Sex differences in training-induced activity of the ubiquitin proteasome system in the dorsal hippocampus and medial prefrontal cortex of male and female mice.

The ubiquitin proteasome system (UPS) is a primary mechanism through which proteins are degraded in cells. UPS activity in the dorsal hippocampus (DH) is necessary for multiple types of memory, including object memory, in male rodents. However, sex differences in DH UPS activation after fear conditioning suggest that other forms of learning may also differentially regulate DH UPS activity in males and females. Here, we examined markers of UPS activity in the synaptic and cytoplasmic fractions of DH and medial prefrontal cortex (mPFC) tissue collected 1 h following object training. In males, training increased phosphorylation of proteasomal subunit Rpt6, 20S proteasome activity, and the amount of PSD-95 in the DH synaptic fraction, as well as proteasome activity in the mPFC synaptic fraction. In females, training did not affect measures of UPS or synaptic activity in the DH synaptic fraction or in either mPFC fraction but increased Rpt6 phosphorylation in the DH cytoplasmic fraction. Overall, training-induced UPS activity was greater in males than in females, greater in the DH than in the mPFC, and greater in synaptic fractions than in cytosol. These data suggest that object training drives sex-specific alterations in UPS activity across brain regions and subcellular compartments important for memory.

Isolation driven changes in Iba1-positive microglial morphology are associated with social recognition memory in adults and adolescents.

Microglia are critical for regulation of neuronal circuits that mature from adolescence to adulthood. The morphological complexity and process length of microglia can indicate different activation states. These states are sensitive to a variety of environmental and stress conditions. Microglia are sensitive to many factors that also regulate social behavior, and in turn, microglial manipulations can impact social function. Brief social isolation is one factor that can lead to robust social changes. Here, we explored the role of microglia in the effects of brief social isolation on social recognition memory. Using morphological measures of Iba1 to index microglial intensity, complexity, and process length, we identified different effects of brief isolation on microglial complexity in the basal region of the amygdala between adults and adolescents alongside overall increases in intensity of Iba1 in several cortical brain regions. Short-term social recognition memory is sensitive to the amount of social engagement, and provides an opportunity to test if social engagement produced by brief isolation enhances social learning in a manner that relies on microglia. We found that brief isolation facilitated social interaction across ages but had opposing effects on short-term social recognition. Isolation increased novel partner investigation in adolescents, which is consistent with better social recognition, but increased familiar partner investigation in adults. Depletion of microglia with PLX3397 prevented these effects of brief isolation in adolescents, and reduced them in adults. These results suggest that distinct changes in microglial function driven by the social environment may differentially contribute to subsequent social recognition memory during development.

Contextual control of conditioned pain tolerance and endogenous analgesic systems.

The mechanisms underlying the transition from acute to chronic pain are unclear but may involve the persistence or strengthening of pain memories acquired in part through associative learning. Contextual cues, which comprise the environment in which events occur, were recently described as a critical regulator of pain memory; both male rodents and humans exhibit increased pain sensitivity in environments recently associated with a single painful experience. It is unknown, however, how repeated exposure to an acute painful unconditioned stimulus in a distinct context modifies pain sensitivity or the expectation of pain in that environment. To answer this question, we conditioned mice to associate distinct contexts with either repeated administration of a mild visceral pain stimulus (intraperitoneal injection of acetic acid) or vehicle injection over the course of 3 days. On the final day of experiments, animals received either an acid injection or vehicle injection prior to being placed into both contexts. In this way, contextual control of pain sensitivity and pain expectation could be tested respectively. When re-exposed to the noxious stimulus in a familiar environment, both male and female mice exhibited context-dependent conditioned analgesia, a phenomenon mediated by endogenous opioid signaling. However, when expecting the presentation of a painful stimulus in a given context, males exhibited conditioned hypersensitivity whereas females exhibited endogenous opioid-mediated conditioned analgesia. These results are evidence that pain perception and engagement of endogenous opioid systems can be modified through their psychological association with environmental cues. Successful determination of the brain circuits involved in this sexually dimorphic anticipatory response may allow for the manipulation of pain memories, which may contribute to the development of chronic pain states.

Unique roles for the anterior and posterior retrosplenial cortices in encoding and retrieval of memory for context.

