If con- firmed in further behavioral models, these findings would suggest that inhibition of intracellular FAAH activity might offer an innovative target for the treatment of anxiety , which is also a feature of marijuana withdrawal .2-AG was identified as a second endocannabinoid substance in 1995 . The multiple roles of this lipid compound in cell metabolism and its high levels in brain tissue— about 200-fold higher than those of anandamide—sug gest that much of cellular 2-AG may be involved in housekeeping functions. The diversity of roles played by this compound also complicates our efforts to establish biochemical route involved in its physiological formation. Nevertheless, one pathway has emerged as the most likely candidate . This pathway starts with the phospholipase-mediated generation of 1, 2- diacylglycerol . This serves as a substrate for two enzymes: DAG kinase, which catalyzes DAG phosphorylationto phosphatidic acid; and DAG lipase , which hydrolyzes DAG to monoacylglycerol . Pharmacological inhibition of phospholipase C and DGL prevent the Ca2+-dependent accumulation of 2-AG in rat cortical neurons, which suggests a key role of this pathway in2-AG generation . However, additional routes of 2-AG synthesis also may exist, including phospho lipase A1 , hormone-sensitive lipase or a lipid phosphatase . In neurons and glia, 2-AG synthesis may be initiated by a rise incytosolic Ca2+ levels. For example, incul tures of rat cortical neurons, the Ca2+ ionophore ionomycin and the glutamate receptor agonist N-methyl d-aspartate stimulate 2-AG productionina Ca2+-dependent manner . Similarly, in freshly dissected hippocampal slices, electrical stimulation of the Schaffer collaterals, a glutama tergic fiber tract that connects neurons in the CA3 and CA1 fields, causes a Ca2+-dependent increase in 2-AG content . This stimulationhas no effect on the levels of non-cannabinoid mono acylglycerols, such as 1-palmitoylglycerol,vertical farming supplies which indicates that 2-AG formation may not be attributed to a broad, non-specific increase in lipid turnover.
Furthermore, electrical stimulationof the Scheffer col laterals does not modify hippocampal anandamide levels, suggesting that the biochemical pathways lead ing to the production of 2-AG and anandamide may be independently controlled . In further support of this idea, activation of D2 receptors, a potent stimulus for anandamide formation in the rat striatum, has no effect on striatal 2-AG levels .Neuronal and glial cells internalize 2-AG through a mechanism apparently similar to that implicated in anandamide transport. Thus, human astrocytoma and other tumor cells accumulate [3 H]anandamide and [ 3 H]2-AG with similar kinetic properties and this pro cess is blocked by the anandamide transport inhibitor AM404 . In addition, anandamide and 2-AG prevent each other’s transport . Nevertheless, there also appear to be differences between anandamide and 2-AG accumulation. For example, [3 H]2-AG inter nalization in astrocytoma cells is reduced by exogenous arachidonic acid, whereas [3 H]anandamide internaliza tion is not. This discrepancy may be explained in two ways: arachidonic acid may directly interfere with a 2- AG carrier distinct from anandamide’s; or the fatty acid may indirectly prevent the facilitated diffusion of 2-AG by inhibiting its enzymatic conversion to arachi donic acid. If the latter explanation is correct, agents that interfere with the arachidonic acid esterification into phospholipids, such as triacsinC , should decrease [ 3 H]2-AG uptake. This was found indeed to be the case, at least inastrocytoma cells . Thus, while anandamide and 2-AG may be internalized through similar transport mechanisms, they appear to differ inhow their intracellular break down can affect the rate of transport into cells.After removal from the external medium, 2-AG is hydrolyzed to arachidonic acid and glycerol. In cell free preparations, FAAH cleaves anandamide and 2- AG at similar rates, which has led to suggest that this enzyme may contribute to the elimination of both com pounds. This appears to be unlikely, however, for three reasons. First, pig brain tissue contains two distinct 2- AG-hydrolase activities, both of which are chromato graphically different from FAAH . Second, inhibition of FAAH activity in intact neurons and astrocytoma cells prevents the hydrolysis of anandamide, but has no effect on 2-AG degradation . Finally, 2-AG hydrolysis is entirely preserved in FAAH-null mice .
