Phosphodiesterase 4 inhibition after retrieval switches the memory fate favoring extinction instead of reconsolidation.

Phosphodiesterase 4 (PDE4), an enzyme expressed in the dorsal hippocampus (DH), hydrolyzes the cAMP, limiting the PKA-induced CREB phosphorylation (pCREB) and BDNF expression. Depending on the brain region, PKA and pCREB mediate reconsolidation or extinction, whereas BDNF is mainly related to extinction facilitation. The mechanisms underpinning the switch between reconsolidation and extinction are relatively unknown. Here, we tested the hypothesis that PDE4 might control these processes. We showed in Wistar rats submitted to contextual fear conditioning that PDE4 inhibition with roflumilast (ROF) within the DH, after a short retrieval, did not change freezing behavior after one day (TestA1). After 10 days, the ROF-treated group significantly reduced the expression of freezing behavior. This effect depended on retrieval, Test A1 exposure, and reinstated after a remainder foot shock, suggesting an extinction facilitation. The ROF effect depended on PKA after retrieval or, protein synthesis after Test A1. After retrieval, ROF treatment did not change the pCREB/CREB ratio in the DH. It enhanced proBDNF expression without changing pre-proBDNF or mature BDNF in the DH after Test A1. The results suggest that the inhibition of PDE4 in the DH after a short retrieval changes the memory sensibility from reconsolidation to extinction via regulating proBDNF expression.

Involvement of cannabinoid receptors and neuroinflammation in early sepsis: Implications for posttraumatic stress disorder.

Sepsis is associated with several comorbidities in survivors, such as posttraumatic stress disorder (PTSD). This study investigated whether rats that survive sepsis develop the generalization of fear memory as a model of PTSD. Responses to interventions that target the endothelin-1 (ET-1)/cannabinoid system and glial activation in the initial stages of sepsis were evaluated. As a control, we evaluated hyperalgesia before fear conditioning. Sepsis was induced by cecal ligation and puncture (CLP) in Wistar rats. CLP-induced sepsis with one or three punctures resulted in fear generalization in the survivors 13 and 20 days after the CLP procedure, a process that was not associated with hyperalgesia. Septic animals were intracerebroventricularly treated with vehicle, the endothelin receptor A (ETA) antagonist BQ123, the cannabinoid CB1 and CB2 receptor antagonists AM251 and AM630, respectively, and the glial blocker minocycline 4 h after CLP. The blockade of either CB1 or ETA receptors increased the survival rate, but only the former reversed fear memory generalization. The endothelinergic system blockade is important for improving survival but not for fear memory. Treatment with the CB2 receptor antagonist or minocycline also reversed the generalization of fear memory but did not increase the survival rate that was associated with CLP. Minocycline treatment also reduced tumor necrosis factor-α levels in the hippocampus suggesting that neuroinflammation is important for the generalization of fear memory induced by CLP. The influence of CLP on the generalization of fear memory was not related to Arc protein expression, a regulator of synaptic plasticity, in the dorsal hippocampus.

The Estrous Cycle Influences the Effects of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Inhibition in the Anxiety-Like Behavior in Rats.

Background: Sex differences in the response to the anxiety-related effects of cannabinoid drugs have been reported, with females being more sensitive than males. Evidence suggests that, according to sex and estrous cycle phase (ECP), the content of the endocannabinoids (eCBs) N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) varies in brain areas involved in the anxiety-like behavior. Methods: Considering the lack of studies evaluating sex and ECP differences in the eCB system in anxiety, using URB597, a fatty acid amide hydrolase inhibitor, or MJN110, a monoacylglycerol lipase inhibitor, we explored the effects of increasing AEA or 2-AG levels, respectively, in cycling and ovariectomized (OVX) female adult Wistar rats, as well as males, subjected to the elevated plus maze. Results: The administration of URB597 (0.1 or 0.3mg/kg; intraperitoneally) either increased or reduced the percentage of open arms time (%OAT) and open arms entries (%OAE), being anxiolytic in diestrus and anxiogenic in estrus. No effects were observed in proestrus or when all ECPs were analyzed together. Both doses produced anxiolytic-like effects in males. In OVX females, the anxiolytic-like effect of URB597 0.1 was associated with low levels of estradiol, whereas the anxiogenic-like effect of URB597 0.3 was spared by estradiol pretreatment. The systemic administration of MJN110 3.0 mg/kg reduced the risk assessment behavior (RAB), suggesting an anxiolytic-like effect independent of the ECP. When considering the ECP, MJN110 3.0 increased the %OAT and reduced the RAB, being anxiolytic in estrus and diestrus. No effects were observed in proestrus. Both doses of MJN110 were anxiogenic in males. In OVX females, the anxiolytic-like effect of MJN110 was dependent on low estradiol levels. Conclusion: Together, our findings support the evidence that females react differently to the effects of cannabinoids in the anxiety-like behavior; in addition, AEA and 2-AG modulation elicits anxiety-like responses that are closely influenced by hormone levels, mainly estradiol.

