Contextual Fear Conditioning Training Research Articles

Memory persistence induced by environmental enrichment is dependent on different brain structures

Environmental enrichment (EE) has been demonstrated to have a beneficial effect on different functions of the central nervous system in several mammal species, being used to improve behavior and cell damage in various neurological and psychiatric diseases. However, little has been investigated on the effect of EE in healthy animals, particularly regarding its impact on memory persistence and the brain structures involved. Therefore, here we verified in male Wistar rats that contextual fear conditioning (CFC) memory persistence, tested 28 days after the CFC training session, was facilitated by 5 weeks of exposure to EE, with no effect in groups tested 7 or 14 days after CFC training. However, a two-week exposure to EE did not affect memory persistence. Moreover, we investigated the role of specific brain regions in mediating the effect of EE on memory persistence. We conducted inactivation experiments using the GABAergic agonist Muscimol to target the basolateral amygdala (BLA), medial prefrontal cortex (mPFC), and CA1 region of the hippocampus (CA1). Inactivation of the BLA immediately and 12 h after CFC training impaired the effect of EE on memory persistence. Similarly, inactivation of the CA1 region and mPFC 12 h after training, but not immediately, also impaired the effect of EE on memory persistence. These results have important scientific implications as they shed new light on the effect of an enriched environment on memory persistence and the brain structures involved, thereby helping elucidate how an environment rich in experiences can modify the persistence of learned information.

Contextual fear conditioning with a time interval induces CREB phosphorylation in the dorsal hippocampus and amygdala nuclei that depend on prelimbic cortex activity.

In temporal associations, a conditioned stimulus (CS) is separated by a time interval from the unconditioned stimulus (US), which activates the prelimbic cortex (PL) to maintain a CS representation over time. However, it is unknown whether the PL participates, besides the encoding, in the memory consolidation, and thus directly, with activity-dependent changes or indirectly, by modulation of activity-dependent changes in other brain regions. We investigated brain regions supporting the consolidation of associations with intervals and the influence of PL activity in this consolidation process. For this, we observed in Wistar rats the effect of pre-training PL inactivation by muscimol in CREB (cAMP response element-binding protein) phosphorylation, which is essential for memory consolidation, in subdivisions of the medial prefrontal cortex (mPFC), hippocampus, and amygdala 3h after the training in the contextual fear conditioning (CFC) or CFC with 5-s interval (CFC-5s), fear associations without or with an interval between the CS and US, respectively. Both the CFC-5s and CFC training increased phosphorylation of CREB in the PL and infralimbic cortex (IL); lateral (LA) and basolateral (BLA) amygdala; dorsal CA1 (dCA1); dorsal (dDG), and ventral dentate gyrus, and the CFC-5s training in the central amygdala (CEA). PL activity was necessary for the CREB phosphorylation in the PL, BLA, CEA, dCA1, and dDG only in animals trained in the CFC-5s. The cingulate cortex, ventral CA1, and ventral subiculum did not have learning-induced phosphorylation of CREB. These results suggest that the mPFC, hippocampus, and amygdala support the consolidation of associations with or without intervals and that PL activity influences consolidation in the dorsal hippocampus and amygdala in temporal associations. Thereby, the PL contributes directly and indirectly by modulation to memory consolidation. The time interval engaged the PL early in recent memory consolidation. Results expanded PL's role beyond the time interval and remote memory consolidation.

Involvement of medial prefrontal cortex canonical Wnt/β-catenin and non-canonical Wnt/Ca2+ signaling pathways in contextual fear memory in male rats

