Compulsive alcohol seeking and relapse: Central role of conditioning factors associated with alleviation of withdrawal states by alcohol.

BACKGROUND AND PURPOSE: Learned associations between environmental stimuli and drugs of abuse represent a major factor in the chronically relapsing nature of drug addiction. In drug dependent subjects these associations must be presumed to include associations linked to reversal of adverse withdrawal states by drug use-"withdrawal-associated learning" (WDL). However, their significance in drug seeking has received little experimental scrutiny. EXPERIMENTAL APPROACH: Using alcohol as a drug of abuse, the behavioural consequences of WDL were investigated in animal models of relapse and compulsive drug seeking by comparing the effects of WD L-associated stimuli versus stimuli associated with alcohol without WDL experience in nondependent and post-dependent rats. Brain sites activated by exposure to the respective stimuli were identified by c-fos immunohistochemistry. KEY RESULTS: (1) WDL-associated stimuli elicited significant alcohol seeking. In rats with WDL experience, stimuli associated with alcohol in the nondependent state no longer elicited robust alcohol seeking. (2) Responding elicited by WDL-associated stimuli, but not stimuli conditioned to alcohol in the nondependent state, was resistant to footshock punishment and increased response effort requirements for presentation of WDL-related stimuli. (3) Stimuli conditioned to alcohol in rats with a dependence but not WDL history did not sustain punished responding or tolerance of increased effort. (4) The central nucleus of the amygdala was identified as a site selectively responsive to WDL stimulus exposure. CONCLUSION AND IMPLICATIONS: Environmental stimuli associated with reversal of adverse withdrawal states by alcohol elicit compulsive-like alcohol seeking and establish WDL as a major, not well-recognized factor, in relapse vulnerability.

Optogenetic reactivation of memory ensembles in the retrosplenial cortex induces systems consolidation.

The neural circuits underlying memory change over prolonged periods after learning, in a process known as systems consolidation. Postlearning spontaneous reactivation of memory-related neural ensembles is thought to mediate this process, although a causal link has not been established. Here we test this hypothesis in mice by using optogenetics to selectively reactivate neural ensembles representing a contextual fear memory (sometimes referred to as engram neurons). High-frequency stimulation of these ensembles in the retrosplenial cortex 1 day after learning produced a recent memory with features normally observed in consolidated remote memories, including higher engagement of neocortical areas during retrieval, contextual generalization, and decreased hippocampal dependence. Moreover, this effect was only present if memory ensembles were reactivated during sleep or light anesthesia. These results provide direct support for postlearning memory ensemble reactivation as a mechanism of systems consolidation, and show that this process can be accelerated by ensemble reactivation in an unconscious state.

Differential fear conditioning generates prefrontal neural ensembles of safety signals.

Fear discrimination is critical for survival, while fear generalization is effective for avoiding dangerous situations. Overgeneralized fear is a typical symptom of anxiety disorders, including generalized anxiety disorder and posttraumatic stress disorder (PTSD). Previous research demonstrated that fear discrimination learning is mediated by prefrontal mechanisms. While the prelimbic (PL) and infralimbic (IL) subdivisions of the medial prefrontal cortex (mPFC) are recognized for their excitatory and inhibitory effects on the fear circuit, respectively, the mechanisms driving fear discrimination are unidentified. To obtain insight into the mechanisms underlying context-specific fear discrimination, we investigated prefrontal neuronal ensembles representing distinct experiences associated with learning to disambiguate between dangerous and similar, but not identical, harmless stimuli. Here, we show distinct quantitative activation differences in response to conditioned and generalized fear experiences, as well as modulation of the neuronal ensembles associated with successful acquisition of context-safety contingencies. These findings suggest that prefrontal neuronal ensembles patterns code functional context-danger and context-safety relationships. The PL subdivision of the mPFC monitors context-danger associations to conditioned fear, whereas differential conditioning sparks additional ensembles associated with the inhibition of generalized fear in both the PL and IL subdivisions of the mPFC. Our data suggest that fear discrimination learning is associated with the modulation of prefrontal subpopulations in a subregion- and experience-specific fashion, and the learning of appropriate responses to conditioned and initially generalized fear experiences is driven by gradual updating and rebalancing of the prefrontal memory representations.

