Targeting the orexin/hypocretin system for the treatment of neuropsychiatric and neurodegenerative diseases: From animal to clinical studies.

Orexins (also known as hypocretins) are neuropeptides located exclusively in hypothalamic neurons that have extensive projections throughout the central nervous system and bind two different G protein-coupled receptors (OX1R and OX2R). Since its discovery in 1998, the orexin system has gained the interest of the scientific community as a potential therapeutic target for the treatment of different pathological conditions. Considering previous basic science research, a dual orexin receptor antagonist, suvorexant, was the first orexin agent to be approved by the US Food and Drug Administration to treat insomnia. In this review, we discuss and update the main preclinical and human studies involving the orexin system with several psychiatric and neurodegenerative diseases. This system constitutes a nice example of how basic scientific research driven by curiosity can be the best route to the generation of new and powerful pharmacological treatments.

New Insights in the Involvement of the Endocannabinoid System and Natural Cannabinoids in Nicotine Dependence.

Nicotine, the main psychoactive component in tobacco smoke, plays a major role in tobacco addiction, producing a high morbidity and mortality in the world. A great amount of research has been developed to elucidate the neural pathways and neurotransmitter systems involved in such a complex addictive behavior. The endocannabinoid system, which has been reported to participate in the addictive properties of most of the prototypical drugs of abuse, is also implicated in nicotine dependence. This review summarizes and updates the main behavioral and biochemical data involving the endocannabinoid system in the rewarding properties of nicotine as well as in nicotine withdrawal and relapse to nicotine-seeking behavior. Promising results from preclinical studies suggest that manipulation of the endocannabinoid system could be a potential therapeutic strategy for treating nicotine addiction.

Anti-inflammatory agents for smoking cessation? Focus on cognitive deficits associated with nicotine withdrawal in male mice.

Nicotine withdrawal is associated with cognitive deficits including attention, working memory, and episodic memory impairments. These cognitive deficits are a hallmark of nicotine abstinence which could be targeted in order to prevent smoking relapse. The underlying mechanisms, however, are poorly understood. In this study, memory impairment was observed in mice 4 days after the precipitation of nicotine withdrawal by the nicotinic antagonist mecamylamine. The presence of cognitive deficits correlated with microglial activation in the hippocampus and the prefrontal cortex. Moreover, an increased expression of neuroinflammatory markers including IL1ß, TNFα and IFNγ was found in both memory-related brain regions. Notably, flow cytometric analysis also revealed an enhancement of TNFα and IFNγ plasmatic levels at the same time point during nicotine withdrawal. Impaired neurogenesis, as shown by reduction in the expression of the endogenous cell proliferation marker Ki67 and the early neuron marker doublecortin, was also associated with nicotine abstinence. Treatment with the non-psychoactive cannabinoid cannabidiol abolished memory impairment of nicotine withdrawal and microglia reactivity, reduced the expression of IL1ß and IFNγ in the hippocampus and the prefrontal cortex, respectively, and normalized Ki67 levels. The nonsteroidal anti-inflammatory drug indomethacin also prevented cognitive deficits and microglial reactivity during withdrawal. These data underline the usefulness of anti-inflammatory agents to improve cognitive performance during early nicotine abstinence.

Facilitation of Contextual Fear Extinction by Orexin-1 Receptor Antagonism Is Associated with the Activation of Specific Amygdala Cell Subpopulations.

Background: Orexins are hypothalamic neuropeptides recently involved in the regulation of emotional memory. The basolateral amygdala, an area orchestrating fear memory processes, appears to be modulated by orexin transmission during fear extinction. However, the neuronal types within the basolateral amygdala involved in this modulation remain to be elucidated. Methods: We used retrograde tracing combined with immunofluorescence techniques in mice to identify basolateral amygdala projection neurons and cell subpopulations in this brain region influenced by orexin transmission during contextual fear extinction consolidation. Results: Treatment with the orexin-1 receptor antagonist SB334867 increased the activity of basolateral amygdala neurons projecting to infralimbic medial prefrontal cortex during fear extinction. GABAergic interneurons expressing calbindin, but not parvalbumin, were also activated by orexin-1 receptor antagonism in the basolateral amygdala. Conclusions: These data identify neuronal circuits and cell populations of the amygdala associated with the facilitation of fear extinction consolidation induced by the orexin-1 receptor antagonist SB334867.