Wnt proteins activate different signaling pathways, such as the canonical Wnt/β-catenin signaling pathway and non-canonical β-catenin-independent signaling pathway and have been related to several functions in central nervous system, including learning and memory. However, whether these signaling pathways are required in the medial prefrontal cortex (mPFC) for fear memory acquisition, consolidation and retrieval remains unclear. To address this question, we submitted male rats to a contextual fear conditioning (CFC) paradigm, and administered canonical Wnt/β-catenin and non-canonical Wnt/Ca2+ signaling pathways inhibitors, DKK1 and SFRP1, respectively, into the prelimbic (PrL) subdivision of the mPFC at different moments and evaluated short-term and long-term memory acquisition, consolidation and retrieval. We found that blocking canonical Wnt/β-catenin and non-canonical Wnt/Ca2+ signaling pathways 15 min before or immediately after CFC training had no effect on STM and LTM of CFC, while their blockade 15 min before the retention test prevented the retrieval of STM and LTM of CFC. These results highlight the importance of the mPFC in fear memory retrieval demonstrating that both canonical Wnt/β-catenin and non-canonical Wnt/Ca2+ signaling pathways participate in this process. To understand how brain systems act on fear memories could provide a new target for the treatment of fear related disorders such as post-traumatic stress disorder and other anxiety disorders.

Effects of different intensities of treadmill exercise on cued fear extinction failure, hippocampal BDNF decline, and Bax/Bcl-2 ratio alteration in chronic−morphine treated male rats

Chronic morphine impairs cued fear extinction, which may contribute to the high prevalence of anxiety disorders and the replase of opiate addiction. This work investigated the effects of forced exercise with different intensities on cued fear extinction impairment and alternations of hippocampal BDNF and apoptotic proteins induced by chronic morphine. Rats were injected with bi-daily doses of morphine or saline for ten days and then received a cued or contextual fear conditioning training, which was followed by fear extinction training for four consecutive days. Cued, but the not contextual fear response was impaired in morphine-treated rats. Then, different saline or morphine-treated rats underwent forced exercise for 4-weeks with light, moderate or high intensities. Subsequently, rats received a cued fear conditioning followed by four days of extinction training, and the expression of hippocampal BDNF and apoptotic proteins was determined. A relatively long time after the last injection of morphine (35 days), rats again showed cued fear extinction failure and reduced hippocampal BDNF, which recovered by light and moderate, but not high exercise. Light and moderate, but not high-intensity treadmill exercise enhanced the up-regulation of Bcl-2 and down-regulation of the Bax proteins in both saline- and morphine-treated rats, which shifted the balance between pro-apoptotic and anti-apoptotic factors in favor of cell survival. These findings highlight the impact of exercise up to moderate intensity in the recovery of cued extinction failure, more likely via BDNF in addicted individuals.

Adaptive Immune Deficiency Impairs Neural Activity After Training and Retrieval.

Neuroimmune interactions have been studied for decades. Several neurodevelopmental disorders have been associated with immune dysfunction. However, the effects of immune system on neuronal function remain unknown. Herein, based on c-Fos protein expression, we characterized the brain areas that are activated after contextual fear conditioning (CFC) training or retrieval in severe combined immune deficiency (SCID) and wild-type mice. Further, we analyzed the interregional correlations of c-Fos activity that are affected by deficiency in adaptive immunity. Results showed significantly lower c-Fos density in learning and memory-associated brain regions of SCID mice after memory retrieval, but not during the CFC training. Moreover, SCID mice exhibited remarkably discordant interregional neuronal activities of learning neuron circuits after CFC training, which could be the cause of inefficient activation of the memory circuit after retrieval. These results provide a new perspective on how adaptive immunity affects neuronal function. Adaptive immune deficiency impairs the coordination of neural activity after training and retrieval, which might be a potential therapeutic target for neurodevelopmental disorders.