Cannabidiol modulates contextual fear memory consolidation in animals with experimentally induced type-1 diabetes mellitus.

OBJECTIVES: In view of the neuroprotective characteristic of cannabidiol (CBD) and its beneficial action on aversive memory in non-diabetic animals, we aimed to investigate in animals with experimentally induced type-1 diabetes mellitus (T1DM) whether CBD treatment would be able to impair the contextual fear memory consolidation, its generalisation and whether the effect would be lasting. We also investigated the CBD effect on anxiety-like responses. METHODS: After T1DM induction, animals received single or more prolonged treatment with CBD and were submitted to the contextual fear conditioning test. As expression of activity-regulated cytoskeletal-associated (Arc) protein is necessary for memory consolidation, we evaluated its expression in the dorsal hippocampus (DH). For evaluating anxiety-related responses, animals were submitted to the elevated plus maze test (EPMT), in which the time and number of entries in the open arms were used as anxiety index. RESULTS: A single injection of CBD impaired the contextual fear memory consolidation and its generalisation, which was evaluated by exposing the animal in a neutral context. This single injection was able to reduce the elevated expression of Arc in the DH from these animals. Interestingly, more prolonged treatment with CBD also impaired the persistence of context-conditioned fear memory and induced an anxiolytic-like effect, as the treated group spent more time in the open arms of the EPMT. CONCLUSION: CBD interferes with contextual fear memory and the dosage regimen of treatment seems to be important. Moreover, we cannot rule out the involvement of emotional aspects in these processes related to fear memory.

Effects of delta-9 tetrahydrocannabinol on fear memory labilization and reconsolidation: A putative role of GluN2B-NMDA receptor within the dorsal hippocampus.

Cannabis preparations could be an effective reconsolidation-based treatment for post-traumatic stress disorder. However, the effects of Δ9-tetrahydrocannabinol (THC) in fear memory labilization, a critical condition for retrieval-induced reconsolidation, are undetermined. We sought to investigate the effect of a conventional and an ultra-low dose of THC in memory labilization of adult male Wistar rats submitted to contextual fear conditioning. Pretreatment with THC 0.002, but not THC 0.3 mg/kg, i. p., before memory retrieval, did not change memory expression during the retrieval but impaired reconsolidation. No treatment changed freezing expression in an unpaired context. Before retrieval, THC 0.3, but not THC 0.002, decreased GluN2A-NMDA expression and the GluN2A/GluN2B ratio in the dorsal hippocampus (DH) 24 h later. No changes were observed immediately after retrieval. Pretreatment with THC 0.3 abolished the reconsolidation-impairing effect of anisomycin injected into the DH, suggesting an impairment in memory labilization. This effect was associated with an increased freezing expression in the unpaired context and was not observed with the THC ultra-low dose. The GluN2B-NMDA antagonism increased fear generalization in the anisomycin-treated group but restored its reconsolidation-impairing effect and reduced fear generalization when animals were pretreated with THC 0.3. GluN2A-NMDA antagonism or inhibition of the ubiquitin-proteasome system in the DH did not interfere with the effects of THC 0.3. Our findings indicate that THC causes a bidirectional effect on fear memory labilization that depends on hippocampal GluN2B-NMDA receptors' involvement in fear memory generalization.

Female but not male rats show biphasic effects of low doses of Δ9-tetrahydrocannabinol on anxiety: can cannabidiol interfere with these effects?

