Laura E. Wise

Inhibition of Fatty-Acid Amide Hydrolase Accelerates Acquisition and Extinction Rates in a Spatial Memory Task

Recent reports have demonstrated that disruption of CB1 receptor signaling impairs extinction of learned responses in conditioned fear and Morris water maze paradigms. Here, we test the hypothesis that elevating brain levels of the endogenous cannabinoid anandamide through either genetic deletion or pharmacological inhibition of its primary catabolic enzyme fatty-acid amide hydrolase (FAAH) will potentiate extinction in a fixed platform water maze task. FAAH (−/−) mice and mice treated with the FAAH inhibitor OL-135, did not display any memory impairment or motor disruption, but did exhibit a significant increase in the rate of extinction. Unexpectedly, FAAH-compromised mice also exhibited a significant increase in acquisition rate. The CB1 receptor antagonist SR141716 (rimonabant) when given alone had no effects on acquisition, but disrupted extinction. Additionally, SR141716 blocked the effects of OL-135 on both acquisition and extinction. Collectively, these results indicate that endogenous anandamide plays a facilitatory role in extinction through a CB1 receptor mechanism of action. In contrast, the primary psychoactive constituent of marijuana, Δ9-tetrahydrocannabinol, failed to affect extinction rates, suggesting that FAAH is a more effective target than a direct acting CB1 receptor agonist in facilitating extinction. More generally, these findings suggest that FAAH inhibition represents a promising pharmacological approach to treat psychopathologies hallmarked by an inability to extinguish maladaptive behaviors, such as post-traumatic stress syndrome and obsessive-compulsive disorder.

Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory

FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2−/− mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.

Hippocampal CB1 receptors mediate the memory impairing effects of Δ9-tetrahydrocannabinol.

It is firmly established that the hippocampus, a brain region implicated in spatial learning, episodic memory, and consolidation, contains a high concentration of CB1 receptors. Moreover, systemic and intrahippocampal administration of cannabinoid agonists have been shown to impair hippocampal-dependent memory tasks. However, the degree to which CB1 receptors in the hippocampus play a specific functional role in the memory disruptive effects of marijuana or its primary psychoactive constituent Δ9-tetrahydrocannabinol (Δ9-THC) is unknown. This study was designed to determine whether hippocampal CB1 receptors play a functional role in the memory disruptive effects of systemically administered cannabinoids, using the radial arm maze, a well characterized rodent model of working memory. Male Sprague–Dawley rats were implanted with bilateral cannulae aimed at the CA1 region of the dorsal hippocampus. The CB1 receptor antagonist, rimonabant, was delivered into the hippocampus before to a systemic injection of either Δ9-THC or the potent cannabinoid analog, CP-55,940. Strikingly, intrahippocampal administration of rimonabant completely attenuated the memory disruptive effects of both cannabinoids in the radial arm maze task, but did not affect other pharmacological properties of cannabinoids, as assessed in the tetrad assay (that is, hypomotility, analgesia, catalepsy, and hypothermia). Infusions of rimonabant just dorsal or ventral to the hippocampus did not prevent Δ9-THC-induced memory impairment, indicating that its effects on mnemonic function were regionally selective. These findings provide compelling evidence in support of the view that hippocampal CB1 receptors play a necessary role in the memory disruptive effects of marijuana.

Repeated low-dose administration of the monoacylglycerol lipase inhibitor JZL184 retains cannabinoid receptor type 1-mediated antinociceptive and gastroprotective effects.

The monoacylglycerol lipase (MAGL) inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) produces antinociceptive and anti-inflammatory effects. However, repeated administration of high-dose JZL184 (40 mg/kg) causes dependence, antinociceptive tolerance, cross-tolerance to the pharmacological effects of cannabinoid receptor agonists, and cannabinoid receptor type 1 (CB1) downregulation and desensitization. This functional CB1 receptor tolerance poses a hurdle in the development of MAGL inhibitors for therapeutic use. Consequently, the present study tested whether repeated administration of low-dose JZL184 maintains its antinociceptive actions in the chronic constriction injury of the sciatic nerve neuropathic pain model and protective effects in a model of nonsteroidal anti-inflammatory drug–induced gastric hemorrhages. Mice given daily injections of high-dose JZL184 (≥16 mg/kg) for 6 days displayed decreased CB1 receptor density and function in the brain, as assessed in [3H]SR141716A binding and CP55,940 [(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]-stimulated guanosine 5′-O-(3-[35S]thio)triphosphate binding assays, respectively. In contrast, normal CB1 receptor expression and function were maintained following repeated administration of low-dose JZL184 (≤8 mg/kg). Likewise, the antinociceptive and gastroprotective effects of high-dose JZL184 underwent tolerance following repeated administration, but these effects were maintained following repeated low-dose JZL184 treatment. Consistent with these observations, repeated high-dose JZL184, but not repeated low-dose JZL184, elicited cross-tolerance to the common pharmacological effects of Δ9-tetrahydrocannabinol. This same pattern of effects was found in a rimonabant [(5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide)]-precipitated withdrawal model of cannabinoid dependence. Taken together, these results indicate that prolonged, partial MAGL inhibition maintains potentially beneficial antinociceptive and anti-inflammatory effects, without producing functional CB1 receptor tachyphylaxis/tolerance or cannabinoid dependence.