These findings suggest that, although 2-AG can be hydrolyzed by FAAH in vitro, different enzyme may be responsible for its degradation in vivo. A possible candidate for this role is MGL, a cytosolic serine hydrolase that cleaves 2- and 1-monoglycerides into fatty acid and glycerol . The molecular cloning of rat brain MGL has recently allowed the testing of this hypothesis . MGL is abundantly expressed in discrete areas of the rat brain—including the hippocampus, cortex, and cerebel lum—where CB1 receptors are also found. Moreover, adenovirus-induced over expression of MGL enhances the hydrolysis of endogenously produced 2-AG in primary cultures of rat brain neurons . Finally, recent experiments indicate that silencing the MGL gene through RNA interference markedly impairs 2-AG degradation in intact HeLa cells . Although these results strongly support a role of MGL in2-AG hydrolysis, the development of additional experimental tools will be needed to demonstrate such a role unambiguously.Recreational stimulant use is a growing concern among young adults, with 4.4% and 5% to 35% of college students endorsing cocaine and recreational amphetamine use, respectively, and 16% of cocaine experimenters developing dependence within 10 years . To develop cost-effective prevention and intervention strategies, it is crucial to identify ultra–high risk recreational users. However, little is known about bio-behavioral markers forecasting trajectory of occasional stimulant use to stimulant use disorder . Previous stimulant use research is predominantly cross-sectional, comparing individuals with chronic stimulant use with healthy individuals; although findings from these studies highlight brain disruptions related to drug use, they cannot disentangle whether disruptions preceded or were a result of chronic use. Young adulthood is a period of increased independence, often providing more opportunities for risky behavior such as drug experimentation. Risky behavior can be defined as actions that may be subjectively desirable but are potentially harmful and is typically quantified in young adults by their degree of substance use, unprotected sex, health habits, and crime engagement .
Risk taking often occurs in clusters of maladaptive behaviors, suggesting underlying impairments in decision making . Decision making involves several brain processes, including learning, inhibition, and outcome assessment,cannabis indoor greenhouse specifically appraising positive or negative valence of choices . Functional magnetic resonance imaging research indicates that individuals with SUD show impaired decision making associated with altered brain activation in executive control and reward processing regions . Decision making is thought to involve a cooperative relationship between an impulsive system activated by immediate rewards and an inhibitory control system. Through learning, the control network allows individuals to resist immediate attraction to rewards in favor of longer-term advantageous outcomes . In SUD, bio-behavioral indices of risk taking suggest an underlying imbalance between the control and impulsive systems. The control system integral to decision making comprises prefrontal cortex , theorized as responsible for learning the relationship between stimuli and outcome, working memory, and inhibiting behavior . SUD samples exhibit frontal lobe impairments associated with compromised decision making and increased risk behavior . For example, cocaine abusers exhibit dorsolateral PFC hypoactivation during response inhibition and prediction of uncertain outcomes ; in cocaine dependence, orbitofrontal cortex and DLPFC attenuation are linked to reduced ability to differentiate between variable monetary gains . Similarly, methamphetamine users inaccurately process success or failure of available options, a pattern associated with orbito frontal cortex/DLPFC hypoactivation. Working in conjunction with frontal regions is striatum, an area associated with reward processing , selecting and initiating actions , and learning . During the Iowa Gambling Task , healthy individuals show stronger striatal activation to wins than to losses , but amphetamine dependent individuals demonstrate hypersensitive striatal responses to rewards . Cocaine and methamphetamine users also exhibit striatal hyperactivation but frontal hypo activation during risky decision-making tasks such as the Iowa Gambling Task and the Balloon Analogue Risk Task that is linked to riskier behavioral performance . This suggests that such neural patterns during decision making pro mote favoring of risky incentives . Evidence from fMRI studies has led researchers to theorize that frontal lobe and striatum form a functional circuit with insular cortex and anterior cingulate cortex ; these regions coordinate to integrate emotional and autonomic information about rewards into goal-oriented behavior . ACC is proposed to be involved in emotion and behavior management based on its neural connections to both the emotion processing limbic system and the cognitive control center, PFC . Insula is proposed to play a role in interoceptive processing, wherein individuals integrate physiological cues to differentiate between risky and safe decisions and transform these cues into conscious feelings and behaviors . ACC and insula hypoactivation is evident in chronic stimulant users in response inhibition and error monitoring during decision making . Evidence for aberrant activity in key components of the PFC-limbic network has led researchers to suggest that weakened ability to accurately process information about options and control behaviors leads to favoring choices that offer immediate, rather than delayed, rewards .
