The Black Box effect: sensory stimulation after learning interferes with the retention of long-term object location memory in rats

Reducing sensory experiences during the period that immediately follows learning improves long-term memory retention in healthy humans, and even preserves memory in patients with amnesia. To date, it is entirely unclear why this is the case, and identifying the neurobiological mechanisms underpinning this effect requires suitable animal models, which are currently lacking. Here, we describe a straightforward experimental procedure in rats that future studies can use to directly address this issue. Using this method, we replicated the central findings on quiet wakefulness obtained in humans: We show that rats that spent 1 h alone in a familiar dark and quiet chamber (the Black Box) after exploring two objects in an open field expressed long-term memory for the object locations 6 h later, while rats that instead directly went back into their home cage with their cage mates did not. We discovered that both visual stimulation and being together with conspecifics contributed to the memory loss in the home cage, as exposing rats either to light or to a cage mate in the Black Box was sufficient to disrupt memory for object locations. Our results suggest that in both rats and humans, everyday sensory experiences that normally follow learning in natural settings can interfere with processes that promote long-term memory retention, thereby causing forgetting in form of retroactive interference. The processes involved in this effect are not sleep-dependent because we prevented sleep in periods of reduced sensory experience. Our findings, which also have implications for research practices, describe a potentially useful method to study the neurobiological mechanisms that might explain why normal sensory processing after learning impairs memory both in healthy humans and in patients suffering from amnesia.

One of the most puzzling phenomena of memory is that we forget, and since its beginning as a scientific discipline, psychology has been trying to find out why and how this happens (Ribot 1882; Ebbinghaus 1885; Müller and Pilzecker 1900; Burnham 1903)? Addressing this question, Jenkins and Dallenbach (1924) published a remarkable study in 1924 suggesting that much forgetting arises from continued mental activity caused by ongoing everyday experiencing that normally follows learning in natural settings. Their intriguing findings were not systematically pursued during the next decades, as the focus shifted to exploring the role of prior or subsequent learning on forgetting; that is, effects of proactive or retroactive interference of highly similar material on memory retention. This research program eventually led into a dead end (Tulving and Madigan 1970; Wixted 2004), and interference research in humans slowed down in the 1970s. In recent years, however, interest about the neurobiological bases of interference began to emerge again (Appleby and Wiskott 2009; Bartko et al. 2010; Blake et al. 2010; Butterly et al. 2012; Luu et al. 2012; Martínez et al. 2012; Winocur et al. 2012; Peters et al. 2013; Alber et al. 2014; Censor et al. 2014; Martínez et al. 2014; McDevitt et al. 2014; Albasser et al. 2015; Eugenia et al. 2016; Koen and Rugg 2016; Ge et al. 2019; Peters and Smith 2020).In their original experiment, Jenkins and Dallenbach (1924) used sleep to reduce the amount of interference after learning. They found that when their participants went about their normal (university campus) day after learning a list of nonsense syllables, their ability to recall the lists 1, 2, 4, or 8 h later was always poorer than when instead they slept during the time between learning and test. Jenkins and Dallenbach (1924) concluded that their results “indicate that forgetting is not so much a matter of the decay of old impressions and associations than a matter of the interference, inhibition, or obliteration of the old by the new.” Their findings were replicated by others, confirming that being asleep, compared with being awake and active, indeed improves memory retention (Van Ormer 1932; Ekstrand 1967). However, it remained an open question whether it is the reduction of sensory stimulation and new learning, which would usually occur during wakefulness, that prevents retroactive interference, or whether a specific, possibly sleep-dependent, memory facilitation process was at play (Ekstrand 1967, 1972).Noting that participants in the sleep condition did not immediately fall asleep in the original experiment, but that they experienced increased quiescence shortly after learning, Minami and Dallenbach (1946) tested the retroactive interference explanation of forgetting more directly, by controlling the amount of stimulation after learning in awake animals. This remarkable experiment used Periplaneta americana (American cockroach) and a little treadmill. After learning to suppress their natural tendency to run into a dark shelter box in a bright open alley (encouraged by an electrical shock received in the dark shelter), the cockroaches were either placed on a running treadmill in a transparent box, or in a normally lit circular transparent resting chamber, where they were not able to fall asleep but experienced notably less activity than the cockroaches on the treadmill. The outcome was that cockroaches who were forced to move presented with more forgetting than those who were not, suggesting that sleep—notwithstanding its possible beneficial effect on memory—may not be necessary to promote memory retention; rather, reducing the amount of stimulation and activity after learning may be critical for attenuating retroactive interference and thus forgetting.Some six decades later, a series of experiments picked up this original line of inquiry. Exploring in humans whether memory for short prose, word lists, or spatial knowledge benefits from reduced stimulation after learning, these studies have invariably replicated the main finding that spending a 10-min retention interval in quiet wakefulness in a dimly lit room after learning leads to better memory for the learned material than participating in unrelated cognitive tasks during the retention interval (Dewar et al. 2007, 2010). Increased memory for the acquired material following quiet wakefulness is long-lasting and can be detected up to 7 d after learning (Dewar et al. 2012; Alber et al. 2014). Even in amnesic patients 10 min of reduced sensory stimulation, compared with participating in cognitive tasks, enhances memory retention for verbal material (Cowan et al. 2004; Dewar et al. 2009, 2010). This lends strong support to the suggestion that the memory loss in amnesia arises from an increased vulnerability to interference shortly after encoding (Warrington and Weiskrantz 1974; Hardt et al. 2013)Similar results have been obtained in rodents in studies exploring the role of perirhinal cortex in object recognition memory. Rats with lesions to the perirhinal cortex typically show robust impairments in object recognition tasks (Brown and Aggleton 2001; Mumby et al. 2002, 2007; Norman and Eacott 2005; Albasser et al. 2015). However, if rats are placed into a dark box during the retention interval between the encoding phase and the test phase of an object recognition task, rats with lesions to perirhinal cortex no longer show a memory deficit and perform as well as intact animals (McTighe et al. 2010). Thus, reduction of sensory stimulation between encoding and test appears to enhance memory for objects even in rats with perirhinal cortex lesions. This finding recapitulates the outcomes of the studies with human patients suffering from amnesia after hippocampal damage.The aim of the current experiments was to determine whether reducing sensory stimulation after encoding would also enhance hippocampus-dependent memory in rats. To do this, we used a spontaneous object exploration task that assesses memory for object locations (Ennaceur and Delacour 1988; Hardt et al. 2010; Migues et al. 2016, 2019). Using this approach, we replicated in rats the basic effect that quiet wakefulness promotes memory retention as previously observed in humans. Specifically, here we show that following learning, everyday activity in the home cage with cage mates impairs object location memory in rats, while reducing sensory stimulation in a dark chamber, without sleep, promotes it.     

Sleep spectral power correlates of prospective memory maintenance

Prospective memory involves setting an intention to act that is maintained over time and executed when appropriate. Slow wave sleep (SWS) has been implicated in maintaining prospective memories, although which SWS oscillations most benefit this memory type remains unclear. Here, we investigated SWS spectral power correlates of prospective memory. Healthy young adult participants completed three ongoing tasks in the morning or evening. They were then given the prospective memory instruction to remember to press “Q” when viewing the words “horse” or “table” when repeating the ongoing task after a 12-h delay including overnight, polysomnographically recorded sleep or continued daytime wakefulness. Spectral power analysis was performed on recorded sleep EEG. Two additional groups were tested in the morning or evening only, serving as time-of-day controls. Participants who slept demonstrated superior prospective memory compared with those who remained awake, an effect not attributable to time-of-day of testing. Contrary to prior work, prospective memory was negatively associated with SWS. Furthermore, significant increases in spectral power in the delta-theta frequency range (1.56 Hz–6.84 Hz) during SWS was observed in participants who failed to execute the prospective memory instructions. Although sleep benefits prospective memory maintenance, this benefit may be compromised if SWS is enriched with delta–theta activity.

Prospective memory refers to the maintenance, retrieval, and execution of a previously formed intention (Einstein and McDaniel 1990). Successful prospective memory is essential for a large number of tasks in daily life, such as remembering to attend a doctor''s appointment, to pick up a prescribed medication after that appointment, and to also pick up other needed items (e.g., groceries) while at the drugstore. The above described hypothetical sequence of events integrates previously studied prospective memory variants including time-based (i.e., maintaining a memory to complete an intention at a prespecified time; e.g., Esposito et al. 2015; Occhionero et al. 2017), activity-based (i.e., maintaining a memory to perform an intention before or after a particular activity; e.g., Occhionero et al. 2020), and cue-based (i.e., relying on external cues to prompt a maintained memory for a set intention; e.g., Scullin and McDaniel 2010; Leong et al. 2019b; Scullin et al. 2019).When it is required that memories be maintained across longer periods of time, prospective memory may become less reliable unless sleep occurs (Scullin and McDaniel 2010; Diekelmann et al. 2013a,b; Grundgeiger et al. 2014; Leong et al. 2019a,b; Scullin et al. 2019). Sleep appears to most strongly aid spontaneous retrieval of cue-based prospective memories (Leong et al. 2019a). Several reports have found that slow wave sleep (SWS) supports spontaneous retrieval of cue-based prospective memory intentions (e.g., Diekelmann et al. 2013a; Leong et al. 2019b), although at least one study found an association with rapid eye movement (REM) sleep instead (Scullin et al. 2019). Cue-based prospective memory is hypothesized to be a type of associative memory that binds prospective components (the prospective memory cue) and retrospective components (maintenance of the memory for the prospective memory intention when presented with the cue; Diekelmann et al. 2013b; Leong et al. 2019a).Rodent and human literature, implementing a variety of invasive and noninvasive brain imaging techniques, show that cortical slow oscillations (SOs; <1 Hz) and fast thalamocortical sleep spindles during SWS facilitate associative memory retention (Niknazar et al. 2015; Latchoumane et al. 2017; Helfrich et al. 2018; Mikutta et al. 2019; Muehlroth et al. 2019), whereas faster oscillations, such as those in the theta frequency band (∼4–7 Hz), may inhibit declarative associative memory (Marshall et al. 2011). We therefore hypothesize that prospective memory performance, like other studied associative memory variants, should benefit from oscillations during SWS (Klinzing et al. 2019). However, it remains unknown which SWS microarchitectural features may facilitate or inhibit prospective memory performance.Here, we aimed to first replicate prior findings that prospective memories are better maintained across a 12-h interval including sleep compared with an equivalent interval of wakefulness (e.g., Scullin and McDaniel 2010). We next explored whether sleep-associated memory maintenance might be linked to SWS microarchitectural features. To our knowledge, this is the first experiment to examine whether SWS oscillations differentiate successful from unsuccessful prospective memory performance. Given the role of hippocampal engagement in both associative memory binding (e.g., Yonelinas et al. 2019) and oscillatory coupling during SWS that supports associative memory (Niknazar et al. 2015; Latchoumane et al. 2017; Helfrich et al. 2018; Mikutta et al. 2019; Muehlroth et al. 2019), we hypothesized that prospective memory performance would be supported by SWS and specifically SOs and sleep spindle activity.     