The rat retrosplenial cortex (RSC) makes critical contributions to learning and memory but these contributions may not be uniform along its rostro-caudal axis. Previous work suggests that event-related and context-related information are differentially encoded by anterior and posterior RSC subregions. Here, we further test this idea using a procedure in which spatial/environmental cues (context) and discrete event memories are acquired separately. All animals received a 5-min pre-exposure to the training context 24 h before contextual fear conditioning where shock was delivered immediately upon being placed in the chamber. Rats were tested for memory for the context the next day. We found that optogenetic inhibition of cells in only the posterior RSC during the pre-exposure phase, when spatial information is encoded, reduced behavioral responding during the subsequent memory test. However, similar inhibition of either the anterior or posterior RSC during shock delivery, when information about both the context and the shock become integrated, impaired memory. Finally, inhibiting cellular activity in only the posterior RSC during memory retrieval during testing reduced responding. Together, these results suggest that while activity in both subregions is needed during the period in which the event-related information becomes integrated with the context representation, the posterior RSC is important for both memory formation and retrieval or expression of memory for information about the context. These results add to a growing literature demonstrating a role for the RSC in integration of multiple aspects of memory, and provide information on how spatial representations reliant on the retrosplenial cortex interact with associative learning.

Optogenetic inhibition of either the anterior or posterior retrosplenial cortex disrupts retrieval of a trace, but not delay, fear memory.

Previous work investigating the role of the retrosplenial cortex (RSC) in memory formation has demonstrated that its contributions are not uniform throughout the rostro-caudal axis. While the anterior region was necessary for encoding CS information in a trace conditioning procedure, the posterior retrosplenial cortex was needed to encode contextual information. Using the same behavioral procedure, we tested if there was a similar dissociation during memory retrieval. First, we found that memory retrieval following trace conditioning results in increased neural activity in both the anterior and posterior retrosplenial cortex, measured using the immediate early gene zif268. Similar increases were not found in either RSC subregion using a delay conditioning task. We then found that optogenetic inhibition of neural activity in either subregion impairs retrieval of a trace, but not delay, memory. Together these results add to a growing literature showing a role for the retrosplenial cortex in memory formation and retention. Further, they suggest that following formation, memory storage becomes distributed to a wider network than is needed for its initial consolidation.

Regulation of learned fear expression through the MgN-amygdala pathway.

Heightened fear responding is characteristic of fear- and anxiety-related disorders, including post-traumatic stress disorder. Neural plasticity in the amygdala is essential for both initial fear learning and fear expression, and strengthening of synaptic connections between the medial geniculate nucleus (MgN) and amygdala is critical for auditory fear learning. However, very little is known about what happens in the MgN-amygdala pathway during fear recall and extinction, in which conditional fear decreases with repeated presentations of the auditory stimulus alone. In the present study, we found that optogenetic inhibition of activity in the MgN-amygdala pathway during fear retrieval and extinction reduced expression of conditional fear. While this effect persisted for at least two weeks following pathway inhibition, it was specific to the context in which optogenetic inhibition occurred, linking MgN-BLA inhibition to facilitation of extinction-like processes. Reduced fear expression through inhibition of the MgN-amygdala pathway was further characterized by similar synaptic expression of GluA1 and GluA2 AMPA receptor subunits compared to what was seen in controls. Inhibition also decreased CREB phosphorylation in the amygdala, similar to what has been reported following auditory fear extinction. We then demonstrated that this effect was reduced by inhibition of GluN2B-containing NMDA receptors. These results demonstrate a new and important role for the MgN-amygdala pathway in extinction-like processes, and show that suppressing activity in this pathway results in a persistent decrease in fear behavior.

Age-Related Memory Impairment and Sex-Specific Alterations in Phosphorylation of the Rpt6 Proteasome Subunit and Polyubiquitination in the Basolateral Amygdala and Medial Prefrontal Cortex.

Aging is marked by an accumulation of damaged and modified brain proteins, and the ubiquitin-proteasome system (UPS) is important for cellular protein degradation. Recent work has established a critical role for the UPS in memory and synaptic plasticity, but the role of the UPS in age-related cognitive decline remains poorly understood. We trained young, middle-aged, and aged male and female rats using trace fear conditioning (TFC) to investigate the effects of age and sex on memory. We then measured markers of UPS-related protein degradation (phosphorylation of the Rpt6 proteasome regulatory subunit and K48-linked polyubiquitination) using western blots. We found that aged males, but not aged females, showed behavioral deficits at memory retrieval. Aged males also displayed reduced phosphorylation of the Rpt6 proteasome subunit and accumulation of K48 in the basolateral amygdala, while aged females displayed a similar pattern in the medial prefrontal cortex. These findings suggest that markers of UPS function are differentially affected by age and sex in a brain region-dependent manner. Together these results provide an important step toward understanding the UPS and circuit-level differences in aging males and females.

