Dorsolateral striatum engagement during reversal learning.

Most experimental preparations demonstrate a role for dorsolateral striatum (DLS) in stimulus-response, but not outcome-based, learning. Here, we assessed DLS involvement in a touchscreen-based reversal task requiring mice to update choice following a change in stimulus-reward contingencies. In vivo single-unit recordings in the DLS showed reversal produced a population-level shift from excited to inhibited neuronal activity prior to choices being made. The larger the shift, the faster mice reversed. Furthermore, optogenetic photosilencing DLS neurons during choice increased early reversal errors. These findings suggest dynamic DLS engagement may facilitate reversal, possibly by signaling a change in contingencies to other striatal and cortical regions.

A novel multichoice touchscreen paradigm for assessing cognitive flexibility in mice.

Cognitive flexibility refers to various processes which enable behaviors to be modified on the basis of a change in the contingencies between stimuli or responses and their associated outcomes. Reversal learning is a form of cognitive flexibility which measures the ability to adjust responding based on a switch in the stimulus-outcome contingencies of, typically two, perceptually distinct stimuli. Reversal tasks have provided valuable insight into the neural basis of cognitive flexibility, implicating brain regions including the lateral orbitofrontal cortex (lOFC) and dorsomedial prefrontal cortex (dmPFC). However, with two-stimulus reversal, it is difficult to determine whether response errors are due excessive perseveration, deficient learning, or other problems with updating. To address this limitation, we developed a mouse three-choice touchscreen-based visual reversal task, in which the contingencies of two stimuli were switched on reversal but a third, simultaneously presented, stimulus was never reinforced. We found that, in male C57BL/6J mice, responding at the previously rewarded stimulus predominated over the newly and never-reinforced stimuli during early reversal. Next, we showed that acute pharmacological inhibition of lOFC, but not dmPFC, impaired early reversal performance, relative