Corticosterone differentially modulates time-dependent fear generalization following mild or moderate fear conditioning training in rats

Stressful and emotionally arousing experiences create strong memories that seem to lose specificity over time. It is uncertain, however, how the stress system contributes to the phenomenon of time-dependent fear generalization. Here, we investigated whether post-training corticosterone (CORT-HBC) injections, given after different training intensities, affect contextual fear memory specificity at several time points. We trained male Wistar rats on the contextual fear conditioning (CFC) task using two footshock intensities (mild CFC, 3 footshocks of 0.3 mA, or moderate CFC, 3x 0.6 mA) and immediately after the training session we administered CORT-HBC systemically. We first tested the animals in a novel context and then in the training context at different intervals following training (2, 14, 28 or 42 days). By measuring freezing in the novel context and then contrasting freezing times shown in both contexts, we inferred contextual fear generalization for each rat, classifying them into Generalizers or Discriminators. Following mild CFC training, the glucocorticoid injection promoted an accurate contextual memory at the recent time point (2 days), and increase the contextual memory accuracy 28 days after training. In contrast, after the moderate CFC training, CORT-HBC facilitated contextual generalization at 14 days, compared to the control group that maintained contextual discrimination at this timepoint. For this training intensity, however, CORT-HBC did not have any effect on recent memory specificity. These findings indicate that treatment with CORT-HBC immediately after the encoding of mild or moderately arousing experiences may differentially modulate memory consolidation and time-dependent fear generalization.

054 Characterization of Sleep Phenotypes and Sleep-Dependent Memory Consolidation in a Mouse Model of Fragile X Syndrome

Abstract Introduction Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by disruption of Fmr1 gene function, leading to intellectual disability. FXS individuals report increased incidence of sleep disruptions such as loss of NREM sleep, irregular sleep/wake cycles, and circadian rhythm disturbances that warrant pharmacological intervention. Since sleep has critical roles in the promotion of memory consolidation, it is unknown whether disrupted cognitive function in FXS is exacerbated by abnormal sleep. We characterized the link between sleep loss phenotypes and cognition in FXS mice (Fmr1 KO). We hypothesized that normalizing sleep in Fmr1 KO mice could improve sleep-dependent cognitive function. Because direct activation of G-protein inward rectifying potassium (GIRK) channels by ML297 has been found to promote NREM sleep, we tested how ML297 affected sleep and memory consolidation phenotypes in Fmr1 KO mice. Methods Wild type (WT) and Fmr1 KO were implanted with electrodes for electroencephalogram/electromyogram (EEG/EMG) recording of wakefulness, NREM and REM sleep. Sleep-dependent memory consolidation was measured using single-trial contextual fear conditioning (CFC). ML297 or vehicle was administered after CFC training to measure the effects on sleep and fear memory consolidation. Results Fmr1 KO mice showed reduced sleep in the hours following CFC learning compared to wild type littermates, and reduced contextual fear memory consolidation. Post-CFC sleep deprivation disrupted memory consolidation in wild type littermates, but not Fmr1 KO mice. Both NREM sleep time and NREM bout length were reduced in Fmr1 KO mice, and preliminary data suggest reduced NREM delta (0.5–4 Hz) power in the prefrontal cortex. These deficits were present at baseline and also following CFC. Post-CFC training administration of ML297 rescued NREM sleep and contextual fear memory deficits in Fmr1 KO mice. Conclusion Our study showed a strong link between NREM sleep loss and cognitive deficits in Fmr1 KO mice. Critically, normalization of NREM sleep through direct activation of GIRK channels rescues cognitive deficits seen in Fmr1 KO mice, suggesting a new therapeutic approach to treating cognitive deficits associated with FXS. Support (if any) This work was supported by a Rackham Merit Fellowship to JDM.

Hippocampal CCR5/RANTES Elevations in a Rodent Model of Post-Traumatic Stress Disorder: Maraviroc (a CCR5 Antagonist) Increases Corticosterone Levels and Enhances Fear Memory Consolidation.