Adolescent exposure to the Spice/K2 cannabinoid JWH-018 impairs sensorimotor gating and alters cortical perineuronal nets in a sex-dependent manner.

The consumption of synthetic cannabinoids during adolescence is reported to be a risk factor for the appearance of psychiatric disorders later in life. JWH-018 was identified as one of the primary psychoactive components present in Spice/K2 preparations. This study evaluated the short- and long-term consequences of exposure to JWH-018 during the adolescence in anxiety-like behavior, fear extinction, and sensorimotor gating in male and female mice. Alterations in anxiety varied depending on the time interval between treatment and behavioral analysis along with sex, while no changes were observed in the extinction of fear memory. A decrease in prepulse inhibition of the startle reflex was revealed in male, but not female, mice at short- and long-term. This behavioral disturbance was associated with a reduction in the number of perineuronal nets in the prelimbic and infralimbic regions of the prefrontal cortex in the short-term. Furthermore, adolescent exposure to JWH-018 induced an activation of microglia and astrocytes in the prefrontal cortex of male mice at both time intervals. A transitory decrease in the expression of GAD67 and CB2 cannabinoid receptors in the prefrontal cortex was also found in male mice exposed to JWH-018. These data reveal that the treatment with JWH-018 during the adolescence leads to long-lasting neurobiological changes related to psychotic-like symptoms, which were sex-dependent.

CB2 cannabinoid receptor expression is increased in 129S1/SvImJ mice: behavioral consequences.

Genetic and environmental factors are implicated in the etiology of neuropsychiatric diseases. Inbred mouse strains, including the 129S1/SvImJ (S1), constitute important models to study the influence of genetic factors in these conditions. S1 mice displayed anxiogenic-like behavior, impaired fear extinction, and increased prepulse inhibition (PPI) of startle reflex compared to C57BL/6J (BL6) mice. Given the role played by the endocannabinoid system (ECS) in these responses, we evaluated the expression of the ECS components in different brain regions in S1 mice. Gene expression levels of the cannabinoid type-1 and type-2 receptors (CB1R and CB2R) and the endocannabinoid metabolizing enzymes varied depending on the brain region evaluated. Notably, CB2R expression markedly increased in the amygdala, prefrontal cortex and hippocampus in S1 mice. Moreover, CB2R blockade with SR144528 partially rescued the anxiogenic phenotype in S1 mice, while CB2R activation with JWH133 potentiated the deficits in fear extinction and the PPI of startle reflex in this mouse strain. These data suggest that CB2R is involved in the behavioral alterations observed in S1 mice and underline the importance of this cannabinoid receptor subtype in the regulation of certain central nervous system disorders.

Amygdalar CB2 cannabinoid receptor mediates fear extinction deficits promoted by orexin-A/hypocretin-1.

Anxiety and stress disorders are often characterized by an inability to extinguish learned fear responses. Orexins/hypocretins are involved in the modulation of aversive memories, and dysregulation of this system may contribute to the aetiology of anxiety disorders characterized by pathological fear. The mechanisms by which orexins regulate fear are unknown. Here we investigated the role of the endogenous cannabinoid system in the impaired fear extinction induced by orexin-A (OXA) in male mice. The selective inhibitor of 2-arachidonoylglycerol (2-AG) biosynthesis O7460 abolished the fear extinction deficits induced by OXA. Accordingly, increased 2-AG levels were observed in the amygdala and hippocampus of mice treated with OXA that do not extinguish fear, suggesting that high levels of this endocannabinoid are related to poor extinction. Impairment of fear extinction induced by OXA was associated with increased expression of CB2 cannabinoid receptor (CB2R) in microglial cells of the basolateral amygdala. Consistently, the intra-amygdala infusion of the CB2R antagonist AM630 completely blocked the impaired extinction promoted by OXA. Microglial and CB2R expression depletion in the amygdala with PLX5622 chow also prevented these extinction deficits. These results show that overactivation of the orexin system leads to impaired fear extinction through 2-AG and amygdalar CB2R. This novel mechanism could be of relevance for the development of novel potential approaches to treat diseases associated with inappropriate retention of fear, such as post-traumatic stress disorder, panic anxiety and phobias.

