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.

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.

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.

Fluorescence laminar optical tomography for brain imaging: system implementation and performance evaluation.

We present our effort in implementing a fluorescence laminar optical tomography scanner which is specifically designed for noninvasive three-dimensional imaging of fluorescence proteins in the brains of small rodents. A laser beam, after passing through a cylindrical lens, scans the brain tissue from the surface while the emission signal is captured by the epi-fluorescence optics and is recorded using an electron multiplication CCD sensor. Image reconstruction algorithms are developed based on Monte Carlo simulation to model light­tissue interaction and generate the sensitivity matrices. To solve the inverse problem, we used the iterative simultaneous algebraic reconstruction technique. The performance of the developed system was evaluated by imaging microfabricated silicon microchannels embedded inside a substrate with optical properties close to the brain as a tissue phantom and ultimately by scanning brain tissue in vivo. Details of the hardware design and reconstruction algorithms are discussed and several experimental results are presented. The developed system can specifically facilitate neuroscience experiments where fluorescence imaging and molecular genetic methods are used to study the dynamics of the brain circuitries.

Activity of the anterior cingulate cortex and ventral hippocampus underlie increases in contextual fear generalization.

Memories for context become less specific with time resulting in animals generalizing fear from training contexts to novel contexts. Though much attention has been given to the neural structures that underlie the long-term consolidation of a context fear memory, very little is known about the mechanisms responsible for the increase in fear generalization that occurs as the memory ages. Here, we examine the neural pattern of activation underlying the expression of a generalized context fear memory in male C57BL/6J mice. Animals were context fear conditioned and tested for fear in either the training context or a novel context at recent and remote time points. Animals were sacrificed and fluorescent in situ hybridization was performed to assay neural activation. Our results demonstrate activity of the prelimbic, infralimbic, and anterior cingulate (ACC) cortices as well as the ventral hippocampus (vHPC) underlie expression of a generalized fear memory. To verify the involvement of the ACC and vHPC in the expression of a generalized fear memory, animals were context fear conditioned and infused with 4% lidocaine into the ACC, dHPC, or vHPC prior to retrieval to temporarily inactivate these structures. The results demonstrate that activity of the ACC and vHPC is required for the expression of a generalized fear memory, as inactivation of these regions returned the memory to a contextually precise form. Current theories of time-dependent generalization of contextual memories do not predict involvement of the vHPC. Our data suggest a novel role of this region in generalized memory, which should be incorporated into current theories of time-dependent memory generalization. We also show that the dorsal hippocampus plays a prolonged role in contextually precise memories. Our findings suggest a possible interaction between the ACC and vHPC controls the expression of fear generalization.

Parameters of hormetic stress and resilience to trauma in rats.

Hormesis is the process by which small stresses build resilience to large stresses. We pre-exposed rats to various parameters of mild-to-moderate stress prior to traumatic stress in the present experiments to assess the potential benefits of hormetic training on resilience to traumatic, uncontrollable stress. Rats underwent varying stress pre-training parameters prior to exposure to uncontrollable traumatic stress in the learned helplessness procedure. The ability to prevent the exaggerated fear responding and escape deficits that normally follow experience with traumatic stress were used as a measure of the benefits of hormetic training. Four experiments examined the effects of number of training sessions, stressor severity and pattern of rest between pre-training stress sessions. Repeated exposure to mild restraint stress or moderate shock stress eliminated both the enhanced fear conditioning and shuttle-escape deficits that result from exposure to traumatic, inescapable shock. The pattern of rest did not contribute to resilience when the pre-exposure stressor was mild, but was vital when the pre-exposure stressor was moderate, with an alternation of stress and rest being the most effective procedure. The data also suggest that the level of resilience may increase with the number of pre-exposure sessions.

GABA-mediated presynaptic inhibition is required for precision of long-term memory.

Though much attention has been given to the neural structures that underlie the long-term consolidation of contextual memories, little is known about the mechanisms responsible for the maintenance of memory precision. Here, we demonstrate a rapid time-dependent decline in memory precision in GABAB(1a) receptor knockout mice. First, we show that GABAB(1a) receptors are required for the maintenance, but not encoding, of a precise fear memory. We then demonstrate that GABAB(1a) receptors are required for the maintenance, but not encoding, of spatial memories. Our findings suggest that GABA-mediated presynaptic inhibition regulates the maintenance of memory precision as a function of memory age.