Post-training reversible inactivation of the hippocampus enhances novel object recognition memory

Research on the role of the hippocampus in object recognition memory has produced conflicting results. Previous studies have used permanent hippocampal lesions to assess the requirement for the hippocampus in the object recognition task. However, permanent hippocampal lesions may impact performance through effects on processes besides memory consolidation including acquisition, retrieval, and performance. To overcome this limitation, we used an intrahippocampal injection of the GABA agonist muscimol to reversibly inactivate the hippocampus immediately after training mice in two versions of an object recognition task. We found that the inactivation of the dorsal hippocampus after training impairs object-place recognition memory but enhances novel object recognition (NOR) memory. However, inactivation of the dorsal hippocampus after repeated exposure to the training context did not affect object recognition memory. Our findings suggest that object recognition memory formation does not require the hippocampus and, moreover, that activity in the hippocampus can interfere with the consolidation of object recognition memory when object information encoding occurs in an unfamiliar environment.The medial temporal lobe plays an important role in recognition memory formation, as damage to this brain structure in humans, monkeys, and rodents impairs performance in recognition memory tasks (for review, see Squire et al. 2007). Within the medial temporal lobe, studies have consistently demonstrated that the perirhinal cortex is involved in this form of memory (Brown and Aggleton 2001; Winters and Bussey 2005; Winters et al. 2007, 2008; Balderas et al. 2008). In contrast, the role of the hippocampus in object recognition memory remains a source of debate. Some studies have reported novel object recognition (NOR) impairments in animals with hippocampal lesions (Clark et al. 2000; Broadbent et al. 2004, 2010), yet others have reported no impairments (Winters et al. 2004; Good et al. 2007). Differences in hippocampal lesion size and behavioral procedures among the different studies have been implicated as the source of discrepancy in these findings (Ainge et al. 2006), but previous studies have not examined the consequences of environment familiarity on the hippocampus dependence of object recognition memory.Previous studies addressing the role of the hippocampus in recognition memory relied on permanent, pre-training lesions (Clark et al. 2000; Broadbent et al. 2004; Winters et al. 2004; Good et al. 2007). Permanent lesions inactivate the hippocampus not only during the consolidation phase, but also during habituation, acquisition, and memory retrieval, potentially confounding interpretation of the results. Furthermore, permanent lesion studies require long surgery recovery times during which extrahippocampal changes may emerge to mask or compensate for the loss of hippocampal function. To overcome these problems, we reversibly inactivated the dorsal hippocampus after training mice in two versions of the object recognition task. We infused muscimol, a γ-aminobutyric acid (GABA) receptor type A agonist, into the dorsal hippocampus immediately after training in an object-place recognition task or immediately following training in a NOR task. Consistent with previous studies (Save et al. 1992; Galani et al. 1998; Mumby et al. 2002; Stupien et al. 2003; Aggleton and Brown 2005), we observed that hippocampal inactivation impairs object-place recognition memory. Interestingly, we observed that the degree of contextual familiarity can influence NOR memory formation. We found that when shorter periods of habituation to the experimental environment were used, hippocampal inactivation enhances long-term NOR memory. In contrast, after extended periods of contextual habituation, long-term recognition memory was unaltered by hippocampal inactivation. Together these results suggest that if familiarization with objects occurs at a stage in which the contextual environment is relatively novel, the hippocampus plays an inhibitory role on the consolidation of object recognition memory. Supporting this view, we observed that object recognition memory is unaffected by hippocampal inactivation when initial exploration of the objects occurred in a familiar environment.     

Memory for individual items is related to nonreinforced preference change

It is commonly assumed that memories contribute to value-based decisions. Nevertheless, most theories of value-based decision-making do not account for memory influences on choice. Recently, new interest has emerged in the interactions between these two fundamental processes, mainly using reinforcement-based paradigms. Here, we aimed to study the role memory processes play in preference change following the nonreinforced cue-approach training (CAT) paradigm. In CAT, the mere association of cued items with a speeded motor response influences choices. Previous studies with this paradigm showed that a single training session induces a long-lasting effect of enhanced preferences for high-value trained stimuli, that is maintained for several months. We hypothesized that CAT increases memory of trained items, leading to enhanced accessibility of their positive associative memories and in turn to preference changes. In two preregistered experiments, we found evidence that memory is enhanced for trained items and that better memory is correlated with enhanced preferences at the individual item level, both immediately and 1 mo following CAT. Our findings suggest that memory plays a central role in value-based decision-making following CAT, even in the absence of external reinforcements. These findings contribute to new theories relating memory and value-based decision-making and set the groundwork for the implementation of novel nonreinforced behavioral interventions that lead to long-lasting behavioral change.

Value-based decision-making and memory are both extensively studied processes in cognitive psychology and cognitive neuroscience (Fellows 2017). Most theories of value-based decision-making have focused on processes related to the incremental learning of value following external reinforcement, but have not explicitly addressed the role of memory per se. Thus, fundamental questions remain regarding interactions between memory and value-based decisions, which have been gaining attention in recent years.Several recent empirical studies have demonstrated interactions between episodic memory and value-based decision-making. For example, memory for past events has been shown to bias value-based decisions (Duncan and Shohamy 2016), differently for choices of novel versus choices of familiar options (Duncan et al. 2019), and choice behavior and fMRI signals during value-based decision-making were better explained by episodic memory for individual past choices than by a standard reinforcement learning model (Bornstein et al. 2017). Another study has found that during sampling of episodic memories of previous choices, the retrieved context influenced present choices, deviating from the predictions of standard reinforcement learning models (Bornstein and Norman 2017). Other studies have demonstrated that the long time known effect of choices on future preferences is related to memory processes (Chammat et al. 2017; DuBrow et al. 2019; Luettgau et al. 2020). At the neural level, the ventromedial prefrontal cortex (vmPFC) and the hippocampus both have been shown to play a role in memory processes and value-based decisions (Weilbächer and Gluth 2017) and recent studies have been further emphasizing that the hippocampus bridges between past experience and future decisions (Bakkour et al. 2019; Biderman et al. 2020).All these studies, and many others, highlighted the interaction between memory and value-based decision-making involving external reinforcements. However, everyday life involves decisions and associations that are not directly reinforced. Thus, it remains unclear whether memory plays a general role in value-based decision-making even without external reinforcements.To better understand the role of memory processes in shaping preferences independently of external reinforcements, we used a novel behavioral change paradigm, named cue-approach training (CAT). In this paradigm, associating images of items with a neutral cue and a speeded motor response results in a consistent preference enhancement without external reinforcement, which is maintained for months (Schonberg et al. 2014; Bakkour et al. 2018; Salomon et al. 2018, 2019; Botvinik-Nezer et al. 2020). During CAT, images of items are consistently paired with a neutral cue and a speeded motor response (“Go items”), while other items are presented without the cue or the response (“NoGo items”). One training session with several presentations of all items leads to long-lasting preference changes, measured as the likelihood of choosing Go over NoGo items that had similar initial subjective values (Schonberg et al. 2014). Results from over 30 samples with this paradigm have demonstrated a replicable effect on various types of stimuli, including snack food items, fruits and vegetables, unfamiliar faces, fractal art images, and positive affective images (Bakkour et al. 2016, 2017; Veling et al. 2017; Zoltak et al. 2017; Bakkour et al. 2018; Salomon et al. 2018, 2019; Botvinik-Nezer et al. 2020), revealing the potential of the CAT paradigm as an experimental platform for value-based decision-making without external reinforcements (Schonberg and Katz 2020).The underlying mechanisms of the change of preferences following CAT are not yet fully understood (Schonberg et al. 2014; Bakkour et al. 2017; Salomon et al. 2019; Botvinik-Nezer et al. 2020; Schonberg and Katz 2020). The long-lasting nature of the effect, which has been shown to last for up to 6 mo following a single training session (Schonberg et al. 2014; Salomon et al. 2018, 2019; Botvinik-Nezer et al. 2020), raises the hypothesis that memory processes are involved in its maintenance. Furthermore, previous studies have found enhanced memory for Go compared with NoGo items with other types of Go–NoGo tasks (Chiu and Egner 2015a,b; Yebra et al. 2019) and for items for which participants have a sense of agency (Murty et al. 2015). One recent study provided preliminary evidence suggesting that memory is involved in preference change following a similar nonreinforced Go/NoGo training task (Chen et al. 2021).We hypothesized that CAT enhances memory of Go items, which in turn leads to preferring these items over NoGo items. Previous neuroimaging findings with CAT that suggested possible interactions between hippocampal fMRI activity and subsequent preferences 1 mo following CAT, provide additional evidence in support of this hypothesis (Botvinik-Nezer et al. 2020). Therefore, here we set out to test the role memory processes play in the behavioral change of preferences following CAT, in the short and in the long term.We propose an underlying mechanism for the CAT effect, in which preference change following CAT results from a boost in memory encoding of positive Go items, which in itself is a consequence of enhanced perceptual processing of Go items (Schonberg et al. 2014; Botvinik-Nezer et al. 2020). We hypothesize that the enhanced encoding of Go items, as well as the greater perceptual activation in response to them, increases accessibility of attributes and associations of these specific Go items (Anderson 1983; Bhatia 2013). Furthermore, we hypothesized that preference changes, reflected in the binary choice phase, are due to the enhanced accessibility of memory associations of the Go items, which tips the scales in favor of the Go items when the associations are positive.In order to test memory for individual items, in the current work we introduced a memory recognition task following CAT. In two independent preregistered experiments and one pilot experiment, memory was evaluated following a long (16 repetitions) or short (a single exposure) CAT training session, before the probe phase that evaluated post-training preferences. We then tested our predictions that (1) memory will be stronger for Go compared with NoGo items following CAT (more accurate and faster responses in the recognition task) and (2) that memory will be related to choices (better remembered Go items will be chosen over worse remembered NoGo items). Since the link between better memory and enhanced choices is hypothesized to be related to positive associated memories, we tested the relationship between memory and choices separately for choices between low-value and choices between high-value items. These hypotheses were tested both in the short term (immediately or a few days after CAT) and in a 1-mo follow-up.     

More than meets the eye: The attentional blink in multisensory environments. Commentary on Kranczioch and Thorne

Temporal fluctuations of attention can influence performance of cognitive tasks substantially. A common paradigm to investigate temporal fluctuations of attention is the attentional blink paradigm. Kranczioch and Thorne (2013) report new evidence for the impact of auditory stimuli on the visual attentional blink in the current issue of Advances in Cognitive Psychology.     

The NMDA antagonist MK-801 disrupts reconsolidation of a cocaine-associated memory for conditioned place preference but not for self-administration in rats