A Functionally Defined In Vivo Astrocyte Population Identified by c-Fos Activation in a Mouse Model of Multiple Sclerosis Modulated by S1P Signaling: Immediate-Early Astrocytes (ieAstrocytes).

Astrocytes have prominent roles in central nervous system (CNS) function and disease, with subpopulations defined primarily by morphologies and molecular markers often determined in cell culture. Here, we identify an in vivo astrocyte subpopulation termed immediate-early astrocytes (ieAstrocytes) that is defined by functional c-Fos activation during CNS disease development. An unbiased screen for CNS cells showing c-Fos activation during experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis (MS), was developed by using inducible, TetTag c-Fos reporter mice that label activated cells with a temporally stable, nuclear green fluorescent protein (GFP). Four-dimensional (3D over time) c-Fos activation maps in the spinal cord were produced by combining tissue clearing (iDISCO) and confocal microscopy that identified onset and expansion of GFP+ cell populations during EAE. More than 95% of the GFP+ cells showed glial fibrillary acidic protein (GFAP) immunoreactivity-in contrast to absent or rare labeling of neurons, microglia, and infiltrating immune cells-which constituted ieAstrocytes that linearly increased in number with progression of EAE. ieAstrocyte formation was reduced by either astrocyte-specific genetic removal of sphingosine 1-phosphate receptor 1 (S1P1) or pharmacological inhibition by fingolimod (FTY720), an FDA-approved MS medicine that can functionally antagonize S1P1. ieAstrocytes thus represent a functionally defined subset of disease-linked astrocytes that are the first and predominant CNS cell population activated during EAE, and that track with disease severity in vivo. Their reduction by a disease-modifying agent supports their therapeutic relevance to MS and potentially other neuroinflammatory and neurodegenerative diseases.

Distinct memory engrams in the infralimbic cortex of rats control opposing environmental actions on a learned behavior.

Conflicting evidence exists regarding the role of infralimbic cortex (IL) in the environmental control of appetitive behavior. Inhibition of IL, irrespective of its intrinsic neural activity, attenuates not only the ability of environmental cues predictive of reward availability to promote reward seeking, but also the ability of environmental cues predictive of reward omission to suppress this behavior. Here we report that such bidirectional behavioral modulation in rats is mediated by functionally distinct units of neurons (neural ensembles) that are concurrently localized within the same IL brain area but selectively reactive to different environmental cues. Ensemble-specific neural activity is thought to function as a memory engram representing a learned association between environment and behavior. Our findings establish the causal evidence for the concurrent existence of two distinct engrams within a single brain site, each mediating opposing environmental actions on a learned behavior.

Chronic fluoxetine dissociates contextual from auditory fear memory.

Fluoxetine is a medication used to treat Major Depressive Disorder and other psychiatric conditions. These experiments studied the effects of chronic fluoxetine treatment on the contextual versus auditory fear memory of mice. We found that chronic fluoxetine treatment of adult mice impaired their contextual fear memory, but spared auditory fear memory. Hippocampal perineuronal nets, which are involved in contextual fear memory plasticity, were unaltered by fluoxetine treatment. These data point to a selective inability to form contextual fear memory as a result of fluoxetine treatment, and they suggest that a blunting of hippocampal-mediated aversive memory may be a therapeutic action for this medication.

A shared neural ensemble links distinct contextual memories encoded close in time.