Hypocretins regulate the anxiogenic-like effects of nicotine and induce reinstatement of nicotine-seeking behavior.

Emerging evidence suggests that the hypocretinergic system is involved in addictive behavior. In this study, we investigated the role of these hypothalamic neuropeptides in anxiety-like responses of nicotine and stress-induced reinstatement of nicotine-seeking behavior. Acute nicotine (0.8 mg/kg, s.c.) induced anxiogenic-like effects in the elevated plus-maze and activated the paraventricular nucleus of the hypothalamus (PVN) as revealed by c-Fos expression. Pretreatment with the hypocretin receptor 1 (Hcrtr-1) antagonist SB334867 or preprohypocretin gene deletion blocked both nicotine effects. In the PVN, SB334867 also prevented the activation of corticotrophin releasing factor (CRF) and arginine-vasopressin (AVP) neurons, which expressed Hcrtr-1. In addition, an increase of the percentage of c-Fos-positive hypocretin cells in the perifornical and dorsomedial hypothalamic (PFA/DMH) areas was found after nicotine (0.8 mg/kg, s.c.) administration. Intracerebroventricular infusion of hypocretin-1 (Hcrt-1) (0.75 nmol/1 mul) or footshock stress reinstated a previously extinguished nicotine-seeking behavior. The effects of Hcrt-1 were blocked by SB334867, but not by the CRF1 receptor antagonist antalarmin. Moreover, SB334867 did not block CRF-dependent footshock-induced reinstatement of nicotine-seeking while antalarmin was effective in preventing this nicotine motivational response. Therefore, the Hcrt system interacts with CRF and AVP neurons in the PVN and modulates the anxiogenic-like effects of nicotine whereas Hcrt and CRF play a different role in the reinstatement of nicotine-seeking. Indeed, Hcrt-1 reinstates nicotine-seeking through a mechanism independent of CRF activation whereas CRF mediates the reinstatement induced by stress.

Central and peripheral consequences of the chronic blockade of CB1 cannabinoid receptor with rimonabant or taranabant.

The endocannabinoid system plays a crucial role in the pathophysiology of obesity. However, the clinical use of cannabinoid antagonists has been recently stopped because of its central side-effects. The aim of this study was to compare the effects of a chronic treatment with the CB(1) cannabinoid antagonist rimonabant or the CB(1) inverse agonist taranabant in diet-induced obese female rats to clarify the biological consequences of CB(1) blockade at central and peripheral levels. As expected, chronic treatment with rimonabant and taranabant reduced body weight and fat content. Interestingly, a decrease in the number of CB(1) receptors and its functional activity was observed in all the brain areas investigated after chronic taranabant treatment in both lean and obese rats. In contrast, chronic treatment with rimonabant did not modify the density of CB(1) cannabinoid receptor binding, and decreased its functional activity to a lower degree than taranabant. Six weeks after rimonabant and taranabant withdrawal, CB(1) receptor density and activity recovered to basal levels. These results reveal differential adaptive changes in CB(1) cannabinoid receptors after chronic treatment with rimonabant and taranabant that could be related to the central side-effects reported with the use of these cannabinoid antagonists.

Effects of the endogenous PPAR-alpha agonist, oleoylethanolamide on MDMA-induced cognitive deficits in mice.

MDMA (3,4-Methylenedioxymethamphetamine) is an amphetamine derivative widely used for recreational purposes. We have recently shown that repeated treatment with high doses of MDMA-induced impairments in the acquisition and recall of an active avoidance task in mice. In this study, we examined whether the endogenous peroxisome proliferator-activated receptor-alpha (PPAR-alpha) agonist, oleoylethanolamide (OEA) protects against these MDMA-induced deficits. Mice were pretreated twice a day with OEA (0, 5, and 25 mg/kg) 30 min before an injection of MDMA (30 mg/kg) or saline during four consecutive days. Twenty-four hours after the last treatment, animals were trained in an active avoidance task for two consecutive weeks. After a 5-day resting period, a recall session was performed. Mice treated with MDMA showed reduced learning and recall of the task when compared with saline-treated controls. OEA at 5 mg/kg ameliorated and at 25 mg/kg worsened this deficit. Dopamine transporter (DAT)-binding sites significantly decreased 4 days after the last MDMA administration and pretreatment with both doses of OEA prevented this effect. In immunohistochemical studies, coexpression of tyrosine-hydroxylase and PPAR-alpha receptors was observed in the striatum and substantia nigra pars compacta of mice. These results suggest that OEA administration can modulate the cognitive deficits induced by MDMA in a DAT-independent manner.