Recent research suggests that drug-related memories are reactivated after exposure to environmental cues and may undergo reconsolidation, a process that can strengthen memories. Conversely, reconsolidation may be disrupted by certain pharmacological agents such that the drug-associated memory is weakened. Several studies have demonstrated disruption of memory reconsolidation using a drug-induced conditioned place preference (CPP) task, but no studies have explored whether cocaine-associated memories can be similarly disrupted in cocaine self-administering animals after a cocaine priming injection, which powerfully reinstates drug-seeking behavior. Here we used cocaine-induced CPP and cocaine self-administration to investigate whether the N-methyl-D-aspartate receptor antagonist (+)-5methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) given just prior to reactivation sessions would suppress subsequent cocaine-primed reinstatement (disruption of reconsolidation). Systemic injection of MK-801 (0.05 or 0.20 mg/kg administered intraperitoneally) in rats just prior to reactivation of the cocaine-associated memory in the CPP context attenuated subsequent cocaine-primed reinstatement, while no disruption occurred in rats that did not receive reactivation in the CPP context. However, in rats trained to self-administer cocaine, systemic administration of MK-801 just prior to either of two different types of reactivation sessions had no effect on subsequent cocaine-primed reinstatement of lever-pressing behavior. Thus, systemic administration of MK-801 disrupted the reconsolidation of a cocaine-associated memory for CPP but not for self-administration. These findings suggest that cocaine-CPP and self-administration do not use similar neurochemical processes to disrupt reconsolidation or that cocaine-associated memories in self-administering rats do not undergo reconsolidation, as assessed by lever-pressing behavior under cocaine reinstatement conditions.The ability to disrupt previously consolidated memories in a reactivation-dependent manner is thought to be due to the disruption of a memory reconsolidation process. This disruption of reconsolidation has been observed in a wide variety of tasks and species (Nader et al. 2000b; Sara 2000; Alberini 2005; Riccio et al. 2006). Early reconsolidation experiments primarily focused on aversive learning paradigms, with an emphasis on disruption of reconsolidation as a potential treatment for posttraumatic stress disorder (Misanin et al. 1968; Nader et al. 2000a; Debiec and Ledoux 2004; Brunet et al. 2008). Only more recently have investigators demonstrated that appetitive memories also undergo reconsolidation; most, but not all (Yim et al. 2006), studies found a disruption of expression for the drug-associated memory, suggesting the potential to target the reconsolidation process as a treatment for drug addiction (Lee et al. 2005; Miller and Marshall 2005; Milekic et al. 2006; Valjent et al. 2006; Brown et al. 2007; Kelley et al. 2007; Sadler et al. 2007; Fricks-Gleason and Marshall 2008; Milton et al. 2008a, b).Miller and Marshall (2005) showed that reconsolidation of cocaine conditioned place preference (CPP) in the rat could be disrupted by either pre- or post-treatment of a phosphorylation inhibitor of extracellular signal-regulated kinase (1/2) (ERK) in a reactivation-dependent manner. Other studies have shown that protein synthesis inhibitors (Milekic et al. 2006), a matrix metalloproteinase (MMP) inhibitor (Brown et al. 2007), a β-noradrenergic receptor antagonist (Bernardi et al. 2006; Robinson and Franklin 2007a; Fricks-Gleason and Marshall 2008), and an N-methyl-D-aspartate (NMDA) receptor antagonist (Kelley et al. 2007; Sadler et al. 2007) can also disrupt the reconsolidation of drug-associated CPP memories. Studies by Lee and colleagues have shown that Zif268 antisense oligodeoxynucleotide infused into the basolateral amygdala prior to reactivation of memory for a cocaine-associated cue (the conditioned stimulus or CS) disrupts the ability of cocaine-associated cues to establish subsequent acquisition of a new instrumental response (Lee et al. 2005), and the ability of a drug-associated cue to induce relapse under a second-order schedule (Lee et al. 2006a). Thus, cocaine-associated memories appear to undergo reconsolidation in both Pavlovian and operant conditioning paradigms.Relapse to drug-seeking or drug-taking behavior can occur after re-exposure to three types of stimuli: the drug itself, drug-associated contextual and discrete cues, and stress; and all of these may promote relapse in humans (for review, see Epstein et al. 2006). Only a few CPP studies (Valjent et al. 2006; Brown et al. 2007) and no self-administration studies to our knowledge have tested whether the drug-associated memory can be rendered susceptible to disruption by pharmacological agents such that subsequent cocaine-primed reinstatement is suppressed. This drug-primed effect is observed in humans, producing relapse (Ludwig et al. 1974; Jaffe et al. 1989), and in rats, producing robust reinstatement of drug-seeking behavior in both CPP and self-administration tasks (McFarland and Ettenberg 1997; McFarland and Kalivas 2001; Sanchez and Sorg 2001; Kalivas and McFarland 2003). The development of a treatment strategy that makes use of the reconsolidation process will ultimately need to be powerful enough to diminish drug-seeking behavior in the presence of sizable doses of the drug itself. Therefore, the primary goal of this study was to determine whether drug-primed reinstatement could be suppressed in rats that have the memory reactivated in the presence of a pharmacological agent in cocaine self-administering rats. Since we previously have demonstrated the ability to disrupt cocaine-primed reinstatement only in animals in which the memory was reactivated using cocaine-induced CPP, we also tested the extent to which the same parameters used to disrupt reconsolidation in a cocaine-induced CPP task would disrupt reconsolidation in a cocaine self-administration task under conditions of drug-induced reinstatement.To examine this question, we chose the noncompetitive NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). MK-801 has been shown to disrupt reconsolidation of spatial tasks (Przybyslawski and Sara 1997), fear tasks (Lee et al. 2006b), amphetamine-induced CPP (Sadler et al. 2007), cocaine-induced CPP (Kelley et al. 2007), and sucrose self-administration (Lee and Everitt 2008). Importantly, the two studies examining CPP using MK-801 did not explore whether MK-801 suppressed drug-seeking behavior in a manner that was dependent on whether the memory was reactivated, leaving open the possibility that it was not a reconsolidation process that was disrupted by MK-801.Here we demonstrate that MK-801 injected prior to cocaine-primed reinstatement of CPP disrupted subsequent cocaine-primed reinstatement of CPP, and this disruption was dependent on CPP contextual reactivation since injection of MK-801 and cocaine in the home cage did not disrupt subsequent cocaine-primed reinstatement of CPP. However, drug-seeking behavior in animals trained for cocaine self-administration was not disrupted when rats were reactivated under the same parameters that disrupted cocaine-induced CPP or when rats were given a reactivation session identical to their self-administration sessions. We thus demonstrate for the first time that memories associated with cocaine-induced CPP and cocaine self-administration are not similarly susceptible to disruption by MK-801.     

Entorhinal cortical Island cells regulate temporal association learning with long trace period

Temporal association learning (TAL) allows for the linkage of distinct, nonsynchronous events across a period of time. This function is driven by neural interactions in the entorhinal cortical–hippocampal network, especially the neural input from the pyramidal cells in layer III of medial entorhinal cortex (MECIII) to hippocampal CA1 is crucial for TAL. Successful TAL depends on the strength of event stimuli and the duration of the temporal gap between events. Whereas it has been demonstrated that the neural input from pyramidal cells in layer II of MEC, referred to as Island cells, to inhibitory neurons in dorsal hippocampal CA1 controls TAL when the strength of event stimuli is weak, it remains unknown whether Island cells regulate TAL with long trace periods as well. To understand the role of Island cells in regulating the duration of the learnable trace period in TAL, we used Pavlovian trace fear conditioning (TFC) with a 60-sec long trace period (long trace fear conditioning [L-TFC]) coupled with optogenetic and chemogenetic neural activity manipulations as well as cell type-specific neural ablation. We found that ablation of Island cells in MECII partially increases L-TFC performance. Chemogenetic manipulation of Island cells causes differential effectiveness in Island cell activity and leads to a circuit imbalance that disrupts L-TFC. However, optogenetic terminal inhibition of Island cell input to dorsal hippocampal CA1 during the temporal association period allows for long trace intervals to be learned in TFC. These results demonstrate that Island cells have a critical role in regulating the duration of time bridgeable between associated events in TAL.

The linkage of temporally discontiguous events, called temporal association learning (TAL), is an essential function for episodic memory formation; for animals, when an event took place, and in what order a series of events occurred is directly linked to adaptation to continuous changes in the environment (Eichenbaum 2000; Tulving 2002a,b; Kitamura et al. 2015a; Kitamura 2017; Pilkiw and Takehara-Nishiuchi 2018). The entorhinal cortical–hippocampal (EC-HPC) network in particular is currently considered to bridge the temporal discontinuity between events (Solomon et al. 1986; Moyer et al. 1990; Wallenstein et al. 1998; McEchron et al. 1999; Eichenbaum 2000; Huerta et al. 2000; Ryou et al. 2001; Takehara et al. 2003; Chowdhury et al. 2005; Esclassan et al. 2009; Morrissey et al. 2012; Suter et al. 2013; Sellami et al. 2017; Wilmot et al. 2019).Two major excitatory inputs to HPC arise from the superficial layers of the EC (Fig. 1A), forming the direct (monosynaptic), and indirect (trisynaptic) pathways (Amaral and Witter 1989; Amaral and Lavenex 2007; Kitamura 2017; Kitamura et al. 2017). While pyramidal cells in EC layer III (ECIII cells) project directly to CA1 (Kohara et al. 2014; Kitamura et al. 2015b), the trisynaptic pathway originates from excitatory Reelin⁺ stellate cells in EC layer II (ECII) projecting directly to DG, CA3, and CA2 (Fig. 1B; Tamamaki and Nojyo 1993; Varga et al. 2010). CalbindinD-28K⁺/Wolfram syndrome 1 (Wfs1)⁺ pyramidal cells, another excitatory neural population in EC layer II called “Island cells,” form cell clusters along the ECII/ECI border (Alonso and Klink 1993; Fujimaru and Kosaka 1996; Klink and Alonso 1997; Kawano et al. 2009; Varga et al. 2010; Kitamura et al. 2014; Ray et al. 2014) and directly project to the GABAergic interneurons of stratum lacunosum (SL-INs) in HPC CA1 and drive feedforward inhibition to HPC CA1 pyramidal cells (Fig. 1B; Kitamura et al. 2014; Surmeli et al. 2016; Kitamura 2017; Ohara et al. 2018; Yang et al. 2018; Zutshi et al. 2018).Open in a separate windowFigure 1.Circuit schematic diagram of the medial entorhinal cortex (MEC)–hippocampal (HPC) circuit. (A) Major projections in the entorhinal cortical (EC)-HPC network. ECIII neurons (green) project directly to CA1. ECII Ocean cells (ECIIo, purple) project to the dentate gyrus (DG) (light blue)/CA3 (pink) initiating the trisynaptic pathway. ECII Island cells (ECIIi, blue) project directly into CA1. (B) ECIII projections (green) excite the distal portions of CA1 pyramidal cell (yellow) dendrites in the stratum moleculare. Island cells (ECIIi, blue) excite the interneurons of stratum lacunosum (SL-INs, red), which in turn inhibit the distal dendrites of CA1 pyramidal cells in SL.Trace fear conditioning (TFC) has been established as one suitable animal model for TAL (Fendt and Fanselow 1999; Maren 2001; Kim and Jung 2006) that can be also used as a translational bridge between animal and human learning (Clark and Squire 1998; Buchel and Dolan 2000; Delgado et al. 2006). Lesion, pharmacological, molecular, and optogenetic manipulation, as well as disease models in medial entorhinal cortex (MEC), demonstrate that MEC is crucial for TFC and temporal learning (Ryou et al. 2001; Woodruff-Pak 2001; Runyan et al. 2004; Esclassan et al. 2009; Gilmartin and Helmstetter 2010; Suh et al. 2011; Morrissey et al. 2012; Shu et al. 2016; Hales et al. 2018; Yang et al. 2018; Heys et al. 2020). Specifically, MECIII inputs into the HPC CA1 pyramidal cells are essential for the formation of TFC (Yoshida et al. 2008; Suh et al. 2011; Kitamura et al. 2014; Kitamura 2017). However, the temporal association function driven by MECIII neurons must be regulated for optimal adaptive memory formation, as too strong an association of a particular pair of events may interfere with associations of other useful pairs, whereas too weak an association for a given pair of events, in terms of weaker impact of events or longer duration of temporal gap between events, would not result in an effective memory (Kitamura et al. 2015a; Marks et al. 2020). In a naturalistic context, this would mean that more distant/quieter sounds, less intense somatic sensations (e.g., pain), or increased temporal distance between any two events would signal that the events are less likely to be causally associated, therefore less relevant, and less likely to be stored and recalled. In fact, successful TFC depends on the strength of event stimuli and duration of temporal gap between events (Stiedl and Spiess 1997; Misane et al. 2005; Kitamura et al. 2014; Kitamura 2017). However, the underlying regulatory mechanism for TAL remains hidden. Previously we demonstrated that feedforward inhibition by Island cells acts as a gating controller for the MECIII inputs to the distal dendrites of HPC CA1 pyramidal cells in stratum moleculare (SM) (Kitamura et al. 2014) to control TFC when weaker (in this case diminished footshock intensity) unconditioned stimuli were delivered for TFC, indicating that Island cell activity controls the temporal association when the strength of two discontinuous events are relatively weaker. However, the way in which the EC-HPC network regulates TFC with a longer trace period still remains unknown. Because the activation of Island cells would result in a net inhibitory effect on the local network in CA1, imposing a tight and specific regulation on associations of events across the temporal gap in TAL (Crestani et al. 2002; Moore et al. 2010; Kitamura et al. 2014, 2015b), we hypothesized that the length of the temporal gap between events would also be modulated by this mechanism. In this study, we examined the role of the regulatory input to this circuit arising specifically from the Island cells in the MECII using apoptotic elimination of Island cells, chemogenetic neural inhibition, and optogenetic terminal inhibition methods within an L-TFC protocol to give a thorough and complete assessment of the circuit involvement while considering each technique''s unique features.     