The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model

AIM The present study tested whether the selective monoacylglycerol lipase (MAGL) inhibitor JZL184 would reduce allodynia and paw edema in the carrageenan test. MAIN METHODS The anti-edematous and anti-allodynic effects of JZL184 were compared to those of PF-3845, an inhibitor of fatty acid amide hydrolase (FAAH), and diclofenac, a non-selective cyclooxygenase inhibitor. Cannabinoid receptor involvement in the anti-edematous and anti-allodynic effects of JZL184 was evaluated by administration of the respective CB1 and CB2 receptor antagonists rimonabant and SR144528 as well as with CB1(-/-) and CB2(-/-) mice. JZL184 (1.6, 4, 16, or 40mg/kg) was administered for six days to assess tolerance. KEY FINDINGS JZL184 administered before or after carrageenan significantly attenuated carrageenan-induced paw edema and mechanical allodynia. Complementary genetic and pharmacological approaches revealed that the anti-allodynic effects of JZL184 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, both the anti-edematous and anti-allodynic effects underwent tolerance following repeated injections of high dose JZL184 (16 or 40mg/kg), but repeated administration of low dose JZL184 (4mg/kg) retained efficacy. SIGNIFICANCE These results suggest that the MAGL inhibitor JZL184 reduces inflammatory nociception through the activation of both CB1 and CB2 receptors, with no evidence of tolerance following repeated administration of low doses.

Synaptic dysfunction in the hippocampus accompanies learning and memory deficits in human immunodeficiency virus type-1 Tat transgenic mice.

BACKGROUND Human immunodeficiency virus (HIV) associated neurocognitive disorders (HAND), including memory dysfunction, continue to be a major clinical manifestation of HIV type-1 infection. Viral proteins released by infected glia are thought to be the principal triggers of inflammation and bystander neuronal injury and death, thereby driving key symptomatology of HAND. METHODS We used a glial fibrillary acidic protein-driven, doxycycline-inducible HIV type-1 transactivator of transcription (Tat) transgenic mouse model and examined structure-function relationships in hippocampal pyramidal cornu ammonis 1 (CA1) neurons using morphologic, electrophysiological (long-term potentiation [LTP]), and behavioral (Morris water maze, fear-conditioning) approaches. RESULTS Tat induction caused a variety of different inclusions in astrocytes characteristic of lysosomes, autophagic vacuoles, and lamellar bodies, which were typically present within distal cytoplasmic processes. In pyramidal CA1 neurons, Tat induction reduced the number of apical dendritic spines, while disrupting the distribution of synaptic proteins (synaptotagmin 2 and gephyrin) associated with inhibitory transmission but with minimal dendritic pathology and no evidence of pyramidal neuron death. Electrophysiological assessment of excitatory postsynaptic field potential at Schaffer collateral/commissural fiber-CA1 synapses showed near total suppression of LTP in mice expressing Tat. The loss in LTP coincided with disruptions in learning and memory. CONCLUSIONS Tat expression in the brain results in profound functional changes in synaptic physiology and in behavior that are accompanied by only modest structural changes and minimal pathology. Tat likely contributes to HAND by causing molecular changes that disrupt synaptic organization, with inhibitory presynaptic terminals containing synaptotagmin 2 appearing especially vulnerable.

Evaluation of fatty acid amides in the carrageenan-induced paw edema model

While it has long been recognized that Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, and other cannabinoid receptor agonists possess anti-inflammatory properties, their well known CNS effects have dampened enthusiasm for therapeutic development. On the other hand, genetic deletion of fatty acid amide hydrolase (FAAH), the enzyme responsible for degradation of fatty acid amides, including endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide; AEA), N-palmitoyl ethanolamine (PEA), N-oleoyl ethanolamine (OEA), and oleamide, also elicits anti-edema, but does not produce any apparent cannabinoid effects. The purpose of the present study was to investigate whether exogenous administration of FAAs would augment the anti-inflammatory phenotype of FAAH (-/-) mice in the carrageenan model. Thus, we evaluated the effects of the FAAs AEA, PEA, OEA, and oleamide in wild-type and FAAH (-/-) mice. For comparison, we evaluated the anti-edema effects of THC, dexamethasone (DEX), a synthetic glucocorticoid, diclofenac (DIC), a nonselective cyclooxygenase (COX) inhibitor, in both genotypes. A final study determined if tolerance to the anti-edema effects of PEA occurs after repeated dosing. PEA, THC, DEX, DIC elicited significant decreases in carrageenan-induced paw edema in wild-type mice. In contrast OEA produced a less reliable anti-edema effect than these other drugs, and AEA and oleamide failed to produce any significant decreases in paw edema. Moreover, none of the agents evaluated augmented the anti-edema phenotype of FAAH (-/-) mice, suggesting that maximal anti-edema effects had already been established. PEA was the most effective FAA in preventing paw edema and its effects did not undergo tolerance. While the present findings do not support a role for AEA in preventing carrageenan-induced edema, PEA administration and FAAH blockade elicited anti-edema effects of an equivalent magnitude as produced by THC, DEX, and DIC in this assay.

