Dysregulation of baseline and learning-dependent protein degradation in the aged hippocampus.

The ubiquitin-proteasome system (UPS) controls the majority of protein degradation in cells and dysregulation of the UPS has been implicated in the pathophysiology of numerous neurodegenerative disorders, including Alzheimer's disease. Further, strong evidence supports a critical role for the UPS in synaptic plasticity and memory formation. However, while proteasome function is known to decrease broadly in the brain across the lifespan, whether it changes in the hippocampus, a region critical for memory storage and among the first impacted in Alzheimer's disease, at rest and following learning in the aged brain remains unknown. Further, which proteins have altered targeting for protein degradation in the aged hippocampus has yet to be explored and whether learning in advanced age interacts with changes in ubiquitin-proteasome function across the lifespan remains unknown. Here, using proteasome activity assays and unbiased proteomic analyses, we report age-dependent changes in proteasome activity and degradation-specific K48 polyubiquitin protein targeting in the hippocampus and retrosplenial cortex of male and female rats across the lifespan. In the hippocampus, the targets of altered protein degradation were involved in transcription and astrocyte structure or G-protein and Interferon signaling in males and females, respectively. Importantly, we found that contextual fear conditioning led to an increase in proteasome activity and K48 polyubiquitin protein targeting in the hippocampus of aged male rats, a result in direct contrast to what was previously reported in young adult animals. Together, these data suggest that changes in protein degradation in the hippocampus across the lifespan may be contributing to age-related memory loss.

Behavioral outputs and overlapping circuits between conditional fear and active avoidance.

Aversive learning can produce a wide variety of defensive behavioral responses depending on the circumstances, ranging from reactive responses like freezing to proactive avoidance responses. While most of this initial learning is behaviorally supported by an expectancy of an aversive outcome and neurally supported by activity within the basolateral amygdala, activity in other brain regions become necessary for the execution of defensive strategies that emerge in other aversive learning paradigms such as active avoidance. Here, we review the neural circuits that support both reactive and proactive defensive behaviors that are motivated by aversive learning, and identify commonalities between the neural substrates of these distinct (and often exclusive) behavioral strategies.

Enhancing fear extinction.

Gradually reducing a source of fear during extinction treatments may weaken negative memories in the long term.

Fear extinction is impaired in aged rats.

Normal aging is accompanied by broad loss of cognitive function in humans and rodents, including declines in cognitive flexibility. In extinction, a conditional stimulus (CS) that was previously paired with a footshock is presented alone. This procedure reliably reduces conditional freezing behavior in young adult rats. Here, we aimed to investigate how normal aging affects extinction learning. Using young (3 months) and aged (20 months) male and female Long Evans rats, we compared extinction (using 20 CS-alone presentations) to a no extinction control (equal exposure to the conditioning chamber without CS presentations) following delay fear conditioning. We found that young animals in the extinction group showed a decrease in freezing following extinction; aged animals did not. We next examined changes in neural activity using expression of the immediate early gene zif268. In young animals, extinction corresponded with decreased expression of zif268 in the basolateral amygdala and anterior retrosplenial cortex; this was not observed in aged animals. Further, aged animals showed increased zif268 expression in each region examined, suggesting that dysfunction in neural activity precedes cognitive deficits. These results demonstrate that aging impacts both extinction learning and neural activity.

Elevated fear states facilitate ventral hippocampal engagement of basolateral amygdala neuronal activity.

Fear memory formation and retention rely on the activation of distributed neural circuits. The basolateral amygdala (BLA) and ventral hippocampus (VH) in particular are two regions that support contextual fear memory processes and share reciprocal connections. The VH → BLA pathway is critical for increases in fear after initial learning, in both fear renewal following extinction learning and during fear generalization. This raises the possibility that functional changes in VH projections to the BLA support increases in learned fear. In line with this, fear can also be increased with alterations to the original content of the memory via reconsolidation, as in fear elevation procedures. However, very little is known about the functional changes in the VH → BLA pathway supporting reconsolidation-related increases in fear. In this study, we used in vivo extracellular electrophysiology to examine the functional neuronal changes within the BLA and in the VH → BLA pathway as a result of fear elevation and standard fear retrieval procedures. Elevated fear expression was accompanied by higher BLA spontaneous firing compared to a standard fear retrieval condition. Across a range of stimulation frequencies, we also found that VH stimulation evoked higher BLA firing following fear elevation compared to standard retrieval. These results suggest that fear elevation is associated with an increased capacity of the VH to drive neuronal activity in the BLA, highlighting a potential circuit involved in strengthening existing fear memories.

Generalization and discrimination of inhibitory avoidance differentially engage anterior and posterior retrosplenial subregions.