Recovery of memory from infantile amnesia is developmentally constrained

Episodic memories formed during infancy are rapidly forgotten, a phenomenon associated with infantile amnesia, the inability of adults to recall early-life memories. In both rats and mice, infantile memories, although not expressed, are actually stored long term in a latent form. These latent memories can be reinstated later in life by certain behavioral reminders or by artificial reactivations of neuronal ensembles activated at training. Whether the recovery of infantile memories is limited by developmental age, maternal presence, or contingency of stimuli presentation remains to be determined. Here, we show that the return of inhibitory avoidance memory in rats following a behavioral reactivation consisting of an exposure to the context (conditioned stimuli [CS]) and footshock (unconditioned stimuli [US]) given in a temporally unpaired fashion, is evident immediately after US and is limited by the developmental age at which the reactivations are presented; however, it is not influenced by maternal presence or the time interval between training and reactivation. We conclude that one limiting factor for infantile memory reinstatement is developmental age, suggesting that a brain maturation process is necessary to allow the recovery of a “lost” infantile memory.

Hippocampus-dependent episodic memories formed early in life are rapidly forgotten. This process of forgetting is evolutionarily conserved and is associated with infantile amnesia, the inability of adults to recall early-life memories (Campbell and Spear 1972; Rovee-Collier 1999; Hayne 2004; Josselyn and Frankland 2012; Callaghan et al. 2014; Madsen and Kim 2016; Alberini and Travaglia 2017).Although they are rapidly forgotten, early-life experiences influence brain functions throughout life (Jacobs and Nadel 1985; Meaney et al. 1988; Sroufe et al. 1990; Brunson et al. 2005; Pryce et al. 2005; Mineka and Zinbarg 2006; Bale et al. 2010; Poulos et al. 2014; Perry and Sullivan 2014) and produce long-lasting biological changes in the brain. For example, aversive early-life experiences regulate the expression of hippocampal glucocorticoid receptor and hypothalamic–pituitary–adrenal (HPA) functions in a persistent fashion (Champagne and Curley 2009). Moreover, threatening experiences in early life can predispose individuals to psychopathologies such as post-traumatic stress disorder (PTSD) and mood and anxiety disorders (Heim and Nemeroff 2001). Consistent with this long-lasting influence on behavior, studies in rodent models have shown that, in contrast to what was previously believed, memories formed in infancy (at postnatal days 16–18 [PN16–PN18]) although not expressed, are not lost. Rather, they are stored over the longer term in a latent form and can reemerge at later times, up to adulthood, following behavioral reactivations or artificial stimulation of the neuronal ensembles activated at learning (Travaglia et al. 2016; Guskjolen et al. 2018; Bessières et al. 2020).In our previous studies based on rat inhibitory avoidance (IA), a paradigm in which the animal learns to avoid a context previously paired with a footshock, we confirmed that an infantile episodic learning event given at PN17 resulted in the typical rapid forgetting. We then found that, however, this memory was stored long term in a latent form, as demonstrated by the observation that it could reemerge following a behavioral reactivation consisting of exposure to the training context (conditioned stimulus [CS]) and a later time footshock of the same intensity that was used during training (unconditioned stimulus [US]) but now given in a distinct context. Reexperiencing either the context or the footshock alone failed to reinstate the infantile memory (Travaglia et al. 2016), suggesting that the return of infantile memory is limited by certain boundaries.Several questions about the conditions for the recovery of latent, infantile episodic memories remain to be addressed. First, is infantile memory reinstatement following the unpaired US presentation temporally regulated? Second, is there an age limit for memory reinstatement? Third, given that other types of learning, such as non-hippocampus-dependent cued conditioning, are regulated by the maternal presence during infancy (Moriceau and Sullivan 2006), is infantile episodic memory reinstatement limited or regulated by the presence of the mother? In this study, we set out to address these questions using IA in infant rats.     

Lifelong reductions of PKMζ in ventral hippocampus of nonhuman primates exposed to early-life adversity due to unpredictable maternal care

Protein kinase Mζ (PKMζ) maintains long-term potentiation (LTP) and long-term memory through persistent increases in kinase expression. Early-life adversity is a precursor to adult mood and anxiety disorders, in part, through persistent disruption of emotional memory throughout life. Here we subjected 10- to 16-wk-old male bonnet macaques to adversity by a maternal variable-foraging demand paradigm. We then examined PKMζ expression in their ventral hippocampi as 7- to 12-yr-old adults. Quantitative immunohistochemistry reveals decreased PKMζ in dentate gyrus, CA1, and subiculum of subjects who had experienced early-life adversity due to the unpredictability of maternal care. Adult animals with persistent decrements of PKMζ in ventral hippocampus express timid rather than confrontational responses to a human intruder. Persistent down-regulation of PKMζ in the ventral hippocampus might reduce the capacity for emotional memory maintenance and contribute to the long-lasting emotional effects of early-life adversity.

Early-life adversity is associated with an increased vulnerability to stress-related disorders that is maintained into adulthood, suggesting a very long-lived effect on emotional memory by the early-life event (Coplan et al. 1996). Although several structural and neurochemical sequelae of early-life adversity have been reported (Teicher et al. 2003; Jackowski et al. 2011), the direct effects of early-life adversity on the molecular substrates maintaining long-term memory storage have not been explored.Accumulating evidence supports a crucial role for the autonomously active, atypical protein kinase C (PKC) isoform protein kinase Mζ (PKMζ) in maintaining synaptic long-term potentiation (LTP), a putative physical substrate for memory, and long-term memory storage (Ling et al. 2002; Pastalkova et al. 2006; Glanzman 2013; Sacktor and Fenton 2018). The autonomous activity of PKMζ is due to its unusual structure that differs from other PKC isoforms (Sacktor et al. 1993). Most PKCs consist of two domains: a catalytic domain and an autoinhibitory regulatory domain that suppresses the catalytic domain. Therefore, most PKCs are inactive until second messengers bind to the regulatory domain and induce a conformational change that releases the autoinhibition. Because second messengers that activate PKCs such as Ca²⁺ or diacylglycerol have short half-lives, most PKCs are only transiently activated.PKMζ, in contrast, consists of an independent PKCζ catalytic domain, and the absence of an autoinhibitory regulatory domain results in autonomous and thus persistent activity once the kinase is synthesized. PKMζ mRNA is transcribed from an internal promoter within the PKCζ/PKMζ gene that is active only in neural tissue (Hernandez et al. 2003). The mRNA is translationally repressed and transported to dendrites of neurons (Muslimov et al. 2004). High-frequency afferent synaptic activity during LTP induction or learning derepresses PKMζ mRNA translation, triggering new synthesis of PKMζ protein (Osten et al. 1996; Hernandez et al. 2003; Tsokas et al. 2016; Hsieh et al. 2017).Once increased, the steady-state amount of PKMζ remains elevated during LTP or long-term memory maintenance. Recent work with quantitative immunohistochemistry (IHC) shows that spatial conditioning induces persistent increases of PKMζ in somatic and selective dendritic compartments of dorsal hippocampal CA1 pyramidal cells that can last at least 1 mo (Hsieh et al. 2021). The persistent increases are preferentially expressed in CA1 pyramidal cells that were activated during the formation of the memory, specifically at the termination zone of the Schaffer collateral/commissural inputs from subfield CA3. In contrast, persistent PKMζ increases are not evident in stratum lacunosum-moleculare, the termination zone that originates in entorhinal cortex that nonetheless is capable of expressing PKMζ. Postsynaptic domain-specific PKMζ expression patterns hint at distinct circuit-specific modifications of cortical–hippocampal synaptic function by maturational and experiential factors.Persistent changes in PKMζ expression are also associated with changes in the capacity for learning and memory across the life span of animals. Decreased memory ability in aged rats is associated with decreased training-induced, persistent PKMζ expression in prelimbic cortex, and increases in PKMζ are crucial for the cognition-enhancing effects of environmental enrichment in the aged animals (Chen et al. 2016). Hara et al. extended the connection between PKMζ and cognitive function to nonhuman primates (NHPs), showing that levels of PKMζ expression in dentate gyrus (DG) axospinous synapses correlate with successful performance on cognitive tasks in young and aged monkeys (Hara et al. 2012). These studies suggest that persistent down-regulation of PKMζ may comprise an important pathophysiological mechanism for cognitive impairment.Here we used a validated NHP model of early-life adversity, maternal variable-foraging demand (VFD), to explore the links between adversity in infancy and PKMζ expression in adulthood (Coplan et al. 1996; Jackowski et al. 2011). Previous studies of the VFD paradigm have revealed that both infants and their mothers exposed to VFD show significant cerebrospinal fluid (CSF) elevations of the stress neuropeptide, corticotropin-releasing factor (CRF). Moreover, the magnitude of CRF change in mothers and infants are positively correlated, suggesting synchronization of maternal–infant stress responses to the VFD stressor (Coplan et al. 2005). From a behavioral standpoint, maternal social rank plays a negligible role in determining an aggregate score of maternal–infant proximity, suggesting preferential attention of mothers to their infants. During the VFD condition, maternal social rank predicts >80% of the variance of maternal–infant proximity, suggesting mothering patterns are interrupted by preferential orientations to social rank; the latter determines food accessibility (Coplan et al. 2015). Dominant females show relative increases in maternal–infant proximity, whereas subordinate females show relative reductions in maternal–infant proximity. Neither pattern of attachment ameliorates an abnormal association between CSF oxytocin concentrations and hypothalamic-pituitary-adrenal (HPA) axis activity (Coplan et al. 2015). Offspring exposed to VFD rearing assessed both as juveniles and as full adults demonstrate persistent increases in CSF CRF concentrations in comparison with controls reared under non-VFD conditions (Coplan et al. 1996, 2001).Our prior neurohistological studies pointed to the DG as a region particularly vulnerable to VFD exposure, as shown by reduced trophic signaling and neurogenesis (Jackowski et al. 2011; Perera et al. 2011; Schoenfeld et al. 2021). We therefore hypothesized that early-life adversity due to unpredictable maternal care (for brevity, subsequently referred to as “early-life adversity”) reduces the persistent expression of PKMζ within the DG of ventral intrahippocampal neurocircuitry that mediates affective memory processing (Fanselow and Dong 2010). We used PKMζ antisera validated by the lack of immunostaining in PKMζ-null mice (Hsieh et al. 2021) to examine PKMζ expression in ventral hippocampus (NHP anterior hippocampus) in both DG granule cell layer and the stratum moleculare of the suprapyramidal blade that receives direct input from entorhinal cortex, as well as other regions encompassing the hippocampal formation, including the hilus, CA3, CA1, and subiculum.To assess behavioral correlates of hippocampal PKMζ expression, we used a stress-inducing paradigm designed specifically for singly housed bonnet macaque male NHPs, which we refer to as the “human exposure response” (Jackowski et al. 2011; Hamel et al. 2017), which is a variation of the paradigm used in human exposure studies by Kalin et al. in rhesus macaques (Kalin and Shelton 1989). On exposure to a direct human presence, singly housed adult male bonnet macaques react with a dichotomy of responses—confrontational versus timid (see the Materials and Methods) (Jackowski et al. 2011). In our macaque colony, groups of fully adult males are necessarily housed individually to prevent injury sustained during male agonistic encounters, whereas adult females and/or juveniles are safely housed in social groups. Because group housing of nursing females and/or juveniles of both sexes elicits a range of behaviors intrinsic to the species’ social repertoire (Rosenblum et al. 2001; Coplan et al. 2015) that complicates behavioral analyses to human exposure, we restricted our current study to male macaques.     

