Neurobiological Effects

Neurobiological Mechanisms – Cellular and Molecular

Much work has focused on the interactions between cannabinoids and brain circuitry in the basal ganglia because of the high density of cannabinoid receptors in this region and the complex effects of cannabinoids on movement. Low doses have activating effects on movement, and high doses have inhibitory effects. CB1 receptors are expressed by the axons of striatal GABAergic medium-spiny neurons. Cannabinoids inhibit GABA-mediated inhibitory postsynaptic potentials in the cell bodies of striatal medium-spiny neurons, possibly by decreasing presynaptic GABA release from medium-spiny axon collaterals. This ultimately could result in a disinhibitory effect by increasing the firing rate of striatal medium-spiny neurons. Similar to their actions in the basal ganglia, cannabinoids in the periaqueductal gray may inhibit presynaptic GABA release or glutamate release and may mediate the well-documented analgesic effects of cannabinoids. In the periaqueductal gray, electrical stimulation produces cannabinoid-mediated analgesia, accompanied by a marked increase in the release of anandamide, suggesting that endogenous anandamide may contribute to this analgesic effect. Similar to the involvement of the basal ganglia in movement and periaqueductal gray in pain, the effects of cannabinoids on reward-related brain structures involve disinhibitory effects. Δ9-THC and synthetic cannabinoids increase the neuronal firing of dopamine neurons in the ventral tegmental area, which can be blocked by CB1 receptor antagonists. The inhibition of GABAergic inhibitory inputs to dopaminergic neurons may increase the firing rate of ventral tegmental area neurons.

FIGURE 8.19 Δ9-THC dose–response curves for intravenous self-administration in squirrel monkeys with no history of exposure to other drugs (n = 3). (A) Numbers of injections per session. (B) Overall rates of responding in the presence of a green light signaling Δ9-THC availability. (C) Total Δ9-THC intake per session. Each panel is presented as a function of the injection dose of Δ9-THC. Each symbol represents the mean of the last three sessions under each Δ9-THC injection dose condition and under a vehicle condition from three monkeys, with the exception of the values for the 1 μg/kg per injection dose, which represent means from two monkeys. ∗p < 0.05, ∗∗p < 0.01, significant differences compared with vehicle conditions. These data show that Δ9-THC is reinforcing in squirrel monkeys. Animals without any deprivation or history of self-administration of other drugs will learn to intravenously self-administer Δ9-THC. The function that relates the unit dose per injection with self-administration shows an inverted U-shaped function, similar to the one seen with other intravenously self-administered drugs in the psychostimulant class. Despite the inverted U-shape function as the unit dose per injection of Δ9-THC increased, the total amount of Δ9-THC self-administered per session increased. [Taken with permission from Justinova Z, Tanda G, Redhi GH, Goldberg SR. Self-administration of Δ9-tetrahydrocannabinol (THC) by drug-naive squirrel monkeys. Psychopharmacology, 2003, (169), 135–140.]

CB1 receptor knockout mouse studies have confirmed a critical role for the CB1 receptor in the behavioral effects of cannabinoids. CB1 knockout mice exhibit no analgesia and no locomotor activity when given Δ9-THC. They also do not intravenously self-administer the CB1 receptor agonist WIN 55,212–2. When these mice are chronically treated with Δ9-THC, they present no cannabinoid withdrawal when administered a CB1 receptor antagonist (Figure 8.23). Other studies have found that anandamide and some synthetic cannabinoids retain their activity in CB1 knockout mice, suggesting that anandamide may act on other receptors aside from CB1 and CB2. One hypothesis is that the effects of anandamide may be mediated by the vanilloid receptor. The vanilloid receptor is a prominent member of the transient receptor potential vanilloid-1 ion channel family. This receptor can be activated by anandamide at concentrations 10 to 20 times higher than those that activate the CB1 receptor.