THC exposure during adolescence does not modify nicotine reinforcing effects and relapse in adult male mice.

RATIONALE: Cannabis use is typically initiated during adolescence, and different studies suggest that adolescent cannabinoid exposure may increase the risk for drug addiction in adulthood. OBJECTIVES: This study investigated the effects of adolescent exposure to the main psychoactive component of cannabis, ∆9-tetrahydrocannabinol (THC), in the reinforcing properties of nicotine in adult male mice. Possible alterations in relapse to nicotine-seeking behaviour in adult animals due to THC adolescent exposure were also evaluated. METHODS: Adolescent mice were exposed to escalating doses of THC from PND35 to PND49. When mice reached adulthood (PND70), surgical procedures were applied for further behavioural evaluation. Nicotine self-administration sessions were conducted consecutively for 10 days. Following extinction, mice were tested for cue- and stress-induced reinstatement of nicotine-seeking behaviour. RESULTS: Adolescent THC treatment did not modify acquisition and extinction of nicotine self-administration in adulthood. Moreover, THC exposure did not alter relapse to nicotine seeking induced by stress or nicotine-associated cues. CONCLUSIONS: These results suggest that a history of exposure to THC during adolescence under these particular conditions does not modify the reinforcing effects and seeking behaviour of nicotine in the adult period.

Prodynorphin gene disruption increases the sensitivity to nicotine self-administration in mice.

The endogenous opioid system has been reported to participate in nicotine behavioural responses. The aim of the study was to determine the contribution of the endogenous peptides derived from prodynorphin in acute and chronic nicotine responses, mainly those related to its addictive properties. Locomotion and nociception were evaluated after acute nicotine administration in prodynorphin knockout mice. In addition, nicotine rewarding properties were investigated in the place-conditioning and the intravenous self-administration paradigms. The somatic signs of nicotine withdrawal were also analysed after the injection of the nicotinic antagonist mecamylamine in nicotine-dependent mice. The hypolocomotor and antinociceptive effects induced by acute nicotine administration were not modified in knockout (KO) animals. Nicotine also produced similar conditioned place preference in both genotypes. However, a shift to the left in the percentage of acquisition of intravenous nicotine-self administration was observed in prodynorphin KO mice. Indeed, a significant increase in the number of KO mice acquiring this operant behaviour was revealed when low doses of nicotine were used. Nicotine physical dependence was similar in wild-type and KO animals. These findings reveal a specific role of endogenous peptides derived from prodynorphin in nicotine self-administration, probably through the modulation of its aversive effects.

Concomitant THC and stress adolescent exposure induces impaired fear extinction and related neurobiological changes in adulthood.

Δ9-tetrahydrocannabinol (THC) consumption during adolescence is reported to be a risk factor for the appearance of psychiatric disorders later in life. The interaction between genetic or environmental events and cannabinoid exposure in the adolescent period can also contribute to exacerbate behavioural deficits in adulthood. Here we investigate the effects of THC treatment as well as the consequences of concomitant THC and stress exposure during adolescence in the extinction of fear memory in adult mice. Adolescent mice treated with THC and exposed to stress exhibit impaired cued fear extinction in adulthood. However, no effect was observed in animals exposed to these two factors separately. Notably, resistance to fear extinction was associated with decreased neuronal activity in the basolateral amygdala (BLA) and the infralimbic prefrontal cortex, suggesting a long-term dysregulation of the fear circuit. These changes in neuronal activation were paralleled with structural plasticity alterations. Indeed, an increase of immature dendritic spines in pyramidal neurons of the BLA was revealed in mice simultaneously exposed to THC and stress. Corticosterone levels were also enhanced after the cued fear conditioning session in the same experimental group. These results show that an interaction between cannabis exposure and stress during adolescence may lead to long-term anxiety disorders characterized by the presence of pathological fear.

Involvement of kappa/dynorphin system in the development of tolerance to nicotine-induced antinociception.