Δ9-tetrahydrocannabinol (THC) is the main phytocannabinoid present in the Cannabis sativa. It can produce dose-dependent anxiolytic or anxiogenic effects in males. THC effects on anxiety have scarcely been studied in females, despite their higher prevalence of anxiety disorders. Cannabidiol, another phytocannabinoid, has been reported to attenuate anxiety and some THC-induced effects. The present study aimed to investigate the behavioral and neurochemical effects of THC administered alone or combined with CBD in naturally cycling female rats tested in the elevated plus-maze. Systemically administered THC produced biphasic effects in females, anxiolytic at low doses (0.075 or 0.1 mg/kg) and anxiogenic at a higher dose (1.0 mg/kg). No anxiety changes were observed in males treated with the same THC dose range. The anxiogenic effect of THC was prevented by co-administration of CBD (1.0 or 3.0 mg/kg). CBD (3.0 mg/kg) caused an anxiolytic effect. At a lower dose (1.0 mg/kg), it facilitated the anxiolytic effect of the low THC dose. The anxiogenic effect of THC was accompanied by increased dopamine levels in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). In contrast, its anxiolytic effect was associated with increased mPFC serotonin concentrations. The anxiolytic effect of CBD was accompanied by increased mPFC serotonin turnover. Together, these results indicate that female rats are susceptible to the biphasic effects of low THC doses on anxiety. These effects could depend on mPFC and NAc dopaminergic and serotoninergic neurotransmissions. CBD could minimize potential THC high-dose side effects whereas enhancing the anxiolytic action of its low doses in females.

Effects of ∆9-tetrahydrocannabinol on aversive memories and anxiety: a review from human studies.

BACKGROUND: Posttraumatic stress disorder (PTSD) may stem from the formation of aberrant and enduring aversive memories. Some PTSD patients have recreationally used Cannabis, probably aiming at relieving their symptomatology. However, it is still largely unknown whether and how Cannabis or its psychotomimetic compound Δ9-tetrahydrocannabinol (THC) attenuates the aversive/traumatic memory outcomes. Here, we seek to review and discuss the effects of THC on aversive memory extinction and anxiety in healthy humans and PTSD patients. METHODS: Medline, PubMed, Cochrane Library, and Central Register for Controlled Trials databases were searched to identify peer-reviewed published studies and randomized controlled trials in humans published in English between 1974 and July 2020, including those using only THC and THC combined with cannabidiol (CBD). The effect size of the experimental intervention under investigation was calculated. RESULTS: At low doses, THC can enhance the extinction rate and reduce anxiety responses. Both effects involve the activation of cannabinoid type-1 receptors in discrete components of the corticolimbic circuitry, which could couterbalance the low "endocannabinoid tonus" reported in PTSD patients. The advantage of associating CBD with THC to attenuate anxiety while minimizing the potential psychotic or anxiogenic effect produced by high doses of THC has been reported. The effects of THC either alone or combined with CBD on aversive memory reconsolidation, however, are still unknown. CONCLUSIONS: Current evidence from healthy humans and PTSD patients supports the THC value to suppress anxiety and aversive memory expression without producing significant adverse effects if used in low doses or when associated with CBD. Future studies are guaranteed to address open questions related to their dose ratios, administration routes, pharmacokinetic interactions, sex-dependent differences, and prolonged efficacy.

Persistence of the extinction of fear memory requires late-phase cAMP/PKA signaling in the infralimbic cortex.