Recent studies suggest that a shared neural ensemble may link distinct memories encoded close in time. According to the memory allocation hypothesis, learning triggers a temporary increase in neuronal excitability that biases the representation of a subsequent memory to the neuronal ensemble encoding the first memory, such that recall of one memory increases the likelihood of recalling the other memory. Here we show in mice that the overlap between the hippocampal CA1 ensembles activated by two distinct contexts acquired within a day is higher than when they are separated by a week. Several findings indicate that this overlap of neuronal ensembles links two contextual memories. First, fear paired with one context is transferred to a neutral context when the two contexts are acquired within a day but not across a week. Second, the first memory strengthens the second memory within a day but not across a week. Older mice, known to have lower CA1 excitability, do not show the overlap between ensembles, the transfer of fear between contexts, or the strengthening of the second memory. Finally, in aged mice, increasing cellular excitability and activating a common ensemble of CA1 neurons during two distinct context exposures rescued the deficit in linking memories. Taken together, these findings demonstrate that contextual memories encoded close in time are linked by directing storage into overlapping ensembles. Alteration of these processes by ageing could affect the temporal structure of memories, thus impairing efficient recall of related information.

Exploring Memory Representations with Activity-Based Genetics.

The brain is thought to represent specific memories through the activity of sparse and distributed neural ensembles. In this review, we examine the use of immediate early genes (IEGs), genes that are induced by neural activity, to specifically identify and genetically modify neurons activated naturally by environmental experience. Recent studies using this approach have identified cellular and molecular changes specific to neurons activated during learning relative to their inactive neighbors. By using opto- and chemogenetic regulators of neural activity, the neurons naturally recruited during learning can be artificially reactivated to directly test their role in coding external information. In contextual fear conditioning, artificial reactivation of learning-induced neural ensembles in the hippocampus or neocortex can substitute for the context itself. That is, artificial stimulation of these neurons can apparently cause the animals to "think" they are in the context. This represents a powerful approach to testing the principles by which the brain codes for the external world and how these circuits are modified with learning.

Memory Retrieval in Mice and Men.

Retrieval, the use of learned information, was until recently mostly terra incognita in the neurobiology of memory, owing to shortage of research methods with the spatiotemporal resolution required to identify and dissect fast reactivation or reconstruction of complex memories in the mammalian brain. The development of novel paradigms, model systems, and new tools in molecular genetics, electrophysiology, optogenetics, in situ microscopy, and functional imaging, have contributed markedly in recent years to our ability to investigate brain mechanisms of retrieval. We review selected developments in the study of explicit retrieval in the rodent and human brain. The picture that emerges is that retrieval involves coordinated fast interplay of sparse and distributed corticohippocampal and neocortical networks that may permit permutational binding of representational elements to yield specific representations. These representations are driven largely by the activity patterns shaped during encoding, but are malleable, subject to the influence of time and interaction of the existing memory with novel information.

A transgenic mouse line for collecting ribosome-bound mRNA using the tetracycline transactivator system.

Acquiring the gene expression profiles of specific neuronal cell-types is important for understanding their molecular identities. Genome-wide gene expression profiles of genetically defined cell-types can be acquired by collecting and sequencing mRNA that is bound to epitope-tagged ribosomes (TRAP; translating ribosome affinity purification). Here, we introduce a transgenic mouse model that combines the TRAP technique with the tetracycline transactivator (tTA) system by expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under control of the tetracycline response element (tetO-TRAP). This allows both spatial control of EGFP-L10a expression through cell-type specific tTA expression, as well as temporal regulation by inhibiting transgene expression through the administration of doxycycline. We show that crossing tetO-TRAP mice with transgenic mice expressing tTA under the Camk2a promoter (Camk2a-tTA) results in offspring with cell-type specific expression of EGFP-L10a in CA1 pyramidal neurons and medium spiny neurons in the striatum. Co-immunoprecipitation confirmed that EGFP-L10a integrates into a functional ribosomal complex. In addition, collection of ribosome-bound mRNA from the hippocampus yielded the expected enrichment of genes expressed in CA1 pyramidal neurons, as well as a depletion of genes expressed in other hippocampal cell-types. Finally, we show that crossing tetO-TRAP mice with transgenic Fos-tTA mice enables the expression of EGFP-L10a in CA1 pyramidal neurons that are activated during a fear conditioning trial. The tetO-TRAP mouse can be combined with other tTA mouse lines to enable gene expression profiling of a variety of different cell-types.