Background: Contextual fear conditioning (CFC) is a rodent model that induces a high and long-lasting level of conditioning associated with traumatic memory formation; this behavioral paradigm resembles many characteristics of posttraumatic stress disorder (PSTD). Chemokines (chemotactic cytokines) play a known role in neuronal migration and neurodegeneration but their role in cognition is not totally elucidated. Aim: We ascertain whether CCR5/RANTES beta chemokines (hippocampus/prefrontal cortex) could play a role in fear memory consolidation (CFC paradigm). We also evaluated whether chronic stress restraint (21 days of restraint, 6-h/day) could regulate levels of these beta chemokines in CFC-trained rats; fear memory retention was determined taking the level of freezing (context and tone) by the animals as an index of fear memory consolidation 24 h after CFC training session; these chemokines (CCR5/RANTES) and IL-6 levels were measured in the hippocampus and prefrontal cortex of chronically stressed rats, 24 h after CFC post-training, and compared with undisturbed CFC-trained rats (Experiment 1). In Experiment 2, rats received 1 mA of footshock during the CFC training session and fear memory consolidation was evaluated at 12 and 24 h after CFC training sessions. We evaluated whether RANTES levels could be differentially regulated at 12 and 24 h after CFC training; in Experiment 3, maraviroc was administered to rats (i.m: 100 mg/Kg, a CCR5 antagonist) before CFC training. These rats were not subjected to chronic stress restraint. We evaluated whether CCR5 blockade before CFC training could increase corticosterone, RANTES, or IL-6 levels and affects fear memory consolidation in the rats 24-h post-testing compared with vehicle CFC-trained rats. Results: Elevations of CCR5/RANTES chemokine levels in the hippocampus could have contributed to fear memory consolidation (24 h post-training) and chronic stress restraint did not affect these chemokines in the hippocampus; there were no significant differences in CCR5/RANTES levels between stressed and control rats in the prefrontal cortex (Experiment 1). In Experiment 2, hippocampal CCR5/RANTES levels increased and enhanced fear memory consolidation was observed 12 and 24 h after CFC training sessions with 1 mA of footshock. Increased corticosterone and CCR5/RANTES levels, as well as a higher freezing percentage to the context, were found at 24 h CFC post-testing in maraviroc-treated rats as compared to vehicle-treated animals (experiment-3). Conversely, IL-6 is not affected by maraviroc treatment in CFC training. Conclusion. Our findings suggest a role for a hippocampal CCR5/RANTES axis in contextual fear memory consolidation; in fact, RANTES levels increased at 12 and 24 h after CFC training. When CCR5 was blocked by maraviroc before CFC training, RANTES (hippocampus), corticosterone levels, and fear memory consolidation were greater than in vehicle CFC-trained rats 24 h after the CFC session.

Relationship between footshock intensity, post-training corticosterone release and contextual fear memory specificity over time

Overgeneralized fear has long been implicated in generalized anxiety and post-traumatic stress disorder, however, time-dependent mechanisms underlying memory retrieval are still not completely understood. Previous studies have revealed that stronger fear conditioning training protocols are associated with both increased post-training corticosterone (CORT) levels and fear responses at later retrieval tests. Here we used contextual fear conditioning (CFC) to investigate the relationship between post-training CORT levels and memory specificity in different retrieval timepoints. Wistar rats were exposed to CFC training with increasing footshock intensities (0.3, 0.6 or 1.0mA) and had their blood collected 30 min afterwards to measure post-training plasma CORT. After 2, 14 or 28 days, rats were tested for memory specificity either in the training or in the novel context. Regression analysis was used to verify linear and non-linear interactions between CORT levels and freezing. Higher footshock intensities increased post-training CORT levels and freezing times during tests in all timepoints. Moreover, stronger trainings elicited faster memory generalization, which was associated with higher CORT levels during memory consolidation. The 0.3mA training maintained memory specificity up to 28 days. Additionally, linear regressions suggest that the shift from specific to generalized memories is underway at 14 days after training. These results are consistent with the hypotheses that stronger training protocols elicit a faster generalization rate, and that this process is associated with increased post-training CORT release.

Coincident Pre- and Post-Synaptic Cortical Remodelling Disengages Episodic Memory from Its Original Context.