FIGURE 8.20 Effects of 0.03 and 0.1 mg/kg naltrexone on responding maintained by (A) Δ9-THC and (B) cocaine (Coc) in monkeys over consecutive sessions and extinction of self-administration behavior by substitution of saline injections for injections of (A) Δ9-THC or (B) cocaine. The number of injections per session during Δ9-THC (4 μg/kg/inj) and cocaine (30 μg/kg/inj) self-administration sessions after pretreatment with vehicle (sessions 1–3 and 9–11) or naltrexone (sessions 4–8) is shown. The number of injections per session during self-administration sessions when saline was substituted for Δ9-THC or cocaine (sessions 4–8) is also shown. The data represent the mean number of injections per session from four (Δ9-THC) and three (cocaine) monkeys. ∗∗p < 0.01, compared with the last Δ9-THC session before naltrexone pretreatment or saline substitution (session 3). These data show that low doses of naltrexone blunt intravenous Δ9-THC self-administration in squirrel monkeys but do not block intravenous cocaine self-administration, suggesting that the release of opioid peptides in the brain contributes to the reinforcing effects of Δ9-THC. [Taken with permission from Justinova Z, Tanda G, Munzar P, Goldberg SR. The opioid antagonist naltrexone reduces the reinforcing effects of Δ9-tetrahydrocannabinol (THC) in squirrel monkeys. Psychopharmacology, 2004, (173), 186–194.]

FIGURE 8.21 Effect of intravenous Δ9-THC, WIN 55,212–2, and heroin on dialysate dopamine in the shell (upper panels) and core (lower panels) of the nucleus accumbens. Rats were pretreated with saline (circles), the CB1 receptor antagonist SR141716A (triangles; 1 mg/kg, s.c.), or the opioid receptor antagonist naloxone (diamonds; 0.1 mg/kg, i.p.). Solid symbols: p < 0.05 compared to baseline values. ∗p < 0.05, compared with corresponding value obtained in the shell of saline-pretreated controls. These data show that Δ9-THC, a synthetic cannabinoid, and heroin all preferentially increase the release of dopamine in the nucleus accumbens shell measured by in vivo microdialysis. The cannabinoid CB1 receptor antagonist SR141716 and opioid receptor antagonist naloxone blocked the cannabinoid-induced increase in the release of dopamine, suggesting a role for endogenous opioid peptides in the dopamine-releasing effects of cannabinoids. [Taken with permission from Tanda G, Pontieri FE, Di Chiara G. Cannabinoid and heroin activation of mesolimbic dopamine transmission by a common μ1 opioid receptor mechanism. Science, 1997, (276), 2048–2050.]

FIGURE 8.22 Sagittal section through a rodent brain that illustrates the pathways and receptor systems implicated in the acute reinforcing actions of cannabinoids. Cannabinoids activate cannabinoid CB1 receptors in the ventral tegmental area, nucleus accumbens, and amygdala via direct actions on interneurons. Cannabinoids facilitate the release of dopamine in the nucleus accumbens via an action either in the ventral tegmental area or nucleus accumbens but are also hypothesized to activate elements independent of the dopamine system. Endogenous cannabinoids may interact with postsynaptic elements in the nucleus accumbens that involve dopamine and opioid peptide systems. The blue arrows represent the interactions within the extended amygdala system. AC, anterior commissure; AMG, amygdala; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; Cer, cerebellum; C-P, caudate putamen; DMT, dorsomedial thalamus; FC, frontal cortex; Hippo, hippocampus; IF, inferior colliculus; LC, locus coeruleus; LH, lateral hypothalamus; N Acc., nucleus accumbens; OT, olfactory tract; PAG, periaqueductal gray; RPn, reticular pontine nucleus; SC, superior colliculus; SNr, substantia nigra pars reticulata; VP, ventral pallidum; VTA, ventral tegmental area. [Taken with permission from Koob GF, Le Moal M. Neurobiology of Addiction. Academic Press, London, 2006.]

Supporting the pharmacological studies with cannabinoid receptor antagonists, CB1 knockout mouse studies have shown significant interactions between the reinforcing and dependence-producing effects of opioids and the cannabinoid system. Morphine-induced conditioned place preference, intravenous morphine self-administration, and morphine-induced dopamine release in the nucleus accumbens are all eliminated in CB1 knockout mice. Δ9-THC-induced conditioned place preference is blocked in μ opioid receptor knockout mice, and Δ9-THC-induced conditioned place aversions are blocked in κ opioid receptor knockout mice. However, such cross-talk between the opioid and cannabinoid systems does not extend to measures of analgesia. Opioid-induced antinociception is not modified in CB1 knockout mice, and the antinociception produced by Δ9-THC is not modified in opioid knockout mice. Knockout studies have provided additional evidence that cannabinoid systems are involved in the reinforcing actions of other drugs of abuse, including alcohol and nicotine.