A cognitive map for object memory in the hippocampus

The hippocampus has been proposed to support a cognitive map, a mental representation of the spatial layout of an environment as well as the nonspatial items encountered in that environment. In the present study, we recorded simultaneously from 43 to 61 hippocampal pyramidal cells as rats performed an object recognition memory task in which novel and repeated objects were encountered in different locations on a circular track. Multivariate analyses of the neural data indicated that information about object identity was represented secondarily to the primary information dimension of object location. In addition, the neural data related to performance on the recognition memory task. The results suggested that objects were represented as points of interest on the hippocampal cognitive map and that this map was useful in remembering encounters with particular objects in specific locations.The hippocampus plays an important role in spatial memory for both humans and rodents (O''Keefe 1999; Burgess et al. 2002). Findings from many studies in rodents indicate that the hippocampus supports memory for locations referenced to external landmarks, a capacity that O''Keefe and Nadel (1978) described over 30 yr ago as a “cognitive map” (using a term they borrowed from Tolman 1948). In the time since that pioneering thesis, it has become clear that the rodent hippocampus is also important for nonspatial memory (Eichenbaum et al. 1999). Damage to the rat hippocampus (defined here as CA fields, dentate gyrus, and subiculum) leads to impairments on nonspatial tasks, including object recognition memory (Clark et al. 2000; Fortin et al. 2004), transitive odor associations (Bunsey and Eichenbaum 1996), memory for temporal order (Fortin et al. 2002; Kesner et al. 2002), and social transmission of food preference (Alvarez et al. 2001; Clark et al. 2002).The circuitry by which information arrives at and exits from the hippocampus is consistent with the idea that the hippocampus is important for both spatial and nonspatial memory. In both rats and macaques, detailed anatomical studies have indicated that spatial information arrives at the hippocampus via the postrhinal cortex (parahippocampal cortex in primates) and the medial entorhinal cortex, whereas nonspatial information takes a path largely through the perirhinal cortex and lateral entorhinal cortex (Witter and Amaral 1991; Suzuki and Amaral 1994; Witter et al. 2000). Thus, the hippocampus is ideally situated to combine spatial and nonspatial information in the service of remembering item–location associations (Manns and Eichenbaum 2006).Single-unit recording studies in the rat hippocampus have largely focused on the spatial correlates of hippocampal pyramidal neuron firing rates. Fewer studies have investigated nonspatial correlates of hippocampal activity during memory tasks for nonspatial items. However, in one such study, Wood et al. (1999) found that some individual hippocampal pyramidal neurons responded to particular odors and that others responded to particular odors in specific locations during an odor recognition memory task. Thus, activity of individual cells appeared to contain information about nonspatial items as well as spatial locations.An important question is how the activity of individual hippocampal neurons combine to represent item–location associations as a neural ensemble. In particular, how is an encounter with an object in a particular location represented in the pattern of spiking among many hippocampal pyramidal neurons? How might this representation relate to memory for the object or for the location? In the present study, we recorded simultaneously from 43 to 61 hippocampal pyramidal cells as rats performed an object recognition memory task in which novel and repeated objects were encountered in different locations on a circular track. Multivariate analyses of the neural data indicated that information about object identity was represented secondarily to the primary information dimension of object location. In addition, the analyses indicated that the neural data related to performance on the recognition memory task. The results suggest that objects were represented as points of interest on the hippocampal cognitive map and that this map was useful in remembering encounters with particular objects in specific locations.     

Gaining the Upper Hand: Evidence of Vertical Asymmetry in Sex-Categorisation of Human Hands

Visual perception is characterised by asymmetries arising from the brain’s preferential response to particular stimulus types at different retinal locations. Where the lower visual field (LVF) holds an advantage over the upper visual field (UVF) for many tasks (e.g., hue discrimination, contrast sensitivity, motion processing), face-perception appears best supported at above-fixation locations (Quek & Finkbeiner, 2014a). This finding is consistent with Previc’s (1990) suggestion that vision in the UVF has become specialised for object recognition processes often required in ”extrapersonal” space. Outside of faces, however, there have been very few investigations of vertical asymmetry effects for higher-level objects. Our aim in the present study was, thus, to determine whether the UVF advantage reported for face-perception would extend to a nonface object – human hands. Participants classified the sex of hand images presented above or below central fixation by reaching out to touch a left or right response panel. On each trial, a briefly presented spatial cue captured the participant’s spatial attention to either the location where the hand was about to appear (valid cue) or the opposite location (invalid cue). We observed that cue validity only modulated the efficiency of the sex-categorisation response for targets in the LVLVF and not the UVF, just as we have reported previously for face-sex categorisation (Quek & Finkbeiner, 2014a). Taken together, the data from these studies provide some empirical support for Previc’s (1990) speculation that object recognition processes may enjoy an advantage in the upper-hemifield.     

Intrahippocampal infusions of anisomycin produce amnesia: Contribution of increased release of norepinephrine,dopamine, and acetylcholine

Intra-amygdala injections of anisomycin produce large increases in the release of norepinephrine (NE), dopamine (DA), and serotonin in the amygdala. Pretreatment with intra-amygdala injections of the β-adrenergic receptor antagonist propranolol attenuates anisomycin-induced amnesia without reversing the inhibition of protein synthesis, and injections of NE alone produce amnesia. These findings suggest that abnormal neurotransmitter responses may be the basis for amnesia produced by inhibition of protein synthesis. The present experiment extends these findings to the hippocampus and adds acetylcholine (ACh) to the list of neurotransmitters affected by anisomycin. Using in vivo microdialysis at the site of injection, release of NE, DA, and ACh was measured before and after injections of anisomycin into the hippocampus. Anisomycin impaired inhibitory avoidance memory when rats were tested 48 h after training and also produced substantial increases in local release of NE, DA, and ACh. In an additional experiment, pretreatment with intrahippocampal injections of propranolol prior to anisomycin and training significantly attenuated anisomycin-induced amnesia. The disruption of neurotransmitter release patterns at the site of injection appears to contribute significantly to the mechanisms underlying amnesia produced by protein synthesis inhibitors, calling into question the dominant interpretation that the amnesia reflects loss of training-initiated protein synthesis necessary for memory formation. Instead, the findings suggest that proteins needed for memory formation are available prior to an experience, and that post-translational modifications of these proteins may be sufficient to enable the formation of new memories.A dominant view of the molecular basis for memory is that the formation of long-term memory for an experience depends on de novo protein synthesis initiated by that experience (Davis and Squire 1984; Frey and Morris 1998; Kandel 2001; Dudai 2002; Nader 2003; Alberini 2008). This view is supported by numerous studies showing that drugs that interfere with protein synthesis by inhibiting translational processes near the time of training produce later amnesia.Despite the centrality of experience-induced protein synthesis in contemporary models of memory formation, the necessity of protein synthesis for memory consolidation and long-term potentiation (LTP) stabilization has been questioned since the beginning of experiments of this type (e.g., Flexner and Goodman 1975; Barraco and Stettner 1976; Flood et al. 1978; Martinez et al. 1981), and continues to be questioned in several recent reviews (Routtenberg and Rekart 2005; Gold 2006, 2008; Radulovic and Tronson 2008; Routtenberg 2008; Rudy 2008). There are many instances of intact memories formed in the presence of extensive inhibition of protein synthesis, and a wide range of behavioral and pharmacological manipulations can rescue memory impaired by protein synthesis inhibitors. For example, amnesia is attenuated in a graded manner by increasing the training trials and foot shock intensity in avoidance tasks (Flood et al. 1975, 1978). Moreover, a wide range of stimulants, such as amphetamine, strychnine, corticosteroids, and caffeine, block amnesia induced by anisomycin (Flood et al. 1978). Like memory, LTP is sometimes insensitive to protein synthesis inhibitors. Simultaneous inhibition of both protein synthesis and degradation does not interfere with induction and maintenance of LTP (Fonseca et al. 2006a). Also, the specific schedule and frequency of test pulses after induction of LTP determine the vulnerability of LTP to anisomycin-induced impairment; anisomycin treatment does not impair LTP unless test pulses at a rate of 1/10 sec were administered during the anisomycin exposure (Fonseca et al. 2006b).Findings that memory and LTP can survive the inhibition of protein synthesis challenge the necessity of specific training- or stimulation-initiated protein synthesis for memory formation and synaptic plasticity. Several actions of protein synthesis inhibitors offer alternative accounts for amnesia produced by these drugs. These include cell sickness (Rudy et al. 2006; Rudy 2008), activation of protein kinases and superinduction of immediate early genes (Radulovic and Tronson 2008), abnormal neural electrical activity (Agnihotri et al. 2004; Xu et al. 2005), and intrusion of neural “noise” that masks the primary changes representing memory formation (Gold 2006). Neural responses to inhibition of protein synthesis such as these may impair memory either secondary to or independent of interference with protein synthesis.Another example of the mechanisms by which inhibition of protein synthesis might impair memory is by altering neurotransmitter functions. This possibility was suggested in early studies (e.g., Flexner and Goodman 1975; Quartermain et al. 1977) and has recently been supported by studies of neurotransmitter release at the site of intra-amygdala injections of anisomycin (Canal et al. 2007). In addition to impairing later memory after inhibitory avoidance training, pretraining injections of anisomycin into the amygdala produced rapid and dramatic increases in release of norepinephrine (NE), dopamine (DA), and serotonin (5-HT) at the sites of injection. The release of NE and DA then plummeted below baselines from 2 to 6 h after anisomycin injections, recovering within 48 h after anisomycin injection. The possibility that these neurochemical changes contribute to anisomycin-induced amnesia was supported by studies showing attenuation of amnesia in rats pretreated with intra-amygdala injections of the β-adrenergic receptor antagonist propranolol, apparently acting to blunt the effects of the large increases in release of NE after anisomycin injection. In addition, amnesia was produced by injections of high doses of norepinephrine into the amygdala.In addition to amnesias produced by anisomycin injections into the amygdala, as above, anisomycin also impairs memory when administered to other memory systems, including the hippocampus, where anisomycin impairs inhibitory avoidance memory (Quevedo et al. 1999; Debiec et al. 2002; Milekic et al. 2006). The present study extends the prior findings (Canal et al. 2007) in several respects. Experiments presented here determine whether anisomycin injections into the hippocampus result in changes in release of the catecholamines, NE and DA, at the site of injection, as seen previously in the amygdala. Additionally, the present experiments determine whether intrahippocampal injections of anisomycin result in increased release of acetylcholine, a neurotransmitter not examined in the previous study. To examine parallels with earlier amygdala findings, a further experiment determines whether intrahippocampal pretreatment with propranolol is effective in attenuating anisomycin-induced amnesia.     

The effect of zolpidem on targeted memory reactivation during sleep

According to the active system consolidation theory, memory consolidation during sleep relies on the reactivation of newly encoded memory representations. This reactivation is orchestrated by the interplay of sleep slow oscillations, spindles, and theta, which are in turn modulated by certain neurotransmitters like GABA to enable long-lasting plastic changes in the memory store. Here we asked whether the GABAergic system and associated changes in sleep oscillations are functionally related to memory reactivation during sleep. We administered the GABA_A agonist zolpidem (10 mg) in a double-blind placebo-controlled study. To specifically focus on the effects on memory reactivation during sleep, we experimentally induced such reactivations by targeted memory reactivation (TMR) with learning-associated reminder cues presented during post-learning slow-wave sleep (SWS). Zolpidem significantly enhanced memory performance with TMR during sleep compared with placebo. Zolpidem also increased the coupling of fast spindles and theta to slow oscillations, although overall the power of slow spindles and theta was reduced compared with placebo. In an uncorrected exploratory analysis, memory performance was associated with slow spindle responses to TMR in the zolpidem condition, whereas it was associated with fast spindle responses in placebo. These findings provide tentative first evidence that GABAergic activity may be functionally implicated in memory reactivation processes during sleep, possibly via its effects on slow oscillations, spindles and theta as well as their interplay.