The anterior retrosplenial cortex encodes event-related information and the posterior retrosplenial cortex encodes context-related information during memory formation.

The retrosplenial cortex (RSC) is extensively interconnected with the dorsal hippocampus and has several important roles in learning and memory. Recent work has demonstrated that certain types of context-dependent learning are selectively impaired when the posterior, but not the anterior, region of the RSC is damaged, suggesting that the role of the RSC in memory formation may not be uniform along its rostro-caudal axis. The current experiments tested the idea that the anterior and posterior portions of the rat RSC contribute to different aspects of memory formation. We first confirmed that brief optogenetic inhibition of either the anterior or posterior RSC resulted in decreased local cellular activity as indexed by immediate early gene zif268 expression and that this decrease was restricted to the target region within RSC. We then found that silencing the anterior or posterior RSC during trace fear training trials had different effects on memory: While inhibiting neural activity in the anterior RSC had a selective impact on behavior evoked by the auditory CS, inhibition of the posterior RSC selectively impaired memory for the context in which training was conducted. These results contribute to a growing literature that supports functionally distinct roles in learning and memory for subregions of the RSC.

Age-Related Memory Impairment Is Associated with Increased zif268 Protein Accumulation and Decreased Rpt6 Phosphorylation.

Aging is associated with cognitive decline, including impairments in the ability to accurately form and recall memories. Some behavioral and brain changes associated with aging are evident as early as middle age, making the understanding of associated neurobiological mechanisms essential to aid in efforts aimed at slowing cognitive decline throughout the lifespan. Here, we found that both 15-month-old and 22-month-old rats showed impaired memory recall following trace fear conditioning. This behavioral deficit was accompanied by increased zif268 protein accumulation relative to 3-month-old animals in the medial prefrontal cortex, the dorsal and ventral hippocampi, the anterior and posterior retrosplenial cortices, the lateral amygdala, and the ventrolateral periaqueductal gray. Elevated zif268 protein levels corresponded with decreases in phosphorylation of the Rpt6 proteasome regulatory subunit, which is indicative of decreased engagement of activity-driven protein degradation. Together, these results identify several brain regions differentially impacted by aging and suggest that the accumulation of proteins associated with memory retrieval, through reduced proteolytic activity, is associated with age-related impairments in memory retention.

SAK3 Administration Improves Spine Abnormalities and Cognitive Deficits in AppNL-G-F/NL-G-F Knock-in Mice by Increasing Proteasome Activity through CaMKII/Rpt6 Signaling.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by neuropathological hallmarks consisting of accumulation of extracellular amyloid-ß (Aß) plaques and intracellular neurofibrillary tangles (NFT). Recently, we have identified a new AD therapeutic candidate, ethyl-8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo [1,2-a] pyridin]-2-ene-3-carboxylate (SAK3), which ameliorates the AD-like pathology in AppNL-F/NL-F knock-in mice. However, the detailed mechanism underlying the therapeutic effects of SAK3 remains unclear. In this study, we found that SAK3 administration improved the reduced proteasome activity through the activation of CaMKII/Rpt6 signaling in AppNL-F/NL-F knock-in (NL-G-F) mice. Moreover, spine abnormalities observed in NL-G-F mice were significantly reversed by SAK3 administration. Along with this, cognitive impairments found in NL-G-F mice were markedly ameliorated by SAK3. In summary, our data suggest that SAK3 administration increases the activity of the proteasome via activation of the CaMKII/Rpt6 signaling pathway, contributing to improvements in spine abnormalities and cognitive deficits in NL-G-F mice. Overall, our findings suggest that SAK3 might be a new attractive drug candidate, representing a new mechanism for the treatment of AD pathology.

Decreased cued fear discrimination learning in female rats as a function of estrous phase.

Relative to males, female rats can show enhanced contextual fear generalization (demonstrating a fear response in a safe or neutral context) dependent on estrogen receptor activation. The current experiment aimed to extend this finding to cued fear conditioning. Females in low-estrogen phases of the estrous cycle showed good discrimination, similar to males, between a conditional stimulus that predicted shock (CS+) and an equally familiar one that did not (CS-), while females in the proestrus (high estrogen) phase demonstrated similar levels of fear between the CS+ and CS-. These results demonstrate that cued fear generalization is similarly influenced by endogenous estrogens.

Age-related memory deficits are associated with changes in protein degradation in brain regions critical for trace fear conditioning.