FIGURE 8.23 Central effects of cannabinoids in mice with (+/+) and without (−/−) CB1 receptors. An intraperitoneal injection of Δ9-THC (or vehicle alone) was given 20 min before the measurements. (A) Latency of escape jumping in the hot-plate test. (B) Spontaneous locomotor activity (number of photocell counts within 10 min). (C) Self-administration of the CB1 receptor agonist WIN 55,212–2. (D) Δ9-THC withdrawal signs. These data show that constitutive knockout of the cannabinoid CB1 receptor (animals born without a CB1 receptor) block the analgesic, locomotor-activating, reinforcing, and dependence-inducing effects of cannabinoids. [Taken with permission from Ledent C, Valverde O, Cossu G, Petitet F, Aubert JF, Beslot F, Bohme GA, Imperato A, Pedrazzini T, Roques BP, Vassart G, Fratta W, Parmentier M. Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice. Science, 1999, (283), 401–404.]

The role of endogenous cannabinoid systems in drug dependence in general is supported by opioid-cannabinoid interactions. Administration of the CB1 receptor antagonist SR141716A blocked morphine self-administration in mice and heroin self-administration in rats. Acute administration of SR141716A blocked the expression of morphine-induced conditioned place preference.

Withdrawal/Negative Affect Stage

Neurobiological Mechanisms of Tolerance

Chronic treatment with Δ9-THC in rodents results in tolerance to its acute behavioral effects, such as analgesia, motor inhibition, and the memory-disruptive effects of cannabis. Such tolerance depends on the dose, duration of treatment, species, and dependent variable measured. For example, tolerance to antinociception and motor inhibition occurs with 3–7 days of treatment, but tolerance to the memory-impairing effects can take weeks. There is significant consensus that the mechanism of such tolerance is pharmacodynamic and not pharmacokinetic. Biochemical measures show that chronic exposure to Δ9-THC produces time-dependent and region-specific downregulation and desensitization of brain cannabinoid receptors, as measured by receptor binding and cannabinoid-induced GTPγS binding. GTPγS is guanine 5’-O-[γ-thio]triphosphate, a G-protein-activating analog of guanosine triphosphate used in G-protein binding studies. These effects were most pronounced in the hippocampus and less dramatic in the basal ganglia. Such receptor-mediated changes could at least partially account for the different time courses and degrees of tolerance to the behavioral effects of repeated cannabis administration. Similar downregulation of brain cannabinoid CB1 receptors was observed in human subjects who chronically smoked cannabis, measured with positron emission tomography. The downregulation correlated with the number of years of cannabis smoking and was selective to cortical brain regions. CB1 receptor density returned to normal levels after approximately 4 weeks of continuously monitored abstinence from cannabis in a secure research unit (for further reading, see Hirvonen et al., 2012).

Neurobiological Mechanisms of Withdrawal

In preclinical animal studies, cannabinoid withdrawal syndromes have been described in both rats and mice. Although some somatic signs have been observed during spontaneous withdrawal from cannabinoids, most studies have precipitated withdrawal using a CB1 antagonist. The most characteristic somatic signs of withdrawal in rodents include a combination of the withdrawal signs observed in opioid and sedative-hypnotic (alcohol) withdrawal. In rats, these various somatic withdrawal signs include wet-dog shakes (this is exactly what it sounds like), scratching, facial rubbing, ptosis, mastication, hunched posture, and ataxia. In mice exposed to chronic Δ9-THC administration, administration of the CB1 receptor antagonist SR14716A produced a robust withdrawal syndrome. The withdrawal signs in mice include wet-dog shakes, facial rubbing, ptosis, hunched posture, front paw tremor, piloerection, and ataxia (these signs are also common in opioid withdrawal).