Sleep supports the consolidation of newly acquired memories (Mednick et al. 2011; Klinzing et al. 2019). According to the active system consolidation theory, new memories and their associated neuronal activation patterns become spontaneously reactivated (replayed) following learning in the sleeping brain (Wilson and McNaughton 1994; Diekelmann and Born 2010). These reactivations allow for the redistribution and integration of the memory representations from hippocampal to neocortical sites for long-term storage (Rasch and Born 2007; Klinzing et al. 2019). Memory reactivation during sleep has been proposed to rely on the synchronized interplay of electrophysiological oscillations characteristic of non–rapid eye movement (NREM) sleep, mainly neocortical slow oscillations (SOs, <1 Hz), thalamocortical spindles (9–15 Hz), and hippocampal ripples (80–200 Hz) (Mölle et al. 2009; Staresina et al. 2015; Helfrich et al. 2018; Ngo et al. 2020). Particularly, sleep spindles and their intricate phase coupling to SO have been suggested to be mechanistically involved in memory consolidation processes during sleep (Ulrich 2016; Antony et al. 2019). It has been proposed that memories become reinstated by spindle events, specifically during the up-state of slow oscillations, allowing for the flow of information between different brain sites as well as the induction of lasting structural and functional plastic changes in the learning-associated neuronal networks (Rosanova and Ulrich 2005; Peyrache and Seibt 2020). In addition to sleep spindles, neocortical and hippocampal theta activity (4–8 Hz) is also phase-locked to SO during NREM sleep (Gonzalez et al. 2018; Cox et al. 2019; Krugliakova et al. 2020), and this coupling has been related to memory consolidation during sleep (Schreiner et al. 2018).A number of neuromodulators seem to be involved in the generation of sleep spindles, SO and associated memory processing, most notably GABA (γ-aminobutyric acid), which is the major inhibitory neurotransmitter (Lancel 1999; Ulrich et al. 2018). Sleep spindles and sleep-dependent memory processing can be boosted by targeting the GABAergic system pharmacologically (Mednick et al. 2013). Zolpidem is one of the most frequently used drugs in this regard, binding to GABA_A receptors at the same location as benzodiazepines, thereby acting as a GABA_A receptor agonist (Lemmer 2007). Zolpidem increases the time spent in slow-wave sleep (SWS) and reduces the amount of rapid eye movement (REM) sleep (Kanno et al. 2000; Uchimura et al. 2006; Zhang et al. 2020). Zolpidem also increases the density and power of sleep spindles (Dijk et al. 2010; Lundahl et al. 2012; Mednick et al. 2013; Niknazar et al. 2015; Zhang et al. 2020) as well as the coupling of spindles to SO (Niknazar et al. 2015; Zhang et al. 2020), and it was further found to enhance declarative memory consolidation during sleep, with postsleep performance improvements being associated with higher spindle density and spindle power as well as with SO–spindle coupling (Kaestner et al. 2013; Mednick et al. 2013; Zhang et al. 2020).However, it remains unclear whether the changes in sleep stages, sleep spindles, and SO–spindle coupling after pharmacological manipulation with zolpidem are functionally related to the mechanisms underlying sleep-dependent memory consolidation such as memory reactivation. Over the last few years, targeted memory reactivation (TMR) has been increasingly applied to manipulate memory reactivation during sleep experimentally by presenting learning-associated reminder cues like odors or sounds (Oudiette and Paller 2013; Hu et al. 2020; Klinzing and Diekelmann 2020). TMR biases sleep-related neuronal replay events toward the reactivated memory contents (Lewis and Bendor 2019) and enhances subsequent recall performance (Rudoy et al. 2009; Diekelmann et al. 2011; Schreiner et al. 2015; Cairney et al. 2018). Although a few studies observed modulations of SOs (Rihm et al. 2014), sleep spindles (Cox et al. 2014), and SO–spindle coupling (Bar et al. 2020) with TMR during sleep, studies on the role of specific neurotransmitters and particularly on the role of GABAergic neurotransmission and associated changes in sleep oscillations for targeted memory reactivation are entirely lacking. One previous study tested the effect of pharmacologically increased GABAergic activity by administering the benzodiazepine clonazepam after cued reactivation of a declarative memory during wakefulness (Rodríguez et al. 2013). Clonazepam increased memory performance when it was administered after reactivation with an incomplete reminder cue, suggesting that increasing GABAergic neurotransmission may enhance the restabilization of reactivated declarative memories in humans during wakefulness.In the present study, we tested the effect of modulating GABAergic activity with zolpidem on targeted memory reactivation during sleep and associated changes in sleep spindles as well as SO–spindle and SO–theta coupling. We hypothesized that zolpidem enhances the beneficial effects of targeted memory reactivation on memory performance and that this enhancement is associated with increases in spindle density, spindle power, SO–spindle coupling, and possibly SO–theta coupling, and the amount of SWS. Participants were trained on a memory task including 30 sound–word associations in the evening (Forcato et al. 2020) and received an oral dose of 10 mg zolpidem (n = 11) or placebo (n = 11) after training before a full night of sleep in the sleep lab (Fig. 1). During the night, incomplete reminder cues (sounds + first syllable of the associated words) were played again via in-ear headphones during SWS. The next morning, participants were trained on an interference memory task to probe the stability of the original memory, which was tested 30 min later.Open in a separate windowFigure 1.Experimental design and memory task. (A) All subjects took part in a training session at ∼22.30, were administered with placebo (n = 11) or 10 mg of zolpidem (n = 11) before going to bed at 23:00, and received targeted memory reactivation during the first SWS period. After ∼8 h of sleep, in the morning, subjects learned an interference task and were tested on the original memory task in a testing session 30 min after the interference task. (B) Training: First, subjects were presented with 30 sound–word associations for learning. For each association, the sound was presented first for 2900 msec. The sound then continued accompanied by the word written on the screen and spoken aloud for 1500 msec. After a 4000-msec break, the next association was presented in the same way. After all associations were presented once, participants completed an immediate cued recall test. For each association, the sound was presented for 2900 msec. The sound then continued accompanied by the first syllable of the associated word for 1500 msec. Participants were then given 5000 msec to say the complete word aloud (sound continued during the entire period). Independently of their response, the correct answer was then presented on the screen and via headphones for 1500 msec. Reactivation: Each sound was first presented alone for an average of 2900 msec; the sound then continued accompanied by the first syllable of each word for another 1500 msec. After a 7000-msec break, the next sound–syllable pair was presented until all 30 pairs had been presented once. Testing: Each sound was presented for 500 msec and then the sound continued and subjects had 5000 msec to say the associated word aloud. After a break of 4000 msec, the procedure continued for the rest of the 30 associations. Adapted from Forcato et al. (2020).     

The basolateral amygdala and nucleus accumbens core mediate dissociable aspects of drug memory reconsolidation

A distributed limbic-corticostriatal circuitry is implicated in cue-induced drug craving and relapse. Exposure to drug-paired cues not only precipitates relapse, but also triggers the reactivation and reconsolidation of the cue-drug memory. However, the limbic cortical-striatal circuitry underlying drug memory reconsolidation is unclear. The aim of this study was to investigate the involvement of the nucleus accumbens core and the basolateral amygdala in the reconsolidation of a cocaine-conditioned stimulus-evoked memory. Antisense oligodeoxynucleotides (ASO) were infused into each structure to knock down the expression of the immediate-early gene zif268, which is known to be required for memory reconsolidation. Control infusions used missense oligodeoxynucleotides (MSO). The effects of zif268 knockdown were measured in two complementary paradigms widely used to assess the impact of drug-paired CSs upon drug seeking: the acquisition of a new instrumental response with conditioned reinforcement and conditioned place preference. The results show that both intranucleus accumbens core and intrabasolateral amygdala zif268 ASO infusions at memory reactivation impaired the reconsolidation of the memory underlying a cocaine-conditioned place preference. However, knockdown of zif268 in the nucleus accumbens at memory reactivation had no effect on the memory underlying the conditioned reinforcing properties of the cocaine-paired CS measured subsequently, and this is in contrast to the marked impairment observed previously following intrabasolateral amygdala zif268 ASO infusions. These results suggest that both the basolateral amygdala and nucleus accumbens core are key structures within limbic cortical-striatal circuitry where reconsolidation of a cue-drug memory occurs. However reconsolidation of memory representations formed during Pavlovian conditioning are differentially localized in each site.Through Pavlovian association with the effects of addictive drugs, a conditioned stimulus (CS) acquires both general motivational and sensory-specific conditioned reinforcing properties (Everitt et al. 2000). These associations contribute to the high likelihood of relapse in addicted individuals, yet the extinction of drug CSs by nonreinforced exposure has proved to be of limited therapeutic utility (Conklin and Tiffany 2002). In abstinent humans, drug CSs evoke salient and persistent memories of drug-taking experiences, inducing craving and relapse (Childress et al. 1988; O''Brien et al. 1992), while in animals they also precipitate relapse to, or reinstatement of, drug-seeking behavior (de Wit and Stewart 1981; Meil and See 1996; Fuchs et al. 1998; Weiss 2000). Thus, disrupting drug-related memories might significantly diminish relapse propensity on subsequent exposure to drug-paired CSs, and thereby promote abstinence.Exposure to a drug-associated CS also triggers a process of memory reconsolidation, which restabilizes the reactivated and labile memory (Nader 2003). While reconsolidation may adaptively update memories (Dudai 2006; Hupbach et al. 2007; Rossato et al. 2007; Lee 2009), its disruption may reduce the impact of intrusive or aberrant memories on behavior subsequently (Lee et al. 2005, 2006; Brunet et al. 2008; Kindt et al. 2009; Taubenfeld et al. 2009). The reconsolidation of CS–cocaine memories has been shown to depend upon protein synthesis and expression of the plasticity-associated immediate-early gene, zif268, in the basolateral amygdala (BLA), since zif268 knockdown at memory reactivation disrupted the acquired conditioned reinforcing properties of the CS measured in drug-seeking tasks days or weeks later (Lee et al. 2005, 2006).Although the BLA has an established role in CS-drug memory reconsolidation, it remains unclear whether other sites within limbic cortical-ventral striatal circuitry participate in this process. The nucleus accumbens core (AcbC) is a primary candidate, as zif268 is up-regulated in the AcbC as well as in the BLA following exposure to cocaine CSs (Thomas et al. 2003). Furthermore, the AcbC, which is strongly implicated in Pavlovian influences on drug seeking and relapse (Cardinal et al. 2002; Kalivas and McFarland 2003), has been shown to be a site where the reconsolidation of a drug conditioned place preference (CPP) memory can be disrupted (Miller and Marshall 2005).Given the evidence of increased zif268 expression in the AcbC following CS-drug memory reactivation, we investigated its requirement in the reconsolidation of cocaine-associated memories. To address this issue, we employed two different but complementary paradigms widely used to measure the conditioned effects of CSs associated with drugs of abuse: the acquisition of a new instrumental response with conditioned reinforcement (ANR) and CPP. These procedures have been used successfully to investigate the mechanisms underlying the reconsolidation of appetitive Pavlovian memories, but it is likely that they depend upon different associative mechanisms (Everitt et al. 1991; White and McDonald 1993) that in turn depend upon different neural loci within limbic cortical-striatal circuitry (Cardinal et al. 2002). Therefore, to enable a full comparison with the functional involvement of the BLA, we investigated the necessity for BLA zif268 expression in drug memory reconsolidation as assessed in the CPP paradigm.     