Direct reactivation of a coherent neocortical memory of context.

Declarative memories are thought to be stored within anatomically distributed neuronal networks requiring the hippocampus; however, it is unclear how neocortical areas participate in memory at the time of encoding. Here, we use a c-fos-based genetic tagging system to selectively express the channelrhodopsin variant, ChEF, and optogenetically reactivate a specific neural ensemble in retrosplenial cortex (RSC) engaged by context fear conditioning. Artificial stimulation of RSC was sufficient to produce both context-specific behavior and downstream cellular activity commensurate with natural experience. Moreover, optogenetically but not contextually elicited responses were insensitive to hippocampal inactivation, suggesting that although the hippocampus is needed to coordinate activation by sensory cues, a higher-order cortical framework can independently subserve learned behavior, even shortly after learning.

The molecular and systems biology of memory.

Learning and memory are two of the most magical capabilities of our mind. Learning is the biological process of acquiring new knowledge about the world, and memory is the process of retaining and reconstructing that knowledge over time. Most of our knowledge of the world and most of our skills are not innate but learned. Thus, we are who we are in large part because of what we have learned and what we remember and forget. In this Review, we examine the molecular, cellular, and circuit mechanisms that underlie how memories are made, stored, retrieved, and lost.

The search for a hippocampal engram.

Understanding the molecular and cellular changes that underlie memory, the engram, requires the identification, isolation and manipulation of the neurons involved. This presents a major difficulty for complex forms of memory, for example hippocampus-dependent declarative memory, where the participating neurons are likely to be sparse, anatomically distributed and unique to each individual brain and learning event. In this paper, I discuss several new approaches to this problem. In vivo calcium imaging techniques provide a means of assessing the activity patterns of large numbers of neurons over long periods of time with precise anatomical identification. This provides important insight into how the brain represents complex information and how this is altered with learning. The development of techniques for the genetic modification of neural ensembles based on their natural, sensory-evoked, activity along with optogenetics allows direct tests of the coding function of these ensembles. These approaches provide a new methodological framework in which to examine the mechanisms of complex forms of learning at the level of the neurons involved in a specific memory.

Transgenically targeted rabies virus demonstrates a major monosynaptic projection from hippocampal area CA2 to medial entorhinal layer II neurons.

The enormous potential of modern molecular neuroanatomical tools lies in their ability to determine the precise connectivity of the neuronal cell types comprising the innate circuitry of the brain. We used transgenically targeted viral tracing to identify the monosynaptic inputs to the projection neurons of layer II of medial entorhinal cortex (MEC-LII) in mice. These neurons are not only major inputs to the hippocampus, the structure most clearly implicated in learning and memory, they also are "grid cells." Here we address the question of what kinds of inputs are specifically targeting these MEC-LII cells. Cell-specific infection of MEC-LII with recombinant rabies virus results in unambiguous labeling of monosynaptic inputs. Furthermore, ratios of labeled neurons in different regions are largely consistent between animals, suggesting that label reflects density of innervation. While the results mostly confirm prior anatomical work, they also reveal a novel major direct input to MEC-LII from hippocampal pyramidal neurons. Interestingly, the vast majority of these direct hippocampal inputs arise not from the major hippocampal subfields of CA1 and CA3, but from area CA2, a region that has historically been thought to merely be a transitional zone between CA3 and CA1. We confirmed this unexpected result using conventional tracing techniques in both rats and mice.

Empathic fear responses in mice are triggered by recognition of a shared experience.