The aim of the present study was to explore the possible role of kappa/dynorphin system in the development of tolerance to nicotine antinociception in mice. First, we observed that kappa-opioid receptor (KOP-r) participates in the acute spinal antinociception produced by nicotine (3 and 5 mg/kg, s.c.) since the pre-treatment with the selective kappa antagonist nor-binaltorphimine (3 mg/kg, i.p.) attenuated this response in the tail-immersion test but not in the hot-plate test nor in locomotor responses. Possible changes in the expression of KOP-r were investigated in tolerant mice to nicotine antinociception by using autoradiography of [3H]CI-977 binding. The density of KOP-r decreased in the spinal cord of tolerant mice. In addition, bi-directional cross-tolerance between nicotine (3 and 5 mg/kg, s.c.) and the selective kappa agonist U50,488H (10 mg/kg, s.c.) was found in the tail-immersion test. Recent evidences indicate that an up-regulation of dynorphin levels in the spinal cord and subsequent activation of NMDA receptors participate in the development of tolerance to opioid and cannabinoid antinociception. In this study, dynorphin content in the lumbar spinal cord was similar in control and nicotine tolerant mice. Furthermore, the administration of the NMDA antagonist MK-801 (0.03 and 0.01 mg/kg, i.p.) before each daily nicotine injection did not modify the development of nicotine tolerance. In summary, these data indicate that KOP-r is directly involved in the development of tolerance to nicotine antinociception by a mechanism independent from dynorphin and NMDA receptors.

Involvement of the endocannabinoid system in drug addiction.

Recent studies have shown that the endocannabinoid system is involved in the common neurobiological mechanism underlying drug addiction. This system participates in the primary rewarding effects of cannabinoids, nicotine, alcohol and opioids, through the release of endocannabinoids in the ventral tegmental area. Endocannabinoids are also involved in the motivation to seek drugs by a dopamine-independent mechanism, demonstrated for psychostimulants and opioids. The endocannabinoid system also participates in the common mechanisms underlying relapse to drug-seeking behaviour by mediating the motivational effects of drug-related environmental stimuli and drug re-exposure. In agreement, clinical trials have suggested that the CB(1) cannabinoid antagonist rimonabant can cause smoking cessation. Thus, CB(1) cannabinoid antagonists could represent a new generation of compounds to treat drug addiction.

MDMA modifies active avoidance learning and recall in mice.

RATIONALE: Several studies have suggested the existence of cognitive deficits after repeated or high doses of 3,4-methylenedioxymethamphetamine (MDMA) in humans and experimental animals. However, the extent of the impairments observed in learning or memory tasks remains unclear. OBJECTIVE: The objective of this study was to evaluate the effects of different dosing regimens of MDMA on the ability of mice to learn and recall an active avoidance task. MATERIALS AND METHODS: Animals were treated with MDMA (0, 1, 3, 10 and 30 mg/kg) under four different experimental conditions, and active avoidance acquisition and recall were evaluated. In experiments 1 and 2, MDMA was administered 1 h before different active avoidance training sessions. In experiments 3 and 4, mice received a repeated treatment with MDMA before or after active avoidance training, respectively. Changes in presynaptic striatal dopamine transporter (DAT) binding sites were evaluated at two different time points in animals receiving a high dose of MDMA (30 mg/kg) or saline twice a day over 4 days. RESULTS: MDMA administered before the active avoidance sessions interfered with the acquisition and the execution of a previously learned task. A repeated treatment with high doses of MDMA administered before training reduced acquisition of active avoidance in mice, while pre-treatment with both high and low doses of MDMA impaired recall of this task. A reduction in DAT binding was observed 4 days but not 23 days after the last MDMA administration. CONCLUSIONS: Acute MDMA modifies the acquisition and execution of active avoidance in mice, while repeated pre-treatment with MDMA impairs acquisition and recall of this task.

Advances in the field of cannabinoid--opioid cross-talk.

A remarkable amount of literature has been generated demonstrating the functional similarities between the endogenous opioid and cannabinoid systems. Anatomical, biochemical and molecular data support the existence of reciprocal interactions between these two systems related to several pharmacological responses including reward, cognitive effects, and the development of tolerance and dependence. However, the assessment of the bidirectionality of these effects has been difficult due to their variety and complexity. Reciprocal interactions have been well established for the development of physical dependence. Cross-tolerance and cross-sensitization, although not always bidirectional, are also supported by a number of evidence, while less data have been gathered regarding the relationship of these systems in cognition and emotion. Nevertheless, the most recent advances in cannabinoid-opioid cross-modulation have been made in the area of drug craving and relapse processes. The present review is focused on the latest developments in the cannabinoid-opioid cross-modulation of their behavioural effects and the possible neurobiological substrates involved.