Introduction: In a variety of behavioral procedures animals will show selective fear responding in shock-associated contexts, but not in other contexts. However, several factors can lead to generalized fear behavior, where responding is no longer constrained to the conditioning context and will transfer to novel contexts. Methods: Here, we assessed memory generalization using an inhibitory avoidance paradigm to determine if generalized avoidance behavior engages the retrosplenial cortex (RSC). Male and female Long Evans rats received inhibitory avoidance training prior to testing in the same context or a shifted context in two distinct rooms; one room that had fluorescent lighting (Light) and one that had red LED lighting (Dark). Results: We found that animals tested in a light context maintained context-specificity; animals tested in the same context as training showed longer latencies to cross and animals tested in the shifted context showed shorter latencies to cross. However, animals tested in the dark generalized their avoidance behavior; animals tested in the same context and animals tested in the shifted context showed similarly-high latencies to cross. We next examined expression of the immediate early gene zif268 and perineuronal nets (PNNs) following testing and found that while activity in the basolateral amygdala corresponded with overall levels of avoidance behaviors, anterior RSC (aRSC) activity corresponded with learned avoidance generally, but posterior RSC (pRSC) activity seemed to correspond with generalized memory. PNN reduction in the RSC was associated with memory formation and retrieval, suggesting a role for PNNs in synaptic plasticity. Further, PNNs did not reduce in the RSC in animals who showed a generalized avoidance behavior, in line with their hypothesized role in memory consolidation. Discussion: These findings suggest that there is differential engagement of retrosplenial subregions along the rostrocaudal axis to generalization and discrimination.

Fear Reduced Through Unconditional Stimulus Deflation Is Behaviorally Distinct From Extinction and Differentially Engages the Amygdala.

Background: Context fear memory can be reliably reduced by subsequent pairings of that context with a weaker shock. This procedure shares similarities with extinction learning: both involve extended time in the conditioning chamber following training and reduce context-elicited fear. Unlike extinction, this weak-shock exposure has been hypothesized to engage reconsolidation-like processes that weaken the original memory. Methods: We directly compared the weak-shock procedure with extinction using male and female Long Evans rats. Results: Both repeated weak-shock exposure and extinction resulted in decreased context freezing relative to animals that received context fear conditioning but no subsequent context exposure. Conditioning with the weak shock was not enough to form a persistent context-shock association on its own, suggesting that the weak-shock procedure does not create a new memory. Weak-shock exposure in a new context can still reduce freezing elicited by the training context, suggesting that it reduces responding through a different process than extinction, which does not transcend context. Finally, reduced fear behavior produced through both extinction and weak-shock exposure was mirrored by reduced zif268 expression in the basolateral amygdala. However, only the weak-shock procedure resulted in changes in lysine-48 polyubiquitin tagging in the synapse of the basolateral amygdala, suggesting that this procedure produced long-lasting changes in synaptic function within the basolateral amygdala. Conclusions: These results suggest that the weak-shock procedure does not rely on the creation of a new inhibitory memory, as in extinction, and instead may alter the original representation of the shock to reduce fear responding.

Maturation of a cortical-amygdala circuit limits sociability in male rats.

Prefrontal cortical maturation coincides with adolescent transitions in social engagement, suggesting that it influences social development. The anterior cingulate cortex (ACC) is important for social interaction, including ACC outputs to the basolateral amygdala (BLA). However, little is known about ACC-BLA sensitivity to the social environment and if this changes during maturation. Here, we used brief (2-hour) isolation to test the immediate impact of changing the social environment on the ACC-BLA circuit and subsequent shifts in social behavior of adolescent and adult rats. We found that optogenetic inhibition of the ACC during brief isolation reduced isolation-driven facilitation of social interaction across ages. Isolation increased activity of ACC-BLA neurons across ages, but altered the influence of ACC on BLA activity in an age-dependent manner. Isolation reduced the inhibitory impact of ACC stimulation on BLA neurons in a frequency-dependent manner in adults, but uniformly suppressed ACC-driven BLA activity in adolescents. This work identifies isolation-driven alterations in an ACC-BLA circuit, and the ACC itself as an essential region sensitive to social environment and regulates its impact on social behavior in both adults and adolescents.

Memory retrieval, reconsolidation, and extinction: Exploring the boundary conditions of post-conditioning cue exposure.

Following fear conditioning, behavior can be reduced by giving many CS-alone presentations in a process known as extinction or by presenting a few CS-alone presentations and interfering with subsequent memory reconsolidation. While the two share procedural similarities, both the behavioral outcomes and the neurobiological underpinnings are distinct. Here we review the neural and behavioral mechanisms that produce these separate behavioral reductions, as well as some factors that determine whether or not a retrieval-dependent reconsolidation process or an extinction process will be in effect.

The lifetime impact of stress on fear regulation and cortical function.

A variety of stressful experiences can influence the ability to form and subsequently inhibit fear memory. While nonsocial stress can impact fear learning and memory throughout the lifespan, psychosocial stressors that involve negative social experiences or changes to the social environment have a disproportionately high impact during adolescence. Here, we review converging lines of evidence that suggest that development of prefrontal cortical circuitry necessary for both social experiences and fear learning is altered by stress exposure in a way that impacts both social and fear behaviors throughout the lifespan. Further, we suggest that psychosocial stress, through its impact on the prefrontal cortex, may be especially detrimental during early developmental periods characterized by higher sociability. This article is part of the Special Issue on 'Fear, Anxiety and PTSD'.