Empathy is an important psychological capacity that involves the ability to recognize and share emotions with others. In humans, empathy for others is facilitated by having had a similar prior experience. It increases with the intensity of distress that observers believe is occurring to others, and is associated with acute emotional responses to witnessing others' distress. We sought to develop a relatively simple and fast mouse model of human empathy that resembled these characteristics. We modeled empathy by measuring the freezing of observer mice to observing the footshock of a subject mouse. Observer mice froze to subject footshocks only when they had a similar shock experience 24 hours earlier. Moreover, this freezing increased with the number of footshocks given to the subject and it was accentuated within seconds after footshock delivery. Freezing was not seen in naïve observers or in experienced observers that observed a subject who was spared footshock. Observers did not freeze to a subject's footshock when they had experienced a swim stress 24 hours prior, demonstrating a specific effect for shared experience, as opposed to a generalized stressor in eliciting observer mouse freezing. We propose that this two-day experimental protocol resembles many aspects of human empathy in a mouse model that is amenable to transgenic analysis of neural substrates for empathy and its impairment in certain clinical disorders.

Inducible control of gene expression with destabilized Cre.

Acute manipulation of gene and protein function in the brain is essential for understanding the mechanisms of nervous system development, plasticity and information processing. Here we describe a technique based on a destabilized Cre recombinase (DD-Cre) whose activity is controlled by the antibiotic trimethoprim (TMP). We show that DD-Cre triggers rapid TMP-dependent recombination of loxP-flanked ('floxed') alleles in mouse neurons in vivo and validate the use of this system for neurobehavioral research.

Selection of distinct populations of dentate granule cells in response to inputs as a mechanism for pattern separation in mice.

The hippocampus is critical for episodic memory and computational studies have predicted specific functions for each hippocampal subregion. Particularly, the dentate gyrus (DG) is hypothesized to perform pattern separation by forming distinct representations of similar inputs. How pattern separation is achieved by the DG remains largely unclear. By examining neuronal activities at a population level, we revealed that, unlike CA1 neuron populations, dentate granule cell (DGC) ensembles activated by learning were not preferentially reactivated by memory recall. Moreover, when mice encountered an environment to which they had not been previously exposed, a novel DGC population-rather than the previously activated DGC ensembles that responded to past events-was selected to represent the new environmental inputs. This selection of a novel responsive DGC population could be triggered by small changes in environmental inputs. Therefore, selecting distinct DGC populations to represent similar but not identical inputs is a mechanism for pattern separation. DOI:http://dx.doi.org/10.7554/eLife.00312.001.

Elimination of dendritic spines with long-term memory is specific to active circuits.

Structural changes in brain circuits active during learning are thought to be important for long-term memory storage. If these changes support long-term information storage, they might be expected to be present at distant time points after learning, as well as to be specific to the circuit activated with learning, and sensitive to the contingencies of the behavioral paradigm. Here, we show such changes in the hippocampus as a result of contextual fear conditioning. There were significantly fewer spines specifically on active neurons of fear-conditioned mice. This spine loss did not occur in homecage mice or in mice exposed to the training context alone. Mice exposed to unpaired shocks showed a generalized reduction in spines. These learning-related changes in spine density could reflect a direct mechanism of encoding or alternately could reflect a compensatory adaptation to previously described enhancement in transmission due to glutamate receptor insertion.

New approaches to neural circuits in behavior.

A fundamental goal of neuroscience is to understand how the brain represents the world. If we consider the neurons of the brain to be one system and the external world to be another system, how do the two systems interact, and by what translational code does the former represent the latter? Recent advances in imaging neural activity, genetically altering specific neural circuits, and genetic tools for the direct manipulation of neural activity are beginning to shed light on this critical question. We review recent advances in these areas that illustrate a path to addressing this fundamental question.

Navigating uncertain waters.

Mice lacking NMDA receptors in the dentate gyrus and CA1 subfields of the hippocampus form spatial memories just as well as wild-type mice, but they disregard them when confounded by ambiguous local cues. Hippocampal NMDA receptors may influence spatial memory more subtly than previously thought.