The view that the neocortex is remotely recruited for long-term episodic memory recall is challenged by data showing that an intense transcriptional and synaptic activity is detected in this region immediately after training. By measuring markers of synaptic activity at recent and remote time points from contextual fear conditioning (CFC), we could show that pre-synaptic changes are selectively detected 1day post-training when the memory is anchored to the training context. Differently, pre- and post-synaptic changes are detected 14days post-training when the memory generalizes to other contexts. Confirming that coincident pre- and post-synaptic remodelling mediates the disengagement of memory from its original context, DREADDs-mediated enhancement of cortical neuron activity during CFC training anticipates expression of a schematic memory and observation of bilateral synaptic remodelling. Together, our data show that the plastic properties of cortical synapses vary over time and specialise in relation to the quality of memory.

Fear conditioning downregulates miR-138 expression in the hippocampus to facilitate the formation of fear memory.

Fear memory is important for the survival of animals and is associated with certain anxiety disorders, such as posttraumatic stress disorder. A thorough understanding of the molecular mechanisms of fear memory, especially associative fear memory, is imperative. MicroRNA-138 (miR-138) is a widely distributed microRNA in the brain and is locally enriched at synaptic sites. The role of miR-138 in the formation of fear memory is still largely unknown. In this study, a contextual fear conditioning (CFC) paradigm, bioinformatic methods, a luciferase assay, real-time PCR and western blot were used to evaluate the detailed effects of miR-138 on fear memory. We found that miR-138 transiently decreased in the dorsal hippocampus (DH) after CFC training. Upregulation or downregulation of miR-138 in the DH with miR-138 agomir or antagomir treatment significantly impaired or enhanced the formation of CFC memory, respectively. Moreover, the effects of miR-138 in the DH on the formation of CFC memory were achieved by changing the expression of the downstream target gene calpain 1 (Capn1). Taken together, both the in-vitro evidence and the in-vivo evidence presented in this study support the involvement of miR-138 in CFC memory formation, at least partly via the regulation of Capn1-mediated synaptic plasticity changes. Therapeutic use of miR-138/Capn1 is promising as an alternative option in the treatment of fear memory-related anxiety disorders.

Alterations in MicroRNA-132/212 Expression Impairs Fear Memory in Goldfish Carassius auratus

Background: Earlier, we showed that nicotinamide (NAM) treatment impairs spatial memory through the downregulation of CREB-Sirt 1-brain-derived neurotrophic factor (Bdnf) signaling cascade. Purpose: In this study, we examine whether NAM treatment alters CREB-regulated genes through ­microRNAs. Method: To test this hypothesis, goldfish (Carassius auratus) were divided into 2 groups: (i) vehicle group (VEH; double distilled water intra-peritoneally [i.p.]) (ii) nicotinamide group (NAM, 1,000 mg/kg, i.p.) and again divided into VEH untrained/trained, NAM untrained/trained. One hour after receiving VEH or NAM, individuals were subject to contextual fear conditioning (CFC) training. After 24 h, both the groups were tested for contextual fear memory. Subsequently, miR-132/212 levels, regulated immediate-early genes (IEGs: C-fos and EGR-1) and Bdnf but not its receptor. ­TrkB1were examined following 0’ and 60’ min after training, and compared with the untrained group. Results: We observed that NAM treatment significantly impaired fear memory. Further, the analysis showed that miR-132 level was not altered, but miR-212 level was significantly upregulated after CFC training only in NAM-treated fish. We also found that NAM treatment downregulated IEGs and Bdnf but not its receptor TrkB1. Conclusions: Present study suggests that NAM-treatment impaired fear memory and control IEGs, Bdnf and TrkB1 expression by differentially regulating miR-132 and 212.

Direct Neuronal Glucose Uptake Is Required for Contextual Fear Acquisition in the Dorsal Hippocampus.

