Prefrontal Neural Ensembles Develop Selective Code for Stimulus Associations within Minutes of Novel Experiences.

Prevailing theories posit that the hippocampus rapidly learns stimulus conjunctions during novel experiences, whereas the neocortex learns slowly through subsequent, off-line interaction with the hippocampus. Parallel evidence, however, shows that the medial prefrontal cortex (mPFC; a critical node of the neocortical network supporting long-term memory storage) undergoes rapid modifications of gene expression, synaptic structure, and physiology at the time of encoding. These observations, along with impaired learning with disrupted mPFC, suggest that mPFC neurons may exhibit rapid neural plasticity during novel experiences; however, direct empirical evidence is lacking. We extracellularly recorded action potentials of cells in the prelimbic region of the mPFC, while male rats received a sequence of stimulus presentations for the first time in life. Moment-to-moment tracking of neural ensemble firing patterns revealed that the prelimbic network activity exhibited an abrupt transition within 1 min after the first encounter of an aversive but not neutral stimulus. This network-level change was driven by ∼15% of neurons that immediately elevated their spontaneous firing rates (FRs) and developed firing responses to a neutral stimulus preceding the aversive stimulus within a few instances of their pairings. When a new sensory stimulus was paired with the same aversive stimulus, about half of these neurons generalized firing responses to the new stimulus association. Thus, prelimbic neurons are capable of rapidly forming ensemble codes for novel stimulus associations within minutes. This circuit property may enable the mPFC to rapidly detect and selectively encode the central content of novel experiences.SIGNIFICANCE STATEMENT During a new experience, a region of the brain, called the hippocampus, rapidly forms its memory and later instructs another region, called the neocortex, that stores its content. Consistent with this dominant view, cells in the neocortex gradually strengthen the selectivity for the memory content over weeks after novel experiences. However, we still do not know precisely when these cells begin to develop the selectivity. We found that neocortical cells were capable of forming the selectivity for ongoing events within a few minutes of new experiences. This finding provides support for an alternative view that the neocortex works with, but not follows, the hippocampus to form new memories.

Distributed representations of temporal stimulus associations across regular-firing and fast-spiking neurons in rat medial prefrontal cortex.

The prefrontal cortex has been implicated in various cognitive processes, including working memory, executive control, decision making, and relational learning. One core computational requirement underlying all these processes is the integration of information across time. When rodents and rabbits associate two temporally discontiguous stimuli, some neurons in the medial prefrontal cortex (mPFC) change firing rates in response to the preceding stimulus and sustain the firing rate during the subsequent temporal interval. These firing patterns are thought to serve as a mechanism to buffer the previously presented stimuli and signal the upcoming stimuli; however, how these critical properties are distributed across different neuron types remains unknown. We investigated the firing selectivity of regular-firing, burst-firing, and fast-spiking neurons in the prelimbic region of the mPFC while rats associated two neutral conditioned stimuli (CS) with one aversive stimulus (US). Analyses of firing patterns of individual neurons and neuron ensembles revealed that regular-firing neurons maintained rich information about CS identity and CS-US contingency during intervals separating the CS and US. Moreover, they further strengthened the latter selectivity with repeated conditioning sessions over a month. The selectivity of burst-firing neurons for both stimulus features was weaker than that of regular-firing neurons, indicating the difference in task engagement between two subpopulations of putative excitatory neurons. In contrast, putative inhibitory, fast-spiking neurons showed a stronger selectivity for CS identity than for CS-US contingency, suggesting their potential role in sensory discrimination. These results reveal a fine-scaled functional organization in the prefrontal network supporting the formation of temporal stimulus associations.NEW & NOTEWORTHY To associate stimuli that occurred separately in time, the brain needs to bridge the temporal gap by maintaining what was presented and predicting what would follow. We show that in rat medial prefrontal cortex, the former function is associated with a subpopulation of putative inhibitory neurons, whereas the latter is supported by a subpopulation of putative excitatory neurons. Our results reveal a distinct contribution of these microcircuit components to neural representations of temporal stimulus associations.

The role of engram cells in the systems consolidation of memory.