When orexins meet cannabinoids: Bidirectional functional interactions.

A growing body of evidence suggests the existence of biochemical and functional interactions between the endocannabinoid and orexin systems. Cannabinoid and orexin receptors have been shown to form heterodimers in agreement with the overlapping distribution of both receptors in several brain areas, and the activation of common intracellular signaling pathways, such as the MAP kinase cascade. The activation of orexin receptors induces the synthesis of the endocannabinoid 2-arachidonoyl glycerol suggesting that the endocannabinoid system participates in some physiological functions of orexins. Indeed, functional interactions between these two systems have been demonstrated in several behavioral responses including nociception, reward and food intake. The present review is focused on the latest developments in cannabinoid-orexin cross-modulation and the implications of this interesting interaction.

CB1 Cannabinoid Receptors Mediate Cognitive Deficits and Structural Plasticity Changes During Nicotine Withdrawal.

BACKGROUND: Tobacco withdrawal is associated with deficits in cognitive function, including attention, working memory, and episodic memory. Understanding the neurobiological mechanisms involved in these effects is crucial because cognitive deficits during nicotine withdrawal may predict relapse in humans. METHODS: We investigated in mice the role of CB1 cannabinoid receptors (CB1Rs) in memory impairment and spine density changes induced by nicotine withdrawal precipitated by the nicotinic antagonist mecamylamine. Drugs acting on the endocannabinoid system and genetically modified mice were used. RESULTS: Memory impairment during nicotine withdrawal was blocked by the CB1R antagonist rimonabant or the genetic deletion of CB1R in forebrain gamma-aminobutyric acidergic (GABAergic) neurons (GABA-CB1R). An increase of 2-arachidonoylglycerol (2-AG), but not anandamide, was observed during nicotine withdrawal. The selective inhibitor of 2-AG biosynthesis O7460 abolished cognitive deficits of nicotine abstinence, whereas the inhibitor of 2-AG enzymatic degradation JZL184 did not produce any effect in cognitive impairment. Moreover, memory impairment was prevented by the selective mammalian target of rapamycin inhibitor temsirolimus and the protein synthesis inhibitor anisomycin. Mature dendritic spines on CA1 pyramidal hippocampal neurons decreased 4 days after the precipitation of nicotine withdrawal, when the cognitive deficits were still present. Indeed, a correlation between memory performance and mature spine density was found. Interestingly, these structural plasticity alterations were normalized in GABA-CB1R conditional knockout mice and after subchronic treatment with rimonabant. CONCLUSIONS: These findings underline the interest of CB1R as a target to improve cognitive performance during early nicotine withdrawal. Cognitive deficits in early abstinence are associated with increased relapse risk.

Nicotine-induced antinociception, rewarding effects, and physical dependence are decreased in mice lacking the preproenkephalin gene.

It has been shown previously that the endogenous opioid system may be involved in the behavioral effects of nicotine. In the present study, the participation of endogenous enkephalins on nicotine responses has been investigated by using preproenkephalin knock-out mice. Acute nicotine-induced hypolocomotion remained unaffected in these mice. In contrast, antinociception elicited in the tail-immersion and hot-plate tests by acute nicotine administration was reduced in mutant animals. The rewarding properties of nicotine were then investigated using the place-conditioning paradigm. Nicotine induced a conditioned place preference in wild-type animals, but this effect was absent in knock-out mice. Accordingly, in vivo microdialysis studies revealed that the enhancement in dopamine extracellular levels in the nucleus accumbens induced by nicotine was also reduced in preproenkephalin-deficient mice. Finally, the somatic expression of the nicotine withdrawal syndrome precipitated in nicotine-dependent mice by mecamylamine was significantly attenuated in mutant animals. In summary, the present results indicate that endogenous opioid peptides derived from preproenkephalin are involved in the antinociceptive and rewarding properties of nicotine and participate in the expression of physical nicotine dependence.