The metabolism of glucose is a nearly exclusive source of energy for maintaining neuronal survival, synaptic transmission and information processing in the brain. Two glucose metabolism pathways have been reported, direct neuronal glucose uptake and the astrocyte-neuron lactate shuttle (ANLS), which can be involved in these functions simultaneously or separately. Although ANLS in the dorsal hippocampus (DH) has been proved to be required for memory consolidation, the specific metabolic pathway involved during memory acquisition remains unclear. The DH and amygdala are two key brain regions for acquisition of contextual fear conditioning (CFC). In 2-NBDG experiments, we observed that 2-NBDG-positive neurons were significantly increased during the acquisition of CFC in the DH. However, in the amygdala and cerebellum, 2-NBDG-positive neurons were not changed during CFC training. Strikingly, microinjection of a glucose transporter (GLUT) inhibitor into the DH decreased freezing values during CFC training and 1 h later, while injection of a monocarboxylate transporter (MCT) inhibitor into the amygdala also reduced freezing values. Therefore, we demonstrated that direct neuronal glucose uptake was the primary means of energy supply in the DH, while ANLS might supply energy in the amygdala during acquisition. Furthermore, knockdown of GLUT3 by a lentivirus in the DH impaired the acquisition of CFC. Taken together, the results indicated that there were two different glucose metabolism pathways in the DH and amygdala during acquisition of contextual fear memory and that direct neuronal glucose uptake in the DH may be regulated by GLUT3.

Uhrf2 deletion impairs the formation of hippocampus-dependent memory by changing the structure of the dentate gyrus.

Ubiquitin-like with PHD and ring finger domains 2 (Uhrf2) is distributed in many brain regions, including the cortex and hippocampus. Decreased Uhrf2 expression is involved in neurodegenerative disease. A recent study showed Uhrf2 deletion impaired spatial memory; however, the mechanism remains elusive. In our study, we determined that Uhrf2+/- and Uhrf2-/- mice had significant learning and memory deficiencies in contextual fear conditioning (CFC) and the novel place recognition test but not in the novel object recognition test. Interestingly, there were no changes in the Uhrf2 protein levels in the hippocampus of C57BL6 mice after CFC training, which suggests Uhrf2 in adult mice may not be related to the formation of CFC long-term memory. Based on Nissl staining, Uhrf2 deletion caused neuropathological changes specifically in the crest of the dentate gyrus (DG), such as cell swelling, a vague outline and confused boundary; however, no changes were identified in the medial prefrontal cortex (mPFC). Transmission electron microscope assay further indicated a series of abnormal ultrastructure changes in neurons and glia in the DG crest. These results suggested that Uhrf2 deletion selectively blocked the development of the DG crest and impaired hippocampus-dependent learning and memory. Our study will facilitate a better understanding of the role of Uhrf2 protein in the central nervous system.

Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation.

Galectin-3, a member of the galectin protein family, has been found to regulate cell proliferation, inhibit apoptosis and promote inflammatory responses. Galectin-3 is also expressed in the adult rat hippocampus, but its role in learning and memory function is not known. Here, we found that contextual fear-conditioning training, spatial training or injection of NMDA into the rat CA1 area each dramatically decreased the level of endogenous galectin-3 expression. Overexpression of galectin-3 impaired fear memory, whereas galectin-3 knockout (KO) enhanced fear retention, spatial memory and hippocampal long-term potentiation. Galectin-3 was further found to associate with integrin α3, an association that was decreased after fear-conditioning training. Transfection of the rat CA1 area with small interfering RNA against galectin-3 facilitated fear memory and increased phosphorylated focal adhesion kinase (FAK) levels, effects that were blocked by co-transfection of the FAK phosphorylation-defective mutant Flag-FAKY397F. Notably, levels of serine-phosphorylated galectin-3 were decreased by fear conditioning training. In addition, blockade of galectin-3 phosphorylation at Ser-6 facilitated fear memory, whereas constitutive activation of galectin-3 at Ser-6 impaired fear memory. Interestingly galectin-1 plays a role in fear-memory formation similar to that of galectin-3. Collectively, our data provide the first demonstration that galectin-3 is a novel negative regulator of memory formation that exerts its effects through both extracellular and intracellular mechanisms.

Age-dependent changes in autophosphorylation of alpha calcium/calmodulin dependent kinase II in hippocampus and amygdala after contextual fear conditioning.

