Contextual memory engrams, and the neuromodulatory influence of the locus coeruleus.

Here, we review the basis of contextual memory at a conceptual and cellular level. We begin with an overview of the philosophical foundations of traversing space, followed by theories covering the material bases of contextual representations in the hippocampus (engrams), exploring functional characteristics of the cells and subfields within. Next, we explore various methodological approaches for investigating contextual memory engrams, emphasizing plasticity mechanisms. This leads us to discuss the role of neuromodulatory inputs in governing these dynamic changes. We then outline a recent hypothesis involving noradrenergic and dopaminergic projections from the locus coeruleus (LC) to different subregions of the hippocampus, in sculpting contextual representations, giving a brief description of the neuroanatomical and physiological properties of the LC. Finally, we examine how activity in the LC influences contextual memory processes through synaptic plasticity mechanisms to alter hippocampal engrams. Overall, we find that phasic activation of the LC plays an important role in promoting new learning and altering mnemonic processes at the behavioral and cellular level through the neuromodulatory influence of NE/DA in the hippocampus. These findings may provide insight into mechanisms of hippocampal remapping and memory updating, memory processes that are potentially dysregulated in certain psychiatric and neurodegenerative disorders.

Reactivating hippocampal-mediated memories during reconsolidation to disrupt fear.

Memories are stored in the brain as cellular ensembles activated during learning and reactivated during retrieval. Using the Tet-tag system in mice, we label dorsal dentate gyrus neurons activated by positive, neutral or negative experiences with channelrhodopsin-2. Following fear-conditioning, these cells are artificially reactivated during fear memory recall. Optical stimulation of a competing positive memory is sufficient to update the memory during reconsolidation, thereby reducing conditioned fear acutely and enduringly. Moreover, mice demonstrate operant responding for reactivation of a positive memory, confirming its rewarding properties. These results show that interference from a rewarding experience can counteract negative affective states. While memory-updating, induced by memory reactivation, involves a relatively small set of neurons, we also find that activating a large population of randomly labeled dorsal dentate gyrus neurons is effective in promoting reconsolidation. Importantly, memory-updating is specific to the fear memory. These findings implicate the dorsal dentate gyrus as a potential therapeutic node for modulating memories to suppress fear.

Hippocampal cells segregate positive and negative engrams.

The hippocampus is involved in processing a variety of mnemonic computations specifically the spatiotemporal components and emotional dimensions of contextual memory. Recent studies have demonstrated cellular heterogeneity along the hippocampal axis. The ventral hippocampus has been shown to be important in the processing of emotion and valence. Here, we combine transgenic and all-virus based activity-dependent tagging strategies to visualize multiple valence-specific engrams in the vHPC and demonstrate two partially segregated cell populations and projections that respond to appetitive and aversive experiences. Next, using RNA sequencing and DNA methylation sequencing approaches, we find that vHPC appetitive and aversive engram cells display different transcriptional programs and DNA methylation landscapes compared to a neutral engram population. Additionally, optogenetic manipulation of tagged cell bodies in vHPC is not sufficient to drive appetitive or aversive behavior in real-time place preference, stimulation of tagged vHPC terminals projecting to the amygdala and nucleus accumbens (NAc), but not the prefrontal cortex (PFC), showed the capacity drive preference and avoidance. These terminals also were able to change their capacity to drive behavior. We conclude that the vHPC contains genetically, cellularly, and behaviorally segregated populations of cells processing appetitive and aversive memory engrams.

Hippocampus and amygdala fear memory engrams re-emerge after contextual fear relapse.

The formation and extinction of fear memories represent two forms of learning that each engage the hippocampus and amygdala. How cell populations in these areas contribute to fear relapse, however, remains unclear. Here, we demonstrate that, in male mice, cells active during fear conditioning in the dentate gyrus of hippocampus exhibit decreased activity during extinction and are re-engaged after contextual fear relapse. In vivo calcium imaging reveals that relapse drives population dynamics in the basolateral amygdala to revert to a network state similar to the state present during fear conditioning. Finally, we find that optogenetic inactivation of neuronal ensembles active during fear conditioning in either the hippocampus or amygdala is sufficient to disrupt fear expression after relapse, while optogenetic stimulation of these same ensembles after extinction is insufficient to artificially mimic fear relapse. These results suggest that fear relapse triggers a partial re-emergence of the original fear memory representation, providing new insight into the neural substrates of fear relapse.