Greater dependence on working memory and restricted familiarity in orangutans compared with rhesus monkeys

The prefrontal cortex is larger than would be predicted by body size or visual cortex volume in great apes compared with monkeys. Because prefrontal cortex is critical for working memory, we hypothesized that recognition memory tests would engage working memory in orangutans more robustly than in rhesus monkeys. In contrast to working memory, the familiarity response that results from repetition of an image is less cognitively taxing and has been associated with nonfrontal brain regions. Across three experiments, we observed a striking species difference in the control of behavior by these two types of memory. First, we found that recognition memory performance in orangutans was controlled by working memory under conditions in which this memory system plays little role in rhesus monkeys. Second, we found that unlike the case in monkeys, familiarity was not involved in recognition memory performance in orangutans, shown by differences with monkeys across three different measures. Memory in orangutans was not improved by use of novel images, was always impaired by a concurrent cognitive load, and orangutans did not accurately identify images seen minutes ago. These results are surprising and puzzling, but do support the view that prefrontal expansion in great apes favored working memory. At least in orangutans, increased dependence on working memory may come at a cost in terms of the availability of familiarity.

The prefrontal cortex is critical for a suite of cognitive control processes that are conspicuous in human cognition (Miller 2000; Rougier et al. 2005; Braver et al. 2009). One such process is working memory, which actively maintains representations in a state of heightened access for further processing (Baddeley and Hitch 1974; Engle 2010). Working memory and cognitive control are positively correlated with measures of general intelligence in humans, implicating the prefrontal cortex as a key neural structure in the evolution of human cognition (Gray et al. 2003; Unsworth and Engle 2007; Cole et al. 2012). Some neuroanatomical studies have found that the prefrontal cortex is larger than would be predicted based on body size and visual cortex volume in apes compared with monkeys (Rilling 2006; Passingham and Smaers 2014). These findings suggest that the prefrontal cortex expanded disproportionately in great apes during primate evolution. Disproportionate expansion of the prefrontal cortex in great apes may have resulted in greater capacity for cognitive control functions, such as working memory, compared with monkeys. Thus, studies directly comparing working memory in monkeys and apes are critical to understanding the evolution of intelligence in primates.The role of cognitive control, and thus the prefrontal cortex, varies among memory systems. For instance, working memory relies heavily on cognitive control, consuming substantial cognitive resources, and is known to depend on frontal brain areas (Goldman-Rakic 1995; Fuster 1997). In contrast, familiarity, which is a strength-based memory signal that codes for whether or not a percept has previously been experienced (Kelley and Jacoby 1998; Yonelinas 2002), relies substantially less on cognitive control, consumes fewer cognitive resources, and has been mostly associated with nonfrontal areas of the brain such as the perirhinal cortex (Bachevalier and Mishkin 1986; Brown and Aggleton 2001; Haskins et al. 2008; Tu et al. 2011; O''Neil et al. 2012). Thus, working memory and familiarity vary in the degree to which they rely on cognitive control, and the degree to which they rely on prefrontal areas of the brain.If a relatively large prefrontal cortex enhances cognitive control and working memory, then we should expect recognition memory tests to engage working memory relatively more in apes than in monkeys. We evaluated this hypothesis by comparing the contributions of working memory and familiarity with recognition memory performance in orangutans and rhesus monkeys. Because the orangutans and monkeys here compared have different experience with cognitive testing, we aimed to compare the relative contributions of working memory and familiarity in each species, rather than the absolute accuracy of the two species in a particular memory test. This follows a logic similar to that used in many comparative anatomical studies; for example, those cited here that found the prefrontal cortex is larger in apes relative to body weight or visual cortex volume, rather than simply absolute volume.The relative contributions of working memory and familiarity to behavior can be measured in visual recognition memory tests. In these tests subjects study a sample image at the beginning of each trial and after a delay they are presented with a test consisting of the recently seen sample image among distractors (Fig. 1). The images used in these tests can either be repeated, such that the subject''s job is to determine which image in a set of familiar images was seen most recently, or the images can be trial unique, such that at test subjects need to discriminate a previously seen image from novel distractors. Working memory is critical for solving tests with repeating images, but much less so for tests using trial unique images, where familiarity plays a much greater role (Brady and Hampton 2018a). Monkeys (Jitsumori et al. 1988; Basile and Hampton 2013a) and apes (Harlow 1944; Hayes and Thompson 1953) are more accurate and better tolerate long delay intervals in tests with trial-unique stimuli, when familiarity can support performance. Experimentally naïve monkeys require comparatively little training to demonstrate proficient use of familiarity as a mnemonic cue, compared with the training required to become proficient in using working memory (Mishkin and Delacour 1975). Active working memory and passive familiarity are independent mnemonic processes that can be doubly dissociated. Working memory is impaired by a concurrent cognitive load imposed during the memory interval, while familiarity is not affected (Logie 1986; Jacoby et al. 1989; Basile and Hampton 2013a; Brady and Hampton 2018a). Completing the double dissociation, equating the familiarity of the sample and distractor images during study impairs choice based on familiarity, but not working memory (Brady and Hampton 2018a). Thus, recognition memory tests may allow us to compare the relative contributions of these two memory processes with recognition performance across species.Open in a separate windowFigure 1.Recognition memory tests with repeating and trial-unique images. (A) In tests with trial-unique images, each image was only used once as a sample or a distractor within a session. (B) When tested with repeating images, the images were the same on each trial. The sample image was pseudorandomly selected each trial such that each image appeared equally often as the sample or as a distractor.One might expect orangutans to show greater dependence on working memory compared with rhesus monkeys for at least two reasons. First, working memory is highly refined in humans and orangutans are more closely related to humans phylogenetically, sharing a common ancestor 13 million to 14 million years ago (Stewart and Disotell 1998), whereas rhesus monkeys and humans shared a common ancestor ∼32 million years ago (Roos and Zinner 2015). Second, orangutans have a relatively larger prefrontal cortex compared with monkeys (Rilling 2006; Passingham and Smaers 2014). We compared the ability of rhesus monkeys and orangutans to maintain images from different sets in working memory. We also determined the extent to which familiarity contributed to recognition memory performance. Across three experiments, we observed striking species differences. We found that in orangutans, recognition memory performance for both repeating and trial-unique images was controlled by working memory. In contrast, monkeys relied on working memory for repeating images, and on familiarity for trial-unique images. Furthermore, monkeys dramatically outperformed orangutans in tests that exceeded the capacity and duration of working memory, and thus depended on familiarity.     

Impairments in fear conditioning in mice lacking the nNOS gene

The fear conditioning paradigm is used to investigate the roles of various genes, neurotransmitters, and substrates in the formation of fear learning related to contextual and auditory cues. In the brain, nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) functions as a retrograde neuronal messenger that facilitates synaptic plasticity, including the late phase of long-term potentiation (LTP) and formation of long-term memory (LTM). Evidence has implicated NO signaling in synaptic plasticity and LTM formation following fear conditioning, yet little is known about the role of the nNOS gene in fear learning. Using knockout (KO) mice with targeted mutation of the nNOS gene and their wild-type (WT) counterparts, the role of NO signaling in fear conditioning was investigated. Plasma levels of the stress hormone corticosterone were measured to determine the relationship between physiological and behavioral response to fear conditioning. Contextual fear learning was severely impaired in male and female nNOS KO mice compared with WT counterparts; cued fear learning was slightly impaired in nNOS KO mice. Sex-dependent differences in both contextual and cued fear learning were not observed in either genotype. Deficits in contextual fear learning in nNOS KO mice were partially overcome by multiple trainings. A relationship between increase in plasma corticosterone levels following footshock administration and the magnitude of contextual, but not cued freezing was also observed. Results suggest that the nNOS gene contributes more to optimal contextual fear learning than to cued fear learning, and therefore, inhibition of the nNOS enzyme may ameliorate context-dependent fear response.Anxiety disorders, such as post-traumatic stress disorder (PTSD), constitute the most prevalent mental illnesses in the United States, costing nearly one-third of the country''s total health bill (Greenberg et al. 1999). The treatment of these disorders requires overcoming complications such as reluctance to seek mental health treatment and an extremely high comorbidity rate with other affective disorders, reaching 80% (Brady 1997; Solomon and Davidson 1997). Emerging evidence suggests that dysfunctions underlying acquired anxiety and PTSD include an abnormal reaction to stress, which is mediated by specific neurochemical and neuroanatomical substrates (Yehuda and McFarlane 1995; Adamec 1997). Pharmacotherapies that target neuronal signaling molecules, such as nitric oxide (NO), may play a role in the treatment of these disorders.In the brain, N-methyl-d-aspartate receptor (NMDAR) activation and calcium influx into the cell activates the neuronal nitric oxide synthase (nNOS) enzyme to produce NO, which has the role of retrograde messenger (Snyder 1992). NO is involved in memory formation and synaptic plastic events such as late-phase long-term potentiation (LTP) (Lu et al. 1999; Arancio et al. 2001; Puzzo et al. 2006). Behavioral evidence in invertebrates (Lewin and Walters 1999; Muller 2000; Kemenes et al. 2002; Matsumoto et al. 2006) and vertebrates (Medina and Izquierdo 1995; Rickard et al. 1998; Ota et al. 2008) suggest that NO has a major role in consolidation of long-term memory (LTM). Recently, studies have shown that site-specific pharmacological blockade of NO signaling in rats impairs contextual (Resstel et al. 2008) and cued (Schafe et al. 2005) fear learning. However, the role of the nNOS gene in fear conditioning has not been investigated.In the present study, fear conditioning was investigated in homozygous nNOS knockout (KO) and wild-type (WT) mice. In the fear-conditioning paradigm, the association of a footshock (unconditioned stimulus; US), with a specific context and a neutral stimulus (auditory cue) results in learned fear. Re-exposure to the conditioning context and to the previously neutral auditory cue (conditioned stimulus; CS) elicits a freezing response in the absence of the aversive US. Thus, the fear-conditioning paradigm includes both contextual and cued fear learning components, which can be measured in separate tests. Fear conditioning recruits both the amygdala (emotional cue learning) and the hippocampus (spatial/contextual learning) (Phillips and LeDoux 1992; Goosens and Maren 2004; Mei et al. 2005). The involvement of these brain regions in fear learning and anxiety has been confirmed by animal and human imaging studies (LeDoux 1998; Rauch et al. 2006).We report that nNOS KO mice showed a severe deficiency in contextual fear learning and a less marked deficit in cued fear learning compared with WT mice after a single fear-conditioning session. This deficiency was partially improved by multiple (four) fear-conditioning sessions. In addition, we observed that plasma levels of corticosterone, the primary stress hormone in rodents, are related to contextual fear learning ability.     