What happens to memories as days, weeks and years go by has long been a fundamental question in neuroscience and psychology. For decades, researchers have attempted to identify the brain regions in which memory is formed and to follow its changes across time. The theory of systems consolidation of memory (SCM) suggests that changes in circuitry and brain networks are required for the maintenance of a memory with time. Various mechanisms by which such changes may take place have been hypothesized. Recently, several studies have provided insight into the brain networks driving SCM through the characterization of memory engram cells, their biochemical and physiological changes and the circuits in which they operate. In this Review, we place these findings in the context of the field and describe how they have led to a revamped understanding of SCM in the brain.

Entorhinal tau pathology disrupts hippocampal-prefrontal oscillatory coupling during associative learning.

A neural signature of asymptomatic preclinical Alzheimer's disease (AD) is disrupted connectivity between brain regions; however, its underlying mechanisms remain unknown. Here, we tested whether a preclinical pathologic feature, tau aggregation in the entorhinal cortex (EC) is sufficient to disrupt the coordination of local field potentials (LFPs) between its efferent regions. P301L-mutant human tau or green fluorescent protein (GFP) was virally overexpressed in the EC of adult rats. LFPs were recorded from the dorsal hippocampus and prelimbic medial prefrontal cortex while the rats underwent trace eyeblink conditioning where they learned to associate 2 stimuli separated by a short time interval. In GFP-expressing rats, the 2 regions strengthened phase-phase and amplitude-amplitude couplings of theta and gamma oscillations during the interval separating the paired stimuli. Despite normal memory acquisition, this learning-related, inter-region oscillatory coupling was attenuated in the tau-expressing rats while prefrontal phase-amplitude theta-gamma cross-frequency coupling was elevated. Thus, EC tau aggregation caused aberrant long-range circuit activity during associative learning, identifying a culprit for the neural signature of preclinical AD stages.

Phasic and tonic neuron ensemble codes for stimulus-environment conjunctions in the lateral entorhinal cortex.

The lateral entorhinal cortex (LEC) is thought to bind sensory events with the environment where they took place. To compare the relative influence of transient events and temporally stable environmental stimuli on the firing of LEC cells, we recorded neuron spiking patterns in the region during blocks of a trace eyeblink conditioning paradigm performed in two environments and with different conditioning stimuli. Firing rates of some neurons were phasically selective for conditioned stimuli in a way that depended on which room the rat was in; nearly all neurons were tonically selective for environments in a way that depended on which stimuli had been presented in those environments. As rats moved from one environment to another, tonic neuron ensemble activity exhibited prospective information about the conditioned stimulus associated with the environment. Thus, the LEC formed phasic and tonic codes for event-environment associations, thereby accurately differentiating multiple experiences with overlapping features.

Engrams and circuits crucial for systems consolidation of a memory.

Episodic memories initially require rapid synaptic plasticity within the hippocampus for their formation and are gradually consolidated in neocortical networks for permanent storage. However, the engrams and circuits that support neocortical memory consolidation have thus far been unknown. We found that neocortical prefrontal memory engram cells, which are critical for remote contextual fear memory, were rapidly generated during initial learning through inputs from both the hippocampal-entorhinal cortex network and the basolateral amygdala. After their generation, the prefrontal engram cells, with support from hippocampal memory engram cells, became functionally mature with time. Whereas hippocampal engram cells gradually became silent with time, engram cells in the basolateral amygdala, which were necessary for fear memory, were maintained. Our data provide new insights into the functional reorganization of engrams and circuits underlying systems consolidation of memory.

Generalizable knowledge outweighs incidental details in prefrontal ensemble code over time.

Memories for recent experiences are rich in incidental detail, but with time the brain is thought to extract latent rules and structures common across past experiences. We show that over weeks following the acquisition of two distinct associative memories, neuron firing in the rat prelimbic prefrontal cortex (mPFC) became less selective for perceptual features unique to each association and, with an apparently different time-course, became more selective for common relational features. We further found that during exposure to a novel experimental context, memory expression and neuron selectivity for relational features immediately generalized to the new situation. These neural patterns offer a window into the network-level processes by which the mPFC develops a knowledge structure of the world that can be adaptively applied to new experiences.