Norepinephrine as a spatial memory reset signal.

Contextual information is represented in the hippocampus (HPC) partially through the recruitment of distinct neuronal ensembles. It is believed that reactivation of these ensembles underlies memory retrieval processes. Recently, we showed that norepinephrine input from phasic locus coeruleus activation induces hippocampal plasticity resulting in the recruitment of new neurons and disengagement from previously established representations. We hypothesize that norepinephrine may provide a neuromodulatory mnemonic switch signaling the HPC to move from a state of retrieval to encoding in the presence of novelty, and therefore, plays a role in memory updating. Here, we tested whether bilateral dorsal dentate gyrus (dDG) infusions of the ß-adrenergic receptor (BAR) agonist isoproterenol (ISO), administered prior to encoding or retrieval, would impair spatial working and reference memory by reverting, the system to encoding (thereby recruiting new neurons) potentially interfering with the retrieval of the previously established spatial ensemble. We also investigated whether dDG infusions of ISO could promote cognitive flexibility by switching the system to encoding when it is adaptive (ie, when new information is presented, eg, reversal learning). We found that intra-dDG infusions of ISO given prior to retrieval caused deficits in working and reference memory which was blocked by pretreatment with the BAR-antagonist, propranolol (PRO). In contrast, ISO administered prior to reversal learning led to improved performance. These data support our hypothesis that norepinephrine serves as a novelty signal to update HPC contextual representations via BAR activation-facilitated recruitment of new neurons. This can be both maladaptive and adaptive depending on the situation.

Chronic activation of fear engrams induces extinction-like behavior in ethanol-exposed mice.

Alcohol withdrawal directly impacts the brain's stress and memory systems, which may underlie individual susceptibility to persistent drug and alcohol-seeking behaviors. Numerous studies demonstrate that forced alcohol abstinence, which may lead to withdrawal, can impair fear-related memory processes in rodents such as extinction learning; however, the underlying neural circuits mediating these impairments remain elusive. Here, we tested an optogenetic strategy aimed at mitigating fear extinction retrieval impairments in male c57BL/6 mice following exposure to alcohol (i.e., ethanol) and forced abstinence. In the first experiment, extensive behavioral extinction training in a fear-conditioned context was impaired in ethanol-exposed mice compared to controls. In the second experiment, neuronal ensembles processing a contextual fear memory in the dorsal hippocampus were tagged and optogenetically reactivated repeatedly in a distinct context in ethanol-exposed and control mice. Chronic activation of these cells resulted in a context-specific, extinction-like reduction in fear responses in both control and ethanol-exposed mice. These findings suggest that while ethanol can impair the retrieval an extinction memory, optogenetic manipulation of a fear engram is sufficient to induce an extinction-like reduction in fear responses.

Social behavior in mice following chronic optogenetic stimulation of hippocampal engrams.

The hippocampus processes both spatial-temporal information and emotionally salient experiences. To test the functional properties of discrete sets of cells in the dorsal dentate gyrus (dDG), we examined whether chronic optogenetic reactivation of these ensembles was sufficient to modulate social behaviors in mice. We found that chronic reactivation of discrete dDG cell populations in male mice largely did not affect social behaviors in an experience-dependent manner. However, we found that social behavior in a female exposure task was increased following chronic optogenetic stimulation when compared to pre-stimulation levels, suggesting that the protocol led to increased social behavior, although alternative explanations are discussed. Furthermore, multi-region analysis of neural activity did not yield detectable differences in immediate-early gene expression or neurogenesis following chronic optogenetic stimulation. Together, these results suggest that the effects of chronic optogenetic stimulation in the dDG on social behaviors are independent of the contextual experience processed by each cellular ensemble.

Odor modulates the temporal dynamics of fear memory consolidation.