The hippocampus and amygdala are essential brain regions responsible for contextual fear conditioning (CFC). The autophosphorylation of alpha calcium-calmodulin kinase II (αCaMKII) at threonine-286 (T286) is a critical step implicated in long-term potentiation (LTP), learning and memory. However, the changes in αCaMKII levels with aging and training in associated brain regions are not fully understood. Here, we studied how aging and training affect the levels of phosphorylated (T286) and proportion of phosphorylated:total αCaMKII in the hippocampus and amygdala. Young and aged mice, naïve (untrained) and trained in CFC, were analysed by immunohistochemistry for the levels of total and phosphorylated αCaMKII in the hippocampus and amygdala. We found that two hours after CFC training, young mice exhibited a higher level of phosphorylated and increased ratio of phosphorylated:total αCaMKII in hippocampal CA3 stratum radiatum. Furthermore, aged untrained mice showed a higher ratio of phosphorylated:total αCaMKII in the CA3 region of the hippocampus when compared to the young untrained group. No effect of training or aging were seen in the central, lateral and basolateral amygdala regions, for both phosphorylated and ratio of phosphorylated:total αCaMKII. These results show that aging impairs the training-induced upregulation of autophosphorylated (T286) αCaMKII in the CA3 stratum radiatum of the hippocampus. This indicates that distinct age-related mechanisms underlie CFC that may rely more heavily on NMDA receptor-dependent plasticity in young age.

HDAC7 Ubiquitination by the E3 Ligase CBX4 Is Involved in Contextual Fear Conditioning Memory Formation.

Histone acetylation, an epigenetic modification, plays an important role in long-term memory formation. Recently, histone deacetylase (HDAC) inhibitors were demonstrated to promote memory formation, which raises the intriguing possibility that they may be used to rescue memory deficits. However, additional research is necessary to clarify the roles of individual HDACs in memory. In this study, we demonstrated that HDAC7, within the dorsal hippocampus of C57BL6J mice, had a late and persistent decrease after contextual fear conditioning (CFC) training (4-24 h), which was involved in long-term CFC memory formation. We also showed that HDAC7 decreased via ubiquitin-dependent degradation. CBX4 was one of the HDAC7 E3 ligases involved in this process. Nur77, as one of the target genes of HDAC7, increased 6-24 h after CFC training and, accordingly, modulated the formation of CFC memory. Finally, HDAC7 was involved in the formation of other hippocampal-dependent memories, including the Morris water maze and object location test. The current findings facilitate an understanding of the molecular and cellular mechanisms of HDAC7 in the regulation of hippocampal-dependent memory.SIGNIFICANCE STATEMENT The current findings demonstrated the effects of histone deacetylase 7 (HDAC7) on hippocampal-dependent memories. Moreover, we determined the mechanism of decreased HDAC7 in contextual fear conditioning (CFC) through ubiquitin-dependent protein degradation. We also verified that CBX4 was one of the HDAC7 E3 ligases. Finally, we demonstrated that Nur77, as one of the important targets for HDAC7, was involved in CFC memory formation. All of these proteins, including HDAC7, CBX4, and Nur77, could be potential therapeutic targets for preventing memory deficits in aging and neurological diseases.

Heterozygous Che-1 KO mice show deficiencies in object recognition memory persistence

Transcriptional regulation is a key process in the formation of long-term memories. Che-1 is a protein involved in the regulation of gene transcription that has recently been proved to bind the transcription factor NF-κB, which is known to be involved in many memory-related molecular events. This evidence prompted us to investigate the putative role of Che-1 in memory processes. For this study we newly generated a line of Che-1+/− heterozygous mice. Che-1 homozygous KO mouse is lethal during development, but Che-1+/− heterozygous mouse is normal in its general anatomical and physiological characteristics. We analyzed the behavioral characteristic and memory performance of Che-1+/− mice in two NF-κB dependent types of memory. We found that Che-1+/− mice show similar locomotor activity and thigmotactic behavior than wild type (WT) mice in an open field. In a similar way, no differences were found in anxiety-like behavior between Che-1+/− and WT mice in an elevated plus maze as well as in fear response in a contextual fear conditioning (CFC) and object exploration in a novel object recognition (NOR) task. No differences were found between WT and Che-1+/− mice performance in CFC training and when tested at 24h or 7days after training. Similar performance was found between groups in NOR task, both in training and 24h testing performance. However, we found that object recognition memory persistence at 7days was impaired in Che-1+/− heterozygous mice. This is the first evidence showing that Che-1 is involved in memory processes.