Cholinergic, but not NMDA, receptors in the lateral entorhinal cortex mediate acquisition in trace eyeblink conditioning.

Anatomical and electrophysiological studies collectively suggest that the entorhinal cortex consists of several subregions, each of which is involved in the processing of different types of information. Consistent with this idea, we previously reported that the dorsolateral portion of the entorhinal cortex (DLE), but not the caudomedial portion, is necessary for the expression of a memory association between temporally discontiguous stimuli in trace eyeblink conditioning (Morrissey et al. (2012) J Neurosci 32:5356-5361). The present study examined whether memory acquisition depends on the DLE and what types of local neurotransmitter mechanisms are involved in memory acquisition and expression. Male Long-Evans rats experienced trace eyeblink conditioning, in which an auditory conditioned stimulus (CS) was paired with a mildly aversive electric shock to the eyelid (US) with a stimulus-free interval of 500 ms. Immediately before the conditioning, the rats received a microinfusion of neuroreactive substances into the DLE. We found that reversible inactivation of the DLE with GABAA receptor agonist, muscimol impaired memory acquisition. Furthermore, blockade of local muscarinic acetylcholine receptors (mACh) with scopolamine retarded memory acquisition while blockade of local NMDA receptors with APV had no effect. Memory expression was not impaired by either type of receptor blocker. These results suggest that the DLE is necessary for memory acquisition, and that acquisition depends on the integrity of local mACh receptor-dependent firing modulation, but not NMDA receptor-dependent synaptic plasticity.

Parvalbumin and GAD65 interneuron inhibition in the ventral hippocampus induces distinct behavioral deficits relevant to schizophrenia.

Hyperactivity within the ventral hippocampus (vHPC) has been linked to both psychosis in humans and behavioral deficits in animal models of schizophrenia. A local decrease in GABA-mediated inhibition, particularly involving parvalbumin (PV)-expressing GABA neurons, has been proposed as a key mechanism underlying this hyperactive state. However, direct evidence is lacking for a causal role of vHPC GABA neurons in behaviors associated with schizophrenia. Here, we probed the behavioral function of two different but overlapping populations of vHPC GABA neurons that express either PV or GAD65 by selectively inhibiting these neurons with the pharmacogenetic neuromodulator hM4D. We show that acute inhibition of vHPC GABA neurons in adult mice results in behavioral changes relevant to schizophrenia. Inhibiting either PV or GAD65 neurons produced distinct behavioral deficits. Inhibition of PV neurons, affecting ∼80% of the PV neuron population, robustly impaired prepulse inhibition of the acoustic startle reflex (PPI), startle reactivity, and spontaneous alternation, but did not affect locomotor activity. In contrast, inhibiting a heterogeneous population of GAD65 neurons, affecting ∼40% of PV neurons and 65% of cholecystokinin neurons, increased spontaneous and amphetamine-induced locomotor activity and reduced spontaneous alternation, but did not alter PPI. Inhibition of PV or GAD65 neurons also produced distinct changes in network oscillatory activity in the vHPC in vivo. Together, these findings establish a causal role for vHPC GABA neurons in controlling behaviors relevant to schizophrenia and suggest a functional dissociation between the GABAergic mechanisms involved in hippocampal modulation of sensorimotor processes.

Diversity of mnemonic function within the entorhinal cortex: a meta-analysis of rodent behavioral studies.