Systems consolidation (SC) theory proposes that recent, contextually rich memories are stored in the hippocampus (HPC). As these memories become remote, they are believed to rely more heavily on cortical structures within the prefrontal cortex (PFC), where they lose much of their contextual detail and become schematized. Odor is a particularly evocative cue for intense remote memory recall and despite these memories being remote, they are highly contextual. In instances such as posttraumatic stress disorder (PTSD), intense remote memory recall can occur years after trauma, which seemingly contradicts SC. We hypothesized that odor may shift the organization of salient or fearful memories such that when paired with an odor at the time of encoding, they are delayed in the de-contextualization process that occurs across time, and retrieval may still rely on the HPC, where memories are imbued with contextually rich information, even at remote time points. We investigated this by tagging odor- and non-odor-associated fear memories in male c57BL/6 mice and assessed recall and c-Fos expression in the dorsal CA1 (dCA1) and prelimbic cortex (PL) 1 or 21 d later. In support of SC, our data showed that recent memories were more dCA1-dependent whereas remote memories were more PL-dependent. However, we also found that odor influenced this temporal dynamic, biasing the memory system from the PL to the dCA1 when odor cues were present. Behaviorally, inhibiting the dCA1 with activity-dependent DREADDs had no effect on recall at 1 d and unexpectedly caused an increase in freezing at 21 d. Together, these findings demonstrate that odor can shift the organization of fear memories at the systems level.

Artificially Enhancing and Suppressing Hippocampus-Mediated Memories.

Emerging evidence indicates that distinct hippocampal domains differentially drive cognition and emotion [1, 2]; dorsal regions encode spatial, temporal, and contextual information [3-5], whereas ventral regions regulate stress responses [6], anxiety-related behaviors [7, 8], and emotional states [8-10]. Although previous studies demonstrate that optically manipulating cells in the dorsal hippocampus can drive the behavioral expression of positive and negative memories, it is unknown whether changes in cellular activity in the ventral hippocampus can drive such behaviors [11-14]. Investigating the extent to which distinct hippocampal memories across the longitudinal axis modulate behavior could aid in the understanding of stress-related psychiatric disorders known to affect emotion, memory, and cognition [15]. Here, we asked whether tagging and stimulating cells along the dorsoventral axis of the hippocampus could acutely, chronically, and differentially promote context-specific behaviors. Acute reactivation of both dorsal and ventral hippocampus cells that were previously active during memory formation drove freezing behavior, place avoidance, and place preference. Moreover, chronic stimulation of dorsal or ventral hippocampal fear memories produced a context-specific reduction or enhancement of fear responses, respectively, thus demonstrating bi-directional and context-specific modulation of memories along the longitudinal axis of the hippocampus. Fear memory suppression was associated with a reduction in hippocampal cells active during retrieval, while fear memory enhancement was associated with an increase in basolateral amygdala activity. Together, our data demonstrate that discrete sets of cells throughout the hippocampus provide key nodes sufficient to bi-directionally reprogram both the neural and behavioral expression of memory.

Locus Coeruleus Phasic, But Not Tonic, Activation Initiates Global Remapping in a Familiar Environment.

Locus coeruleus (LC) neurons, the source of hippocampal norepinephrine (NE), are activated by novelty and changes in environmental contingencies. Based on the role of monoamines in reconfiguring invertebrate networks, and data from mammalian systems, a network reset hypothesis for the effects of LC activation has been proposed. We used the cellular compartmental analysis of temporal FISH technique based on the cellular distribution of immediate early genes to examine the effect of LC activation and inactivation, on regional hippocampal maps in male rats, when LC activity was manipulated just before placement in a second familiar (A/A) and/or novel environment (A/B). We found that bilateral phasic, but not tonic, activation of LC reset hippocampal maps in the A/A condition, whereas silencing the LC with clonidine before placement in the A/B condition blocked map reset and a familiar map emerged in the dentate gyrus, proximal and distal CA1, and CA3c. However, CA3a and CA3b encoded the novel environment. These results support a role for phasic LC responses in generating novel hippocampal sequences during memory encoding and, potentially, memory updating. The silencing experiments suggest that novel environments may not be recognized as different by dentate gyrus and CA1 without LC input. The functional distinction between phasic and tonic LC activity argues that these parameters are critical for determining network changes. These data are consistent with the hippocampus activating internal network representations to encode novel experiential episodes and suggest LC input is critical for this role.SIGNIFICANCE STATEMENT Burst activation of the broadly projecting novelty signaling system of the locus coeruleus initiates new network representations throughout the hippocampus despite unchanged external environments. Tonic activation does not alter network representations in the same condition. This suggests differences in the temporal parameters of neuromodulator network activation are critical for neuromodulator function. Silencing this novelty signaling system prevented the appearance of new network representations in a novel environment. Instead, familiar representations were expressed in a subset of hippocampal areas, with another subset encoding the novel environment. This "being in two places at once" argues for independent functional regions within the hippocampus. These experiments strengthen the view that internal states are major determinants of the brain's construction of environmental representations.