Impairment of contextual fear extinction by chronic nicotine and withdrawal from chronic nicotine is associated with hippocampal nAChR upregulation

Chronic nicotine and withdrawal from chronic nicotine have been shown to be major modulators of fear learning behavior. Moreover, recent studies from our laboratory have shown that acute nicotine impaired fear extinction and safety learning in mice. However, the effects of chronic nicotine and withdrawal on fear extinction are unknown. Therefore, the current experiments were conducted to investigate the effects of chronic nicotine as well as withdrawal from chronic nicotine on contextual fear extinction in mice. C57BL6/J mice were given contextual fear conditioning training and retention testing during chronic nicotine administration. Mice then received contextual fear extinction either during chronic nicotine or during withdrawal from chronic nicotine. Our results showed that contextual fear extinction was impaired both during chronic nicotine administration and subsequent withdrawal. However, it was also observed that the effects of prior chronic nicotine disappeared after 72 h in withdrawal, a timeline that closely matches with the timing of the chronic nicotine-induced upregulation of hippocampal nicotinic acetylcholine receptor (nAChR) density. Additional experiments found that 4 days, but not 1 day, of continuous nicotine administration upregulated hippocampal nAChRs and impaired contextual fear extinction. These effects disappeared following 72 h withdrawal. Overall, these experiments provide a potential link between nicotine-induced upregulation of hippocampal nAChRs and fear extinction deficits observed in patients with anxiety disorders, which may lead to advancements in the pharmacological treatment methods for this disorder.

Possible involvement of hippocampal immediate–early genes in contextual fear memory deficit induced by cranial irradiation

Cranial irradiation can trigger adverse effects on brain functions, including cognitive ability. However, the cellular and molecular mechanisms underlying radiation-induced cognitive impairments remain still unknown. Immediate–early genes (IEGs) are implicated in neuronal plasticity and the related functions (i.e., memory formation) in the hippocampus. The present study quantitatively assessed changes in the mRNA and protein levels of the learning-induced IEGs, including Arc, c-fos, and zif268, in the mouse hippocampus after cranial irradiation using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry, respectively. Mice (male, 8-week-old C57BL/6) received whole-brain irradiation with 0 or 10Gy of gamma-ray and, 2weeks later, contextual fear conditioning (CFC) was used to induce IEGs. In the CFC task, mice evaluated 2weeks after irradiation exhibited significant memory deficits compared with sham (0Gy)-irradiated controls. The levels of mRNA encoding IEGs were significantly upregulated in the hippocampus 10 and 30min after CFC training. The mRNA levels in the irradiated hippocampi were significantly lower than those in the sham-irradiated controls. The IEG protein levels were significantly increased in all hippocampal regions, including the hippocampal dentate gyrus, cornu ammonis (CA)1, and CA3, after CFC training. The CFC-induced upregulation of Arc and c-fos in 10Gy-irradiated hippocampi was significantly lower than that in sham-irradiated controls, although there were no significant differences in the protein levels of the learning-induced zif268 between sham-irradiated and 10Gy-irradiated hippocampi. Thus, cranial irradiation with 10Gy of gamma-ray impairs the induction of hippocampal IEGs (particularly Arc and c-fos) via behavioral contextual fear memory, and this disturbance may be associated with the memory deficits evident in mice after cranial irradiation, possibly through the dysregulation of neuronal plasticity during memory formation.