Precipitated cannabinoid withdrawal also has some motivational characteristics. Precipitated Δ9-THC withdrawal causes anxiety-like effects in animal models of anxiety. Spontaneous withdrawal from an acute injection of Δ9-THC can produce an elevation in brain reward thresholds (Figure 8.24). Administration of the CB1 receptor antagonist SR141716A in rats that received long-term cannabinoid agonist treatment (HU-210) resulted in anxiety-like responses in the defensive withdrawal test, in which the time that elapsed to exit a small enclosure and enter a larger space is measured in rodents. Notably, however, acute Δ9-THC administration itself can be considered aversive in rodents, reflected by acute cannabinoid-induced conditioned place aversions at moderate to high doses.

FIGURE 8.24 Diminished brain stimulation reward (elevated reward thresholds) during withdrawal from an acute 1.0 mg/kg dose of Δ9-THC in rats. Withdrawal from Δ9-THC significantly shifted the reward function to the right. These data show that acute withdrawal from THC can also produce "dysphoric-like" responses in animals measured by brain stimulation reward. Notice this is a rate-frequency measure of reward thresholds. [Taken with permission from Gardner EL, Vorel SR. Cannabinoid transmission and reward-related events. Neurobiology of Disease, 1998, (5), 502–533.]

Neurobiological mechanisms that may be involved in the motivational withdrawal syndrome include decreased activity in the mesocorticolimbic dopamine system and activation of brain corticotropin-releasing factor (CRF) systems in the extended amygdala. Precipitated withdrawal from chronic cannabinoids decreases the firing of ventral tegmental dopamine neurons and decreases extracellular dopamine levels in the nucleus accumbens (Figure 8.25). Precipitated withdrawal increases extracellular levels of CRF in the central nucleus of the amygdala, and the anxiogenic-like effects of precipitated Δ9-THC withdrawal are blocked by a CRF receptor antagonist (Figure 8.26).

The CB1 receptor antagonist SR141716A can precipitate both somatic and motivational opioid withdrawal in morphine-dependent rats. Naloxone-precipitated opioid withdrawal is also decreased in CB1 knockout mice. The cannabinoid withdrawal syndrome is also decreased in μ opioid receptor knockout mice and preproenkephalin knockout mice. This interaction is also bidirectional, in which the opioid antagonist naloxone induces partial cannabinoid withdrawal in rats treated chronically with the synthetic CB1 agonist HU-210 (Figure 8.27). This appears to be an interaction with μ opioid receptors. μ Opioid receptor knockout mice chronically treated with Δ9-THC also show blunted Δ9-THC-precipitated withdrawal. In summary, endogenous opioid peptides derived from preproenkephalin appear to be important for the Δ9-THC withdrawal syndrome, and a certain level of endogenous cannabinoid tone appears to contribute to opioid dependence.

FIGURE 8.25 Time course of the effects of administration of the CB1 cannabinoid receptor antagonist SR141716A on dopamine output in dialysate samples from the nucleus accumbens shell and wet-dog shakes in rats chronically treated with Δ9-THC. The bars represent the number of wet-dog shakes observed every 10 min after SR141716A administration. ∗p < 0.05, compared with the corresponding time point in chronic saline-injected rats challenged with SR141716A. These data show that precipitated withdrawal from chronic Δ9-THC decreased the release of dopamine in the nucleus accumbens measured by in vivo microdialysis. This is similar to the effects seen with withdrawal from all drugs of abuse and represents a common element of drug dependence. [Taken with permission from Tanda G, Loddo P, Di Chiara G, Dependence of mesolimbic dopamine transmission on delta9-tetrahydrocannabinol, European Journal of Pharmacology, 1999, (376), 23–26.].