Are There Place Cells in the Avian Hippocampus?

Birds possess a hippocampus that serves many of the same spatial and mnemonic functions as the mammalian hippocampus but achieves these outcomes with a dramatically different neuroanatomical organization. The properties of spatially responsive neurons in birds and mammals are also different. Much of the contemporary interest in the role of the mammalian hippocampus in spatial representation dates to the discovery of place cells in the rat hippocampus. Since that time, cells that respond to head direction and cells that encode a grid-like representation of space have been described in the rat brain. Research with homing pigeons has discovered hippocampal cells, including location cells, path cells, and pattern cells, that share some but not all properties of spatially responsive neurons in the rodent brain. We have recently used patterns of immediate-early gene expression, visualized by the catFISH method, to investigate how neurons in the hippocampus of brood-parasitic brown-headed cowbirds respond to spatial context. We have found cells that discriminate between different spatial environments and are re-activated when the same spatial environment is re-experienced. Given the differences in habitat and behaviour between birds and rodents, it is not surprising that spatially responsive cells in their hippocampus and other brain regions differ. The enormous diversity of avian habitats and behaviour offers the potential for understanding the general principles of neuronal representation of space.

Attenuated Late-Phase Arc Transcription in the Dentate Gyrus of Mice Lacking Egr3.

The dentate gyrus (DG) engages in sustained Arc transcription for at least 8 hours following behavioral induction, and this time course may be functionally coupled to the unique role of the DG in hippocampus-dependent learning and memory. The factors that regulate long-term DG Arc expression, however, remain poorly understood. Animals lacking Egr3 show less Arc expression following convulsive stimulation, but the effect of Egr3 ablation on behaviorally induced Arc remains unknown. To address this, Egr3-/- and wild-type (WT) mice explored novel spatial environments and were sacrificed either immediately or after 5, 60, 240, or 480 minutes, and Arc expression was quantified by fluorescence in situ hybridization. Although short-term (i.e., within 60 min) Arc expression was equivalent across genotypes, DG Arc expression was selectively reduced at 240 and 480 minutes in mice lacking Egr3. These data demonstrate the involvement of Egr3 in regulating the late protein-dependent phase of Arc expression in the DG.

Context-Dependent Egr1 Expression in the Avian Hippocampus.

In mammals, episodic memory and spatial cognition involve context-specific recruitment of unique ensembles in the hippocampal formation (HF). Despite their capacity for sophisticated spatial (e.g., for migration) and episodic-like (e.g., for food-caching) memory, the mechanisms underlying contextual representation in birds is not well understood. Here we demonstrate environment-specific Egr1 expression as male brown-headed cowbirds (Molothrus ater) navigate environments for food reward, showing that the avian HF, like its mammalian counterpart, recruits distinct neuronal ensembles to represent different contexts.

Role of the kappa-opioid receptor system in stress-induced reinstatement of nicotine seeking in rats.

RATIONALE: The correlation between stress and smoking is well established. The mechanisms that underlie this relationship are, however, unclear. Recent data suggest that the kappa-opioid system is involved in the mediation of negative affective states associated with stress thereby promoting drug addiction and relapse. Pharmacological treatments targeting the kappa-opioid system and this mechanism may prove to be useful therapeutics for nicotine addiction in the future. OBJECTIVES: We sought to determine whether there was a stress-specific role of the kappa-opioid system in nicotine seeking behavior. METHOD: Groups of male Long Evans rats were trained to self-administer nicotine intravenously; their operant responding for nicotine was extinguished prior to tests of reinstatement. Pretreatment with systemic injections of the kappa-opioid receptor (KOR) antagonist nor-binaltorphimine (nor-BNI) was given prior to tests of stress (systemic injections of yohimbine (YOH)) or cue-induced reinstatement of nicotine seeking. Systemic injections of the KOR agonist U50,488 were also given in a test for reinstatement of nicotine seeking. RESULTS: Nor-BNI pretreatment at 1h and 24h prior to testing was able to block YOH-induced, but not cue-induced reinstatement of nicotine seeking. U50,488 reinstated nicotine seeking behavior in a dose-dependent manner. CONCLUSIONS: These findings support the hypothesis that the kappa-opioid system is involved in relapse to nicotine seeking induced by stress, but not by conditioned cues. KOR antagonists such as nor-BNI may therefore be useful novel therapeutic agents for decreasing the risk of stress-induced drug relapse.