Developmental differences in amygdala projection neuron activation associated with isolation-driven changes in social preference.

Adolescence is a developmental period characterized by brain maturation and changes in social engagement. Changes in the social environment influence social behaviors. Memories of social events, including remembering familiar individuals, require social engagement during encoding. Therefore, existing differences in adult and adolescent social repertoires and environmentally-driven changes in social behavior may impact novel partner preference, associated with social recognition. Several amygdala subregions are sensitive to the social environment and can influence social behavior, which is crucial for novelty preference. Amygdala neurons project to the septum and nucleus accumbens (NAc), which are linked to social engagement. Here, we investigated how the social environment impacts age-specific social behaviors during social encoding and its subsequent impact on partner preference. We then examined changes in amygdala-septal and -NAc circuits that accompany these changes. Brief isolation can drive social behavior in both adults and adolescents and was used to increase social engagement during encoding. We found that brief isolation facilitates social interaction in adolescents and adults, and analysis across time revealed that partner discrimination was intact in all groups, but there was a shift in preference within isolated and non-isolated groups. We found that this same isolation preferentially increases basal amygdala (BA) activity relative to other amygdala subregions in adults, but activity among amygdala subregions was similar in adolescents, even when considering conditions (no isolation, isolation). Further, we identify isolation-driven increases in BA-NAc and BA-septal circuits in both adults and adolescents. Together, these results provide evidence for changes in neuronal populations within amygdala subregions and their projections that are sensitive to the social environment that may influence the pattern of social interaction within briefly isolated groups during development.

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.

Examining a role for the retrosplenial cortex in age-related memory impairment.

Aging is often characterized by changes in the ability to form and accurately recall episodic memories, and this is especially evident in neuropsychiatric conditions including Alzheimer's disease and dementia. Memory impairments and cognitive decline associated with aging mirror the impairments observed following damage to the retrosplenial cortex, suggesting that this region might be important for continued cognitive function throughout the lifespan. Here, we review lines of evidence demonstrating that degeneration of the retrosplenial cortex is critically involved in age-related memory impairment and suggest that preservation of function in this region as part of a larger circuit that supports memory maintenance will decrease the deleterious effects of aging on memory processing.

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.

Developmental Shifts in Amygdala Activity during a High Social Drive State.

Amygdala abnormalities characterize several psychiatric disorders with prominent social deficits and often emerge during adolescence. The basolateral amygdala (BLA) bidirectionally modulates social behavior and has increased sensitivity during adolescence. We tested how an environmentally-driven social state is regulated by the BLA in adults and adolescent male rats. We found that a high social drive state caused by brief social isolation increases age-specific social behaviors and increased BLA neuronal activity. Chemogenetic inactivation of BLA decreased the effect of high social drive on social engagement. High social drive preferentially enhanced BLA activity during social engagement; however, the effect of social opportunity on BLA activity was greater during adolescence. While this identifies a substrate underlying age differences in social drive, we then determined that high social drive increased BLA NMDA GluN2B expression and sensitivity to antagonism increased with age. Further, the effect of a high social drive state on BLA activity during social engagement was diminished by GluN2B blockade in an age-dependent manner. These results demonstrate the necessity of the BLA for environmentally driven social behavior, its sensitivity to social opportunity, and uncover a maturing role for BLA and its GluN2B receptors in social engagement.SIGNIFICANCE STATEMENT Social engagement during adolescence is a key component of healthy development. Social drive provides the impetus for social engagement and abnormalities underlie social symptoms of depression and anxiety. While adolescence is characterized by transitions in social drive and social environment sensitivity, little is known about the neural basis for these changes. We found that amygdala activity is uniquely sensitive to social environment during adolescence compared with adulthood, and is required for expression of heightened social drive. In addition, the neural substrates shift toward NMDA dependence in adulthood. These results are the first to demonstrate a unique neural signature of higher social drive and begin to uncover the underlying factors that heighten social engagement during adolescence.

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.

Contributions of the rodent cingulate-retrosplenial cortical axis to associative learning and memory: A proposed circuit for persistent memory maintenance.

While the anterior cingulate (ACC) and retrosplenial (RSC) cortices have been extensively studied for their role in spatial navigation, less is known about how they contribute to associative learning and later memory recall. The limited work that has been conducted on this topic suggests that each of these cortical regions makes distinct, but similar contributions to associative learning and memory. Here, we review evidence from the rodent literature demonstrating that while ACC activity seems to be necessary at remote time points associated with imprecise or generalized memories, the role of the RSC seems to be uniform over time. Together, the lines of evidence reviewed here suggest that the ACC and RSC likely function together to support memory formation and maintenance following associative learning.