist such as D9-THC has a detrimental effect on memory, then the antagonist may possibly enhance memory through disinhibition of homeostatic control. Indeed, there has been one report of memory enhancement with SR 141716A in the maternal-pup rat model (128). Replication of these findings in more conventional memory models, or preferably in models of memory deficits, would indicate a potential therapeutic use of cannabinoid antagonists.
The recreational use of marijuana and our understanding of its health consequences have remained largely unchanged from that described in the last edition. However, there are two important epidemiological considerations. First, marijuana use is steadily increasing after years of continual decline. Secondly, a large segment of the population is still using this drug. Consistent use of high quantities of marijuana has detrimental effects on social and interpersonal skills and impedes the normal development of young individuals. The adverse consequences of infrequent use of marijuana are less clearly defined. Concerns remain regarding marijuana-induced pulmonary damage and possible lung cancer. Despite the absence of definitive studies demonstrating a direct role of marijuana in producing detrimental effects on reproduction and development, there is sufficient evidence to discourage marijuana use during pregnancy. As for long-term effects induced by marijuana, reports of other disorders have been described in selected groups of individuals but have not been confirmed in the general population.
The therapeutic potential of cannabinoids continues to attract interest. Unfortunately, few synthetic analogs with therapeutic potential have emerged that are devoid of cannabinoid behavioral effects. The one promising, non-psychoactive agent is currently undergoing extensive evaluation as an neuroprotective agent (97). Drug development strategy is to avoid agents that produce a wide spectrum of effects, even if they are not severely toxic to the patient. With the advent of potent new cannabinoid derivatives, it may be possible in the near future to develop cannabinoids that lack undesirable side effects.
Tremendous progress has been made in the past five years regarding our understanding of the mechanism of action of cannabinoids. A cannabinoid receptor has been characterized using traditional in vitro binding methodology, and its distribution has been mapped throughout the central nervous system. This receptor has also been cloned and appears to be a member of the G-protein-associated receptors. Moreover, a putative endogenous ligand (anandamide, identified from porcine brain) appears to share most of the pharmacological properties of D9-THC. Hopefully, our understanding of the cannabinoid system in the brain will continue to grow in an exponential fashion.
Preparation of his chapter was supported in part by National Institute on Drug Abuse grants DA-03672 and DA-09789.