Oxycodone dose-dependently imparts conditioned reinforcing properties to discrete sensory stimuli in rats.

The aim of this study was to explore the psychopharmacological characteristics of opioid-induced conditioned reinforcement using oxycodone, a potent mu-opioid receptor agonist with known abuse potential. In differed groups of rats, passive intravenous infusions of oxycodone (100 infusions/3 h×6 sessions in total; 0, 0.01, 0.05 and 0.1 mg/kg/inf) were paired with an audio-visual stimulus and, subsequently, operant responding maintained by this conditioned stimulus was tested in extinction conditions. It was found that the oxycodone-paired stimulus maintained operant responding and that this effect was dependent on the number of conditioning sessions and on the conditioning dose. Responding maintained the oxycodone-paired stimulus could also be reinstated by both foot-shock stress and by oxycodone priming (0.25 mg/kg, SC). A conditioned place preference experiment (3 drug and 3 vehicle injections over 6 days; oxycodone: 0, 0.25, 2 and 5 mg/kg, SC) confirmed that stimuli associated with lower doses of oxycodone induce conditioned approach. Finally, two control experiments performed with chlordiazepoxide ruled out an interpretation of the oxycodone data based on drug-induced amnesia, and confirmed that operant responding for a drug-conditioned stimulus is observed only when the drug possesses unconditioned reinforcing properties. Therefore, the intravenous conditioned reinforcement procedure appears a useful method to study how opioid drugs impart reinforcing value to discrete environmental stimuli.

Previous maternal experience affects accumbal dopaminergic responses to pup-stimuli.

The present study investigated the release of dopamine from the nucleus accumbens (shell) in response to pup-stimuli in the absence of lactation and maternal behaviors at time of sample collection. Subjects were female rats given maternal experiences through prior parturitions, recent pup-induced sensitization, or a combination of both. Nulliparous (N) or multiparous (M, had 2 prior litters but cycling) female rats either received pup-sensitization (S+) until they responded maternally in their homecage or no pup-sensitization (S-), thus, there were four groups: NS- (n=5), NS+ (n=6), MS- (n=5), and MS+ (n=8). Four hours after removal of pups (from homecage for S+ groups), all females were placed into the microdialysis chamber for sample collection. After baseline collection, four foster pups were given to the females. In this paradigm females show little to no maternal behavior in the test chamber. Samples (collected every 8 min) were analyzed for dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) with electrochemical detection using HPLC. Relative to the inexperienced NS- females, the experienced NS+, MS- and MS+ females displayed significantly increased DA levels only during the first 8 min of pup-exposure. The more experience a female had with pups, the greater was the DA response (p<.05). The results suggest that enhanced responding to pups following previous maternal experiences may be mediated through accumbal DA.

Prenatal restraint stress and motherless rearing disrupts expression of plasticity markers and stress-induced corticosterone release in adult female Sprague-Dawley rats.

This study investigated the effects of prenatal stress and complete maternal deprivation, using the artificial rearing (AR) paradigm, on the expression of neural plasticity markers and hypothalamic-pituitary-adrenal (HPA) axis responsivity to stress. Rats were exposed to stress during gestation (day 10-21) and postnatally were either artificially reared (AR) or mother reared (MR). AR involves complete separation of the pup from both the dam and the litter throughout the pre-weaning period. In adulthood, we measured levels of corticosterone (CORT) in response to restraint stress. Also, we examined the expression of synaptophysin (SYN) and brain-derived neurotrophic factor (BDNF) in the medial prefrontal cortex (MPFC) and the nucleus accumbens (Nacc), areas of the brain that mediate behavioral activation and attention, among other behaviors. Earlier work on the same rats indicated that these behavioral endpoints, such as locomotor activity and sensorimotor gating, are affected by our prenatal and postnatal manipulations. Prenatal stress decreased CORT at 20 and 90 min post-stressor in MR, but not in AR, animals. Also, in comparison to MR groups, AR decreased SYN and BDNF expression in the MPFC and Nacc. Additional somatosensory 'licking-like' stroking stimulation partially reversed the effects of AR. Prenatal stress did not have a robust main effect but affected the impact of the postnatal rearing condition on SYN expression and stress-induced CORT. These results suggest that both prenatal and postnatal adversities have an influence on HPA axis responsivity and alter the expression of plasticity related neuronal proteins.