Brain aging is accompanied by an accumulation of damaged proteins, which results from deterioration of cellular quality control mechanisms and decreased protein degradation. The ubiquitin-proteasome system (UPS) is the primary proteolytic mechanism responsible for targeted degradation. Recent work has established a critical role of the UPS in memory and synaptic plasticity, but the role of the UPS in age-related cognitive decline remains poorly understood. Here, we measured markers of UPS function and related them to fear memory in rats. Our results show that age-related memory deficits are associated with reductions in phosphorylation of the Rpt6 proteasome regulatory subunit and corresponding increases in lysine-48 (K48)-linked ubiquitin tagging within the basolateral amygdala. Increases in K48 polyubiquitination were also observed in the medial prefrontal cortex and dorsal hippocampus. These data suggest that protein degradation is a critical component of age-related memory deficits. This extends our understanding of the relationship between the UPS, aging, and memory, which is an important step toward the prevention and treatment of deficits associated with normal cognitive aging and memory-related neurodegenerative diseases.

The dorsal hippocampus mediates synaptic destabilization and memory lability in the amygdala in the absence of contextual novelty.

The recall of a previously formed fear memory triggers a process through which synapses in the amygdala become "destabilized". This labile state at retrieval may be critical for the plasticity required to modify, update, or disrupt long-term memories. One component of this process involves the rapid internalization of calcium impermeable AMPA receptors (CI-AMPAR). While some recent work has focused on the details of modifying amygdala synapses, much less is known about the environmental factors that control memory updating and the important circuit level processes. Synchrony between the hippocampus and amygdala increases during memory retrieval and stable memories can sometimes be made labile with hippocampal manipulations. Recent work shows that memory lability at retrieval is influenced by the novelty of the retrieval environment, and detection of this novelty likely relies on the dorsal hippocampus (DH). Our goal was to determine how local activity in the DH contributes to memory lability and synaptic destabilization in the amygdala during retrieval when contextual novelty is introduced. We found that contextual novelty during retrieval is necessary for alterations in amygdala activity and CI-AMPAR internalization. In the absence of novelty, suppression of local activity in the DH prior to learning allowed for retrieval-dependent CI-AMPAR internalization in the amygdala. We next tested whether the changes in AMPAR internalization were accompanied by differences in memory lability. We found that a memory was made labile when activity within the DH was disrupted in the absence of contextual novelty. These results suggest that the DH is important for encoding contextual information during learning that regulates retrieval-dependent memory modification in the amygdala.

GluR2 endocytosis-dependent protein degradation in the amygdala mediates memory updating.

Associations learned during Pavlovian fear conditioning are rapidly acquired and long lasting, providing an ideal model for studying long-term memory formation, storage, and retrieval. During retrieval, these memories can "destabilize" and become labile, allowing a transient "reconsolidation" window during which the memory can be updated, suggesting that reconsolidation could be an attractive target for the modification of memories related to past traumatic experiences. This memory destabilization process is regulated by protein degradation and GluR2-endocytosis in the amygdala. However, it is currently unknown if retrieval-dependent GluR2-endocytosis in the amygdala is critical for incorporation of new information into the memory trace. We examined whether the addition of new information during memory retrieval required GluR2-endocytosis to modify the original memory. The presentation of two foot shocks of weaker intensity during retrieval resulted in GluR2 endocytosis-dependent increase in fear responding on a later test, suggesting modification of the original memory. This increase in fear expression was associated with increased protein degradation and zif268 expression in the same population of cells in the amygdala, indicating increased destabilization processes and cellular activity, and both were lost following blockade of GluR2-endocytosis. These data suggest that the endocytosis of GluR2-containing AMPA receptors in the amygdala regulates retrieval-induced strengthening of memories for traumatic events by modulating cellular destabilization and activity.

Cortico-limbic connectivity changes following fear extinction and relationships with trait anxiety.

Fear extinction is a powerful model of adaptive and anxiety-related maladaptive fear inhibition. This learning process is dependent upon plastic interactions between the amygdala, the anterior midcingulate cortex (aMCC), the hippocampus, and the ventromedial prefrontal cortex (vmPFC). With regard to the amygdala, the basolateral (BLA) and centromedial amygdala (CMA) serve unique roles in fear extinction. In a large sample (N = 91), the current study examined pre- to post-extinction changes in resting state functional connectivity (RSFC) of fear inhibition and expression pathways. We also examined how trait anxiety and extinction performance were associated with extinction-related changes within these neural pathways. We found stronger pre- to post-extinction RSFC in pathways known to play a role in the down-regulation of fear responses (BLA-hippocampus, aMCC-hippocampus, CMA-hippocampus, CMA-aMCC). We also found that trait anxiety was associated with strengthening of a BLA-aMCC circuit supporting fear expression following extinction learning. Furthermore, we found that physiological indices of poorer extinction learning were linked to weaker pre- to post-extinction RSFC of a BLA-hippocampus pathway important for fear extinction consolidation. Our results highlight the network changes that occur during extinction, the separable role of CMA and BLA-based circuitry and a key pathway linked to risk for anxiety pathology.