The entorhinal cortex (EC) has been shown to be an integral piece of the hippocampal memory system. It sits in a unique position within the brain with strong, intricate, reciprocal connectivity with the hippocampus as well as a vast array of neocortical regions. Topographical patterns of afferent and efferent projections suggest that the EC can be divided into the medial and lateral regions, each of which can be further divided into dorsal, intermediate, and lateral bands. These EC sub-regions, with variable anatomical features, indicate a multifaceted role of the EC in memory processing. The present article reviews rodent behavioral studies which tested the effect of manipulation to EC sub-regions in several different memory paradigms. An analysis of the specific targets of EC manipulations reveals an important role of the caudomedial EC for spatial memory. In recognition memory paradigms, damage to the lateral EC impairs recognition of the combined information of objects, locations, and environmental contexts relevant to the content of an experience; whereas damage to medial EC preferentially impairs the recognition of the spatial arrangement of objects relevant to the spatial location of an experience. Fewer studies have examined the impact of EC manipulations on contextual memory and temporal associative memory, the results of which are fairly conflicting and possible confounds are explored. Our summary provides further support for the functional dissociation within the EC for learning and memory and generates several ideas for future investigations.

Unilateral lateral entorhinal inactivation impairs memory expression in trace eyeblink conditioning.

Memory in trace eyeblink conditioning is mediated by an inter-connected network that involves the hippocampus (HPC), several neocortical regions, and the cerebellum. This network reorganizes after learning as the center of the network shifts from the HPC to the medial prefrontal cortex (mPFC). Despite the network reorganization, the lateral entorhinal cortex (LEC) plays a stable role in expressing recently acquired HPC-dependent memory as well as remotely acquired mPFC-dependent memory. Entorhinal involvement in recent memory expression may be attributed to its previously proposed interactions with the HPC. In contrast, it remains unknown how the LEC participates in memory expression after the network disengages from the HPC. The present study tested the possibility that the LEC and mPFC functionally interact during remote memory expression by examining the impact of pharmacological inactivation of the LEC in one hemisphere and the mPFC in the contralateral hemisphere on memory expression in rats. Memory expression one day and one month after learning was significantly impaired after LEC-mPFC inactivation; however, the degree of impairment was comparable to that after unilateral LEC inactivation. Unilateral mPFC inactivation had no effect on recent or remote memory expression. These results suggest that the integrity of the LEC in both hemispheres is necessary for memory expression. Functional interactions between the LEC and mPFC should therefore be tested with an alternative design.

Functional dissociation within the entorhinal cortex for memory retrieval of an association between temporally discontiguous stimuli.

Anatomical connectivity and single neuron coding suggest a segregation of information representation within lateral (LEC) and medial (MEC) portions of the entorhinal cortex, a brain region serving as the primary input/output of the hippocampus and maintaining widespread connections to many association cortices. The present study aimed to expand this idea by examining whether these two subregions differentially contribute to memory retrieval for an association between temporally discontiguous stimuli. We found that reversible inactivation of the LEC, but not the MEC, severely impaired the retrieval of the recently and remotely acquired memory in rat trace eyeblink conditioning, in which a stimulus-free interval was interposed between the conditioned and unconditioned stimulus. Conversely, inactivation of the LEC had no effect on retrieval in delay eyeblink conditioning, where two stimuli were presented without an interval. Therefore, the LEC, but not the MEC, plays a long-lasting role in the retrieval of a memory for an association between temporally discontiguous stimuli.

Enduring deficits in contextual and auditory fear conditioning after adolescent, not adult, social instability stress in male rats.

Adolescence is a time of developmental changes and reorganization in the brain and stress systems, thus, adolescents may be more vulnerable than adults to the effects of chronic mild stressors. Most studies, however, have not directly compared stress experienced in adolescence to the same stress experience in adulthood. In the present study, adolescent (n=46) and adult (n=48) male rats underwent 16 days of social instability stress (daily 1h isolation and change of cage partners) or were non-stress controls. Rats were then tested on the strength of acquired contextual and cued fear conditioning, as well as extinction learning, beginning either the day after the stress procedure or 3 weeks later. No difference was found among the groups during the Training Phase of conditioning. Irrespective of the time between the social stress experience and fear conditioning, rats stressed in adolescence had decreased context and cue memory, and cue generalization compared to control rats, as measured by the percentage of time spent freezing in tests. Social instability stress in adulthood had no effect on any measure of fear conditioning. The results support the hypothesis that adolescence is a time of heightened vulnerability to stressors.

Increased Entorhinal-Prefrontal Theta Synchronization Parallels Decreased Entorhinal-Hippocampal Theta Synchronization during Learning and Consolidation of Associative Memory.