Pharmacological modulation of acute trauma memories to prevent PTSD: considerations from a developmental perspective.

Estimates of the lifetime prevalence of posttraumatic stress disorder (PTSD) in American adults range from 6.4% to 6.8%. PTSD is associated with increased risk for comorbid major depression, substance use disorder, suicide, and a variety of other mental and physical health conditions. Given the negative sequelae of trauma/PTSD, research has focused on identifying efficacious interventions that could be administered soon after a traumatic event to prevent or reduce the subsequent incidence of PTSD. While early psychosocial interventions have been shown to be relatively ineffective, early (secondary) pharmacological interventions have shown promise. These pharmacological approaches are largely based on the hypothesis that disruption of altered stress hormone levels and the consequent formation of trauma memories could protect against the development of PTSD. The present manuscript reviews the literature regarding the role of peri-traumatic stress hormones as risk factors for the development of PTSD and reviews evidence for the efficacy of exogenously modulating stress hormone levels to prevent/buffer the development of PTSD symptoms. Whereas prior literature has focused primarily on either child or adult studies, the present review incorporates both child and adult studies in a developmental approach to understanding risk for PTSD and how pharmacological modulation of acute memories may buffer the development of PTSD symptoms.

Sex differences in the generalization of fear as a function of retention intervals.

In previous studies using male rodents, context change disrupted a fear response at a short, but not a long, retention interval. Here, we examined the effects of context changes on fear responses as a function of time in male and female rats. Males displayed context discrimination at all intervals, whereas females exhibited generalization by 5 d. Ovariectomized females with no hormone replacement displayed context discrimination at 5 d, whereas those receiving 17ß-estradiol generalized their fear response to a neutral context. These results demonstrate that fear generalization for contextual cues occurs faster in female rats and is mediated, in part, by estrogens.

Remembering another aspect of forgetting.

Although forgetting is most often thought of in terms of declines in performance (response loss or impairment), another class of memory phenomena, the forgetting of stimulus attributes, has begun to attract experimental attention. In non-human animals, the loss of memory for stimulus features is reflected in the flattening of stimulus generalization gradients as well as in the attenuation of the disrupting effect of a shift in context at testing. In both cases, a delay between the learning episode and testing results in increased responding in the presence of previously ineffective stimuli. Thus, previously discriminable cues become more functionally interchangeable. The implications of the forgetting of attributes for some theoretical issues of memory loss and for methodological strategies have been noted earlier. However, relatively little is known about the neurobiological mechanisms underlying stimulus attribute forgetting, and why some memories are maintained while others are not. In this paper we review the evidence for the forgetting of stimulus attributes, discuss recent findings identifying neurobiological underpinnings of forgetting and generalization of fear responses, and discuss relevant clinical implications of fear generalization.

Enhancing brain adenosine signaling with the nucleoside transport blocker NBTI (S-(4-nitrobenzyl)-6-theoinosine) mimics the effects of inescapable shock on later shuttle-escape performance in rats.

Experience with unsignaled, inescapable shock represents a profound challenge to brain metabolic function and physiology. The authors have argued that behavioral impairment following this traumatic stress is a consequence of enhanced brain adenosine signaling, which promotes metabolic recovery by profoundly inhibiting neural activation. The authors tested this hypothesis by artificially increasing extracellular brain adenosine concentration by blocking uptake transport with NBTI in rats given only restraint stress in five experiments. NBTI impaired shuttle-escape performance in the manner of inescapable shock in a dose-dependent manner and acted synergistically with an ineffective number of inescapable shocks to maximally impair test performance. These deficits produced by inescapable shock and NBTI were reversed by the nonselective adenosine receptor antagonist caffeine, and the highly selective A-sub(2A) receptor antagonist CSC (8-(3-chloro-styrl)caffeine). The highly selective A-sub-1 receptor antagonist DPCPX (8-Cyclopentyl-1,3-Dipropylxanthine) failed to improve performance in rats preexposed to inescapable shock or pretreated with NBTI. These data suggest that enhanced adenosine signaling at a brain A-sub(2A) receptor impairs escape performance following inescapable shock in the learned helplessness paradigm.