Fear extinction is a form of new learning that inhibits expression of the original fear memory without erasing the conditioned stimulus-unconditioned stimulus association. Much is known about the mechanisms that underlie the acquisition of extinction, but the way in which fear extinction is maintained has been scarcely explored. Evidence suggests that protein kinase A (PKA) in the frontal cortex might be related to the persistence of extinction. Phosphodiesterase-4 (PDE4) specifically hydrolyzes cyclic adenosine monophosphate (cAMP). The present study evaluated the effect of the selective PDE4 inhibitor roflumilast (ROF; 0.01, 0.03, and 0.1 mg/kg given i.p.) on acquisition and consolidation of the extinction of fear memory in male Wistar rats in a contextual fear conditioning paradigm. When administered before acquisition, 0.1 mg/kg ROF disrupted short-term (1 day) extinction recall. In contrast, 0.03 mg/kg ROF administration in the late consolidation phase (3 h after extinction learning) but not in the early phase immediately after learning improved long-term extinction recall at 11 days, suggesting potentiation of the persistence of extinction. This effect of ROF requires the first (day 1) exposure to the context. A similar effect was observed when 9 ng ROF or 30 µM 8-bromoadenosine 3',5'-cAMP (PKA activator) was directly infused in the infralimbic cortex (IL), a brain region necessary for memory extinction. The PKA activity-dependent ROF-induced effect in the IL was correlated with an increase in its brain-derived neurotrophic factor (BDNF) protein expression, while blockade of PKA with 10 µM H89 in the IL abolished the ROF-induced increase in BDNF expression and prevented the effect of ROF on extinction recall. These effects were not associated with changes in anxiety-like behavior or general exploratory behavior. Altogether, these findings suggest that cAMP-PKA activity in the IL during the late consolidation phase after extinction learning underlies the persistence of extinction.

Role of prelimbic cortex PKC and PKMζ in fear memory reconsolidation and persistence following reactivation.

The persistence of newly acquired memories is supported by the activity of PKMζ, an atypical isoform of protein kinase C (PKC). Whether the activity of conventional and atypical PKC isoforms contributes to reactivated memories to persist is still unknown. Similarly, whether memory reactivation is a prerequisite for interventions to be able to change memory persistence is scarcely investigated. Based on the above, we examined the role of conventional and atypical PKC isoforms in the prelimbic cortex in reconsolidation and persistence of a reactivated contextual fear memory in male Wistar rats. It is shown that (i) inhibiting the PKC activity with chelerythrine or the PKMζ activity with ZIP impaired the persistence of a reactivated memory for at least 21 days; (ii) ZIP given immediately after memory reactivation affected neither the reconsolidation nor the persistence process. In contrast, when given 1 h later, it impaired the memory persistence; (iii) chelerythrine given immediately after memory reactivation impaired the reconsolidation; (iv) omitting memory reactivation prevented the chelerythrine- and ZIP-induced effects: (v) the ZIP action is independent of the time elapsed between its administration and the initial memory test. The results indicate that prelimbic cortex PKC and PKMζ are involved in memory reconsolidation and persistence.

A time-dependent contribution of hippocampal CB1 , CB2 and PPARγ receptors to cannabidiol-induced disruption of fear memory consolidation.

BACKGROUND AND PURPOSE: In preclinical studies, cannabidiol (CBD) mitigates fear memories by facilitating their extinction or interfering with their generalization and reconsolidation. The brain regions and mechanisms underlying these effects, and their temporal window, are still poorly understood. Here, we have investigated related questions in the dorsal hippocampus (DH) during contextual fear consolidation. EXPERIMENTAL APPROACH: Adult male Wistar rats received CBD (10-30 pmol) intra-DH immediately, 1 or 3 hr after fear conditioning. Effects of CBD on consolidation were inferred behaviourally and by analysing expression of the activity-regulated, cytoskeleton-associated (Arc) protein. The contribution of anandamide, CB1 , CB2 , 5-HT1A , A2A , and PPARγ receptors was also assessed. KEY RESULTS: CBD impaired memory consolidation when given immediately or 1 hr after fear conditioning, but not after 3 hr. Expression of Arc protein in DH was reduced by systemic CBD treatment in both cases. Immediately after fear conditioning, CBD effects were abolished by CB1 or CB2 receptor blockade, partly reduced by 5-HT1A or A2A antagonism, and remained unchanged after antagonism of PPARγ receptors. One hour after fear conditioning, CBD effects were prevented only by PPARγ receptor antagonism. Also, inhibition of fatty acid amide hydrolase by URB597, impaired memory consolidation when infused immediately, but not 1 hr after fear conditioning. CONCLUSIONS AND IMPLICATIONS: CBD disrupts memory consolidation up to 1 hr after fear conditioning, allowing an extended window of opportunity to mitigate aversive memories after their acquisition. Our results suggest time-dependent participation of anandamide, CB1 , CB2 and PPARγ receptors in the DH, during this process.