Combination of Rimonabant and Donepezil Prolongs Spatial Memory Duration

The observations that the cannabinoid1(CB1) receptor antagonist/inverse agonist, rimonabant, and the selective noncompetitive inhibitor of acetylcholinesterase (AChE), donepezil, improve performance in a variety of animal memory models, suggest that these neurochemical systems play integral roles in cognition. The present study tested whether each of these agents administered alone or in combination will prolong the duration of spatial memory. Rats were trained in a two-phase radial-arm maze procedure, consisting of acquisition and retrieval tests, which were separated by an 18 h delay. Each drug was administered 30 min before the acquisition phase, immediately after the acquisition phase, or 30 min before the retrieval test to assess acquisition/consolidation, consolidation, and retrieval mnemonic processes, respectively. Rimonabant or donepezil administered before the acquisition phase, but not immediately after acquisition or before retrieval, led to a significant decrease in the number of errors committed during the retrieval test. Combined administration of subthreshold doses of rimonabant and donepezil that had no discernable effects on performance when given alone, enhanced memory. These results taken together demonstrate that the delay radial-arm maze task is sufficiently sensitive to detect memory enhancing effects of these drugs. Moreover, these findings suggest that combined administration of subthreshold doses of rimonabant and donepezil can improve memory and may represent a novel approach to treat cognitive deficits associated with neurodegenerative disorders.

Dual fatty acid amide hydrolase and monoacylglycerol lipase blockade produces THC-like Morris water maze deficits in mice.

Acute administration of Δ(9)-tetrahydrocannabinol (THC) or exposure to marijuana smoke impairs short-term spatial memory in water maze tasks through a CB(1) receptor mechanism of action. N-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) are endogenous cannabinoids that are predominantly metabolized by the respective enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Although the MAGL inhibitor JZL184 enhances short-term synaptic plasticity, it has yet to be evaluated in the Morris water maze. Previous research demonstrated that simultaneous, complete blockade of FAAH and MAGL produces full blown THC-like effects. Thus, in the following studies we tested whether dual blockade of FAAH and MAGL would impair learning in a repeated acquisition Morris water maze task. Mice treated with the dual FAAH/MAGL inhibitor JZL195 (20 mg/kg) as well as JZL184-treated FAAH -/- mice displayed robust deficits in Morris water maze performance that were similar in magnitude to THC-treated mice. While 20 or 40 mg/kg impaired water maze performance in FAAH -/- mice, only the high dose of JZL184 disrupted performance in FAAH +/+ mice. The memory impairing effects of JZL184 were blocked by the CB(1) receptor antagonist rimonabant. Neither JZL184 nor JZL195 impaired performance in a cued version of the water maze task, arguing against the notion that sensorimotor or motivational deficits accounted for the impaired acquisition performance. JZL184 increased 2-AG levels in the hippocampus, prefrontal cortex, and cerebellum to a similar degree in FAAH -/- and +/+ mice. FAAH -/- mice, regardless of drug treatment, possessed elevated AEA levels in each brain region assessed. The results of this study reveal that concomitant increases in AEA and 2-AG disrupt short-term spatial memory performance in a manner similar to that of THC.

Clozapine discrimination with a low training dose distinguishes atypical from typical antipsychotic drugs in rats

Abstract  Rationale: Previous drug discrimination studies with clozapine have not reliably distinguished between atypical and typical antipsychotics. Objectives: The present study was conducted to determine whether low-dose clozapine drug discrimination could distinguish atypical from typical antipsychotics. Methods: Rats were trained to discriminate 1.25 mg/kg clozapine from vehicle in a two-lever drug discrimination procedure. Results: Generalization testing revealed full substitution with the atypical antipsychotics olanzapine (90.3% maximum generalization), sertindole (99.8%), and risperidone (87.1%) and partial substitution for quetiapine (seroquel, 66.4%) and the typical antipsychotics haloperidol (56.8%) and thioridazine (74.3%). Remoxipride (23.1%) and the typical antipsychotics chlorpromazine (27.9%) and fluphenazine (29.5%) did not reliably substitute for clozapine. Conclusions: In contrast to previous clozapine drug discrimination studies with higher training doses, the atypical antipsychotics olanzapine, sertindole, and risperidone reliably substituted for clozapine while typical antipsychotics did not. These results suggest that low-dose clozapine drug discrimination may be a more sensitive assay for distinguishing atypical from typical antipsychotic drugs.