FIGURE 8.26 (A) Effects of a single injection of the cannabinoid receptor agonist HU-210 (100 mg/kg) in rats on corticotropin-releasing factor (CRF) release from the central nucleus of the amygdala. HU-210 lowered CRF release. Vehicle injections did not alter CRF release. Administration of the CB1 receptor antagonist SR141716A did not modify CRF release. (B) Effects of SR141716A (3 mg/kg) on CRF release from the central nucleus of the amygdala in animals pretreated with HU-210 (100 mg/kg) for 14 days. Cannabinoid withdrawal induced by SR141716A was associated with increased CRF release. Vehicle injections did not alter CRF release. (C) Mean of summed cannabinoid withdrawal scores 0, 10, 30, and 60 min after SR141716A injection in rats treated with HU-210 or its vehicle for 14 days. SR141716A induced a mild behavioral syndrome in drug-naive rats that received long-term pretreatment with vehicle (SR141716A) and a clear withdrawal syndrome in animals pretreated with HU-210 (long-term HU-210 + SR141716A). Rats pretreated with the cannabinoid (long-term HU-210) that received vehicle on the test day did not exhibit withdrawal signs. Drug-naive control animals that received vehicle injections were indistinguishable from the long-term HU-210 treatment group, and cannabinoid-naive rats did not exhibit observable changes in behavior after a single injection of HU-210. (D) Anatomical location of the microdialysis probes in animals subjected to SR141716A-induced cannabinoid withdrawal. These data show an increase in CRF release in the central nucleus of the amygdala during precipitated withdrawal measured by in vivo microdialysis. Notice that the increase in CRF release in the central nucleus of the amygdala represents another common element of drug dependence. [Taken with permission from Rodriguez de Fonseca F, Carrera MRA, Navarro M, Koob GF, Weiss F. Activation of corticotropin-releasing factor in the limbic system during cannabinoid withdrawal. Science, 1997, (276), 2050–2054.]

FIGURE 8.27 Naloxone administration induced a partial cannabinoid withdrawal syndrome in male rats chronically exposed to either the cannabinoid receptor agonist or morphine (two 75 mg morphine pellets implanted subcutaneously for 72 h).The CB1 receptor antagonist induced a partial cannabinoid withdrawal syndrome in morphine-dependent animals.∗p<0.05, ∗∗p<0.01, significant differences from saline-treated animals . [Taken with permission from Navarro M, Carrera MRA, Fratta W, Valverde O, Cossu G, Fattore L, Chowen JA, Gomez R, Del Arco I, Villanua MA, Maldonado R, Koob GF, Rodriguez de Fonseca F. Functional interaction between opioid and cannabinoid receptors in drug self-administration. Journal of Neuroscience, 2001, 21: 5344-5350.] These data show that caanabinoid withdrawal can be precipitated by opioid receptor antagonists in cannabinoid-dependent rats, and opioid withdrawal can be precipitated by cannabinoid receptor antagonists in opioid-dependent rats. These results suggest some “cross-talk” in the neuroadaptations between the endogenous cannabinoid and endogenous opioid peptide systems during the chronic activation of their respective receptors.

Preoccupation/Anticipation Stage

Neurobiological Mechanisms – Cellular and Molecular

As described in the other sections, animal models of reinstatement have been used to support hypotheses about the neural substrates of relapse. Treatments that trigger the reinstatement of drug seeking can indicate the likely neural substrates of “craving.” Little work has been done with cannabinoids in the reinstatement model. One study involved rats previously trained to intravenously self-administer the synthetic cannabinoid receptor agonist WIN 55,212–2 under a fixed-ratio 1 schedule of reinforcement and found that intraperitoneal priming injections of a previously self-administered CB1 receptor agonist reinstated cannabinoid-seeking behavior following extinction. Cues paired with WIN 55,212–2 self-administration can also reinstate cannabinoid seeking. The selective CB1 receptor antagonist SR 141716A completely prevented cannabinoid-seeking behavior triggered by WIN 55,212–2, but this antagonist did not reinstate responding when administered alone. The opioid receptor agonist heroin facilitates cannabinoid-seeking behavior, and the opioid receptor antagonist naloxone blocks the cannabinoid-induced reinstatement of cannabinoid-seeking behavior. Such results suggest an endogenous opioid contribution to cannabinoid reinstatement.

Cannabinoids can produce reinstatement of drug seeking for other drugs of abuse. Systemic injections of cannabinoids can reinstate cocaine, alcohol, nicotine, and heroin seeking. A CB1 receptor antagonist blocks the reinstatement of drug seeking in response to cocaine and cocaine-associated cues, nicotine, alcohol, and heroin.

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