Context memory formation requires activity-dependent protein degradation in the hippocampus.

Numerous studies have indicated that the consolidation of contextual fear memories supported by an aversive outcome like footshock requires de novo protein synthesis as well as protein degradation mediated by the ubiquitin-proteasome system (UPS). Context memory formed in the absence of an aversive stimulus by simple exposure to a novel environment requires de novo protein synthesis in both the dorsal (dHPC) and ventral (vHPC) hippocampus. However, the role of UPS-mediated protein degradation in the consolidation of context memory in the absence of a strong aversive stimulus has not been investigated. In the present study, we used the context preexposure facilitation effect (CPFE) procedure, which allows for the dissociation of context learning from context-shock learning, to investigate the role of activity-dependent protein degradation in the dHPC and vHPC during the formation of a context memory. We report that blocking protein degradation with the proteasome inhibitor clasto-lactacystin ß-lactone (ßLac) or blocking protein synthesis with anisomycin (ANI) immediately after context preexposure significantly impaired context memory formation. Additionally, we examined 20S proteasome activity at different time points following context exposure and saw that the activity of proteasomes in the dHPC increases immediately after stimulus exposure while the vHPC exhibits a biphasic pattern of proteolytic activity. Taken together, these data suggest that the requirement of increased proteolysis during memory consolidation is not driven by processes triggered by the strong aversive outcome (i.e., shock) normally used to support fear conditioning.

Input from the medial geniculate nucleus modulates amygdala encoding of fear memory discrimination.

Generalization of fear can involve abnormal responding to cues that signal safety and is common in people diagnosed with post-traumatic stress disorder. Differential auditory fear conditioning can be used as a tool to measure changes in fear discrimination and generalization. Most prior work in this area has focused on elevated amygdala activity as a critical component underlying generalization. The amygdala receives input from auditory cortex as well as the medial geniculate nucleus (MgN) of the thalamus, and these synapses undergo plastic changes in response to fear conditioning and are major contributors to the formation of memory related to both safe and threatening cues. The requirement for MgN protein synthesis during auditory discrimination and generalization, as well as the role of MgN plasticity in amygdala encoding of discrimination or generalization, have not been directly tested. GluR1 and GluR2 containing AMPA receptors are found at synapses throughout the amygdala and their expression is persistently up-regulated after learning. Some of these receptors are postsynaptic to terminals from MgN neurons. We found that protein synthesis-dependent plasticity in MgN is necessary for elevated freezing to both aversive and safe auditory cues, and that this is accompanied by changes in the expressions of AMPA receptor and synaptic scaffolding proteins (e.g., SHANK) at amygdala synapses. This work contributes to understanding the neural mechanisms underlying increased fear to safety signals after stress.

Corrigendum: Psychopaths Show Enhanced Amygdala Activation during Fear Conditioning.

[This corrects the article on p. 348 in vol. 7, PMID: 27014154.].

Updating Procedures Can Reorganize the Neural Circuit Supporting a Fear Memory.

Established memories undergo a period of vulnerability following retrieval, a process termed 'reconsolidation.' Recent work has shown that the hypothetical process of reconsolidation is only triggered when new information is presented during retrieval, suggesting that this process may allow existing memories to be modified. Reconsolidation has received increasing attention as a possible therapeutic target for treating disorders that stem from traumatic memories, yet little is known about how this process changes the original memory. In particular, it is unknown whether reconsolidation can reorganize the neural circuit supporting an existing memory after that memory is modified with new information. Here, we show that trace fear memory undergoes a protein synthesis-dependent reconsolidation process following exposure to a single updating trial of delay conditioning. Further, this reconsolidation-dependent updating process appears to reorganize the neural circuit supporting the trace-trained memory, so that it better reflects the circuit supporting delay fear. Specifically, after a trace-to-delay update session, the amygdala is now required for extinction of the updated memory but the retrosplenial cortex is no longer required for retrieval. These results suggest that updating procedures could be used to force a complex, poorly defined memory circuit to rely on a better-defined neural circuit that may be more amenable to behavioral or pharmacological manipulation. This is the first evidence that exposure to new information can fundamentally reorganize the neural circuit supporting an existing memory.