Memories are thought to be encoded as a distributed representation in the neocortex. The medial prefrontal cortex (mPFC) has been shown to support the expression of memories that initially depend on the hippocampus (HPC), yet the mechanisms by which the HPC and mPFC access the distributed representations in the neocortex are unknown. By measuring phase synchronization of local field potential (LFP) oscillations, we found that learning initiated changes in neuronal communication of the HPC and mPFC with the lateral entorhinal cortex (LEC), an area that is connected with many other neocortical regions. LFPs were recorded simultaneously from the three brain regions while rats formed an association between an auditory stimulus (CS) and eyelid stimulation (US) in a trace eyeblink conditioning paradigm, as well as during retention 1 month following learning. Over the course of learning, theta oscillations in the LEC and mPFC became strongly synchronized following presentation of the CS on trials in which rats exhibited a conditioned response (CR), and this strengthened synchronization was also observed during remote retention. In contrast, CS-evoked theta synchronization between the LEC and HPC decreased with learning. Our results suggest that communication between the LEC and mPFC are strengthened with learning whereas the communication between the LEC and HPC are concomitantly weakened, suggesting that enhanced LEC-mPFC communication may be a neuronal correlate for theoretically proposed neocortical reorganization accompanying encoding and consolidation of a memory.

Estimating aggression from emotionally neutral faces: which facial cues are diagnostic?

The facial width-to-height ratio, a size-independent sexually dimorphic property of the human face, is correlated with aggressive behaviour in men. Furthermore, observers' estimates of aggression from emotionally neutral faces are accurate and are highly correlated with the facial width-to-height ratio. We investigated whether observers use the facial width-to-height ratio to estimate propensity for aggression. In experiments 1a-1c, estimates of aggression remained accurate when faces were blurred or cropped, manipulations that reduce featural cues but maintain the facial width-to-height ratio. Accuracy decreased when faces were scrambled, a manipulation that retains featural information but disrupts the facial width-to-height ratio. In experiment 2, computer-modeling software identified eight facial metrics that correlated with estimates of aggression; regression analyses revealed that the facial width-to-height ratio was the only metric that uniquely predicted these estimates. In experiment 3, we used a computer-generated set of faces varying in perceived threat (Oosterhof and Todorov, 2008 Proceedings of the National Academy of Sciences of the USA 105 11087-11092) and found that as emotionally neutral faces became more 'threatening', the facial width-to-height ratio increased. Together, these experiments suggest that the facial width-to-height ratio is an honest signal of propensity for aggressive behaviour.

Motivational and situational factors and the relationship between testosterone dynamics and human aggression during competition.

Men engage in aggression at a cost to extrinsic reward, and this behaviour is associated with a rise in testosterone. To characterize the factors underlying aggression, men were assigned to one of the four experimental conditions of a computer game in which they were provoked (points were stolen from them or not) and/or received reward for aggression (received points for aggression or not). Men who were provoked but did not receive reward for aggression enjoyed the task the most, demonstrated an increase in salivary testosterone, and were more likely to choose a competitive versus non-competitive task than men in the other experimental conditions. Moreover, individual differences in aggressive behaviour among these men were positively correlated with the extent to which they enjoyed the task and with testosterone fluctuations. These results indicate that costly aggressive behaviour is intrinsically rewarding, perhaps to regulate future interactions, and that testosterone may be a physiological marker of such reward value.

Individual differences in activity predict locomotor activity and conditioned place preference to amphetamine in both adolescent and adult rats.

Individual and developmental differences in novelty seeking have been implicated in differential sensitivity to psychostimulants in rodents, but findings are mixed. The extent to which age differences in activity in a novel arena depended on test duration was examined by comparing adolescent and adult rats after 5 and after 60 min of testing (session 1). Rats were tested again after amphetamine or saline administration 24h later (session 2) to examine whether activity in a novel arena predicts sensitivity to locomotor-activating effects of amphetamine. Data from two experiments were used to examine consistency of the findings. Only activity in 60 min sessions produced a consistent age difference (adolescent