Cross-sectional studies of occasional stimulant users report decision-making impairments that parallel findings in stimulant-dependent individuals, including 1) weakened inhibitory control and reduced cognitive flexibility ; 2) neuropsychological impairments in executive functions ; and 3) frontal, striatal, and insular attenuation during a Risky Gains Task paired with reduced ability to differentiate between safe and risky decisions . Several research groups have recognized limitations of cross-sectional addiction research and have shifted toward a longitudinal approach to understand the transition to prob lematic substance use . Structural MRI studies show that decreased brain volume in fronto central regions at age 14 years predicts binge drinking at age 16 and that frontostriatal regions are linked to heightened stimulant use in OSUs 1 to 2 years later . However, fMRI has been less applied to predict the development of SUD. The current longitudinal study used follow-up clinical and drug use data from OSUs 3 years after an fMRI scan to determine whether baseline behavioral and blood oxygen level–dependent responses during the RGT 1) differentiated young adults who became problem stimulant users from those who desisted from stimulant use during the 3-year interim and 2) predicted cumulative baseline and follow-up stimulant and marijuana use across OSUs, regardless of clinical status , to address concerns regarding significant rates of marijuana and stimulant co-use . Analyses compared BOLD activity related to specific task requirements: decision contrasts compared BOLD activity during risk-taking choice trials versus safe choice trials; outcome contrasts compared BOLD activity on trials where each subject took a risk and subsequently earned a win or a loss. Categorical hypotheses were tested based on prior bio-behavioral findings in stimulant- dependent individuals: 1) PSUs would exhibit riskier task performance than DSUs; 2) PSUs would show greater striatal BOLD signals than DSUs to out comes, particularly in response to risky wins; and 3) PSUs would exhibit lower PFC, insular, and cingulate BOLD signals during decision making. Because dimensional analyses were exploratory, no a priori hypotheses were tested.The RGT has been previously described by our experimental group . On each trial, participants were shown the numbers 20, 40, and 80 in increasing order, which represented the number of cents to be added to their total. Participants were informed that 20 was always the “safe option” but that they had the option to wait 1 second to receive 40 cents or to wait an additional second to receive 80 cents. They were also informed of the potential that 40 or 80 would appear in red font, denoting actual losses of money from the total score, with 40 and 80 being explicitly called “risky options.” Unknown to subjects, 240 and 280 outcome frequencies were predefined so that the final gain was identical regard less of whether they selected 20, 40, or 80 cents. That is, there was no actual advantage to selecting risky options compared with safe options. Participants were told that a positive value needed to be collected via an index finger button press within a subsequent 1-second window. A press outside of that time frame would result in a loss. The 1- second length was chosen to allow slow-responding individuals to collect an option. Auditory and visual feedback followed each choice. The task consisted of 96 trials lasting 3.5 seconds each. Three trial types were presented in a preset randomized order: 54 rewarded trials, 24 punished 240 trials, and 18 punished 280 trials. In addition to potential losses due to slow responses or non-responses on rewarded trials, the RGT design led to each participant receiving a different amount of 240 and 280 trials.