Object recognition memory and the rodent hippocampus

In rodents, the novel object recognition task (NOR) has become a benchmark task for assessing recognition memory. Yet, despite its widespread use, a consensus has not developed about which brain structures are important for task performance. We assessed both the anterograde and retrograde effects of hippocampal lesions on performance in the NOR task. Rats received 12 5-min exposures to two identical objects and then received either bilateral lesions of the hippocampus or sham surgery 1 d, 4 wk, or 8 wk after the final exposure. On a retention test 2 wk after surgery, the 1-d and 4-wk hippocampal lesion groups exhibited impaired object recognition memory. In contrast, the 8-wk hippocampal lesion group performed similarly to controls, and both groups exhibited a preference for the novel object. These same rats were then given four postoperative tests using unique object pairs and a 3-h delay between the exposure phase and the test phase. Hippocampal lesions produced moderate and reliable memory impairment. The results suggest that the hippocampus is important for object recognition memory.Recognition memory refers to the ability to judge a previously encountered item as familiar and depends on the integrity of the medial temporal lobe (Squire et al. 2007). Tasks that assess recognition memory (and object recognition memory in particular) have become increasingly useful tools for basic and preclinical research investigating the neural basis of memory (Winters et al. 2008). Perhaps the best known of these tasks is the novel object recognition task (NOR) (also known as the visual paired-comparison task in studies with humans and monkeys).Studies of the NOR task in humans with hippocampal damage (McKee and Squire 1993; Pascalis et al. 2004) and in monkeys with selective damage to the hippocampus (Pascalis and Bachevalier 1999; Zola et al. 2000; Nemanic et al. 2004) have resulted in clear and consistent findings. Damage limited to the hippocampus is sufficient to produce impaired recognition memory (Squire et al. 2007, Box 1). In rats and mice, the NOR task has become particularly popular and is currently a benchmark task for assessing recognition memory. Yet despite its widespread use in rodents, the findings are rather mixed. For example, in the rat, although there is agreement that the perirhinal cortex is critically important for normal NOR performance, there is less agreement about the hippocampus (for review, see Winters et al. 2008). Although some of the discrepancies between studies may be attributed to differences in lesion size and in the length of the retention delay (Broadbent et al. 2004), these factors cannot account for all the findings (Squire et al. 2007).Whereas most studies have investigated the effects of hippocampal lesions on postoperative NOR performance, there is also interest in the effects of hippocampal lesions on memory for previously encountered objects. For a number of tasks, hippocampal lesions produce temporally graded retrograde amnesia, such that memory acquired recently is impaired and memory acquired more remotely is spared (for review, see Squire et al. 2004; Frankland and Bontempi 2005). In the case of the single study of retrograde memory that has involved the NOR task, recognition memory was impaired when a 5-wk interval intervened between training and hippocampal surgery (Gaskin et al. 2003). It remains possible that memory might be spared if a longer delay was imposed between training and surgery.The aim of the present study was to assess both the anterograde and retrograde effects of hippocampal lesions on recognition memory using the NOR task. To thoroughly assess the effects of hippocampal lesions we used (1) large groups of animals, (2) multiple tests of NOR memory, (3) a scoring method that allowed object preference to be determined on a second-by-second basis during the recognition tests, and (4) a novel training protocol that permitted the evaluation of recognition memory even after a retention interval as long as 10 wk.     

The amygdala is not necessary for unconditioned stimulus inflation after Pavlovian fear conditioning in rats

The basolateral complex (BLA) and central nucleus (CEA) of the amygdala play critical roles in associative learning, including Pavlovian conditioning. However, the precise role for these structures in Pavlovian conditioning is not clear. Recent work in appetitive conditioning paradigms suggests that the amygdala, particularly the BLA, has an important role in representing the value of the unconditioned stimulus (US). It is not known whether the amygdala performs such a function in aversive paradigms, such as Pavlovian fear conditioning in rats. To address this issue, Experiments 1 and 2 used temporary pharmacological inactivation of the amygdala prior to a US inflation procedure to assess its role in revaluing shock USs after either overtraining (Experiment 1) or limited training (Experiment 2), respectively. Inactivation of the BLA or CEA during the inflation session did not affect subsequent increases in conditioned freezing observed to either the tone conditioned stimulus (CS) or the conditioning context in either experiment. In Experiment 3, NBQX infusions into the BLA impaired the acquisition of auditory fear conditioning with an inflation-magnitude US, indicating that the amygdala is required for associative learning with intense USs. Together, these results suggest that the amygdala is not required for revaluing an aversive US despite being required for the acquisition of fear to that US.Pavlovian fear conditioning in rats is a behavioral model used to investigate the neurobiology underlying the development and maintenance of fear learning and memory (Grillon et al. 1996; LeDoux 1998, 2000; Bouton et al. 2001; Maren 2001b, 2005; Kim and Jung 2006). In this model, an innocuous conditioned stimulus (CS), such as a tone, is paired with an aversive unconditioned stimulus (US), such as a footshock. After one or more pairings, the rat learns that the CS predicts the US. As a consequence, CS presentations alone elicit a conditioned fear response (CR), which includes increases in heart rate, arterial blood pressure, hypoalgesia, potentiated acoustic startle, stress hormone release, and freezing (somatomotor immobility).The amygdala has been identified as one of the major regions in which fear memories are encoded and stored. Within the amygdala, the basolateral complex of the amygdala (BLA; consisting of the lateral, basolateral, and basomedial nuclei) and the central nucleus of the amygdala (CEA) receive convergent CS and US information and are involved in the acquisition of fear memories (LeDoux 1998, 2000; Fendt and Fanselow 1999; Davis and Whalen 2001; Maren 2001b; Schafe et al. 2001; Fanselow and Gale 2003; Wilensky et al. 2006; Zimmerman et al. 2007). In addition, the CEA has an important role in the expression of fear CRs (Fendt and Fanselow 1999; LeDoux 2000; Davis and Whalen 2001; Maren 2001b; Fanselow and Gale 2003). In support of this, many studies have shown that either permanent or temporary lesions of the BLA or CEA prevent the acquisition and/or expression of fear memories (Helmstetter 1992; Helmstetter and Bellgowan 1994; Campeau and Davis 1995; Maren et al. 1996a,b; Killcross et al. 1997; Muller et al. 1997; Walker and Davis 1997; Cousens and Otto 1998; Maren 1998, 1999, 2001a,b; Wilensky et al. 1999, 2000, 2006; Goosens and Maren 2001, 2003; Nader et al. 2001; Fanselow and Gale 2003; Gale et al. 2004; Koo et al. 2004; Zimmerman et al. 2007).In addition to its role in encoding CS–US associations during conditioning, recent work suggests that the amygdala is also involved in representing properties of the US itself. For example, temporary or permanent lesions of the BLA reduce both decrements in conditioned responding after devaluation of a food US (Hatfield et al. 1996; Killcross et al. 1997; Blundell et al. 2001; Balleine et al. 2003; Everitt et al. 2003; Pickens et al. 2003; Holland 2004) and increments in conditional responding after inflation of a shock US (Fanselow and Gale 2003). Moreover, recent electrophysiological studies in primates indicate that amygdala neurons represent the value of both aversive and appetitive outcomes (Paton et al. 2006; Belova et al. 2007, 2008; Salzman et al. 2007). These studies suggest that one function of the BLA is to represent specific properties of biologically significant events, such as the food or shock USs that are typically used in Pavlovian conditioning paradigms. By this view, the BLA may represent specific sensory properties of USs that shape the nature of learned behavioral responses to the US (Balleine and Killcross 2006) and allow CSs to gain access to the incentive value of the US (Everitt et al. 2003).In contrast to this view, we recently reported that rats with neurotoxic BLA lesions exhibit normal US revaluation after Pavlovian fear conditioning (Rabinak and Maren 2008). In this study, auditory fear conditioning (75 CS–US trials) with a moderate footshock (1 mA) was followed by several exposures (five US-alone trials) to an intense footshock (3 mA) during an inflation session. Both intact rats and rats with BLA lesions exhibit a robust increase in conditional freezing to the auditory CS during a subsequent retention test (Rabinak and Maren 2008). Control experiments suggested that this was due to a revaluation of the US with which the CS was associated, rather than nonassociative sensitization of fear engendered by exposure to intense shock. These data reveal that the BLA may not be necessary for representing properties of shock USs during Pavlovian fear conditioning. To address these issues further, we have examined the consequence of reversible pharmacological manipulations of the amygdala during US inflation on conditional fear responses established with either extensive or limited training.     

Recognition memory: Adding a response deadline eliminates recollection but spares familiarity

A current controversy in memory research concerns whether recognition is supported by distinct processes of familiarity and recollection, or instead by a single process wherein familiarity and recollection reflect weak and strong memories, respectively. Recent studies using receiver operating characteristic (ROC) analyses in an animal model have shown that manipulations of the memory demands can eliminate the contribution of familiarity while sparing recollection. Here it is shown that a different manipulation, specifically the addition of a response deadline in recognition testing, results in the opposite performance pattern, eliminating the contribution of recollection while sparing that of familiarity. This dissociation, combined with the earlier findings, demonstrates that familiarity and recollection are differentially sensitive to specific memory demands, strongly supporting the dual process view.Receiver operating characteristic (ROC) analysis holds the promise of dissecting the contributions to recognition memory of episodic recollection and familiarity (Yonelinas 2001), and this method can be applied equally well to examine these memory processes in animals as well as humans (Fortin et al. 2004; Sauvage et al. 2008). According to the dual process model, recollection is indexed by the asymmetry of the ROC function whereas familiarity is measured by the degree of curvilinearity of that function, and correspondingly, these two parameters can vary independently (Yonelinas 2001). However, there is controversy about this interpretation of ROC components. Some have argued that the asymmetry and curvilinearity of the ROC function both reflect the strength of memories mediated by a single process (Wixted 2007), and correspondingly, these components of the ROC increase or decrease together in stronger or weaker memories, respectively (Squire et al. 2007).A resolution of this controversy can be advanced by examining whether the ROC asymmetry and curvilinearity are independently influenced by task manipulations that favor either recollection or familiarity, consistent with dual process theory, or instead are similarly influenced by conditions that affect memory strength. Recent data from an animal model of recognition have shown that adding a demand for remembering associations between independent stimuli eliminates the ROC curvilinearity without affecting the asymmetry, consistent with the dual process view (Sauvage et al. 2008; for discussion of associative recognition, see Mayes et al. 2007). However, in order to provide compelling evidence of independence of the two ROC components, it is also critical to show that other memory demands that favor familiarity produce the opposite pattern, elimination of the ROC asymmetry while sparing its curvilinearity. Together these findings would constitute a double dissociation between the two parameters of the ROC function that cannot be explained by a single process theory.As originally conceived in models proposed in the 1970s, familiarity is characterized as a perceptually driven, pattern matching process that is completed rapidly, whereas recollection is characterized as a conceptually driven, organizational process that requires more time (Mandler 1972; Atkinson and Juola 1973, 1974; for reviews, see Yonelinas 2002; Mandler 2008). Consistent with this view, the results of several studies that employ response deadlines in the test phase report that familiarity is more rapid than recollection. For example, forcing people to make speeded recognition responses has little effect on simple yes–no recognition but strikingly reduces performance when subjects must remember where or when an item was studied (Yonelinas and Jacoby 1994; Gronlund et al. 1997; Hintzman et al. 1998). Other studies that require subjects to oppose familiarity and recollection reveal a two-component temporal function that includes a rapidly available familiarity process and a slower recollective process (Dosher 1984; Gronlund and Ratcliff 1989; Hintzman and Curran 1994; McElree et al. 1999). In addition, studies that measure brain evoked response potentials (ERPs) have revealed two distinct ERP modulations commonly observed during recognition: a mid-frontal negativity onsetting about 400 msec after stimulus onset that is associated with familiarity, and a parietally distributed positivity beginning about 500 msec after stimulus onset that is associated with recollection (Smith 1993; Duzel et al. 1997; Curran 2004; Duarte et al. 2006; Woodruff et al. 2006; but see Voss and Paller 2009).Dual process theory predicts that applying an appropriate early response deadline should allow sufficient time for contribution of familiarity but not that of recollection, and so should reduce the ROC asymmetry while sparing its curvilinearity, opposite to the already observed effects of associative memory demands that favor recollection (Sauvage et al. 2008). Confirmation of this prediction combined with the previous findings of the opposite effects in associative recognition would constitute a double dissociation between the features of recollection and familiarity. This result would therefore strongly support the conclusion that the asymmetry and curvilinearity are independent parameters of the ROC function that are differentially linked to features of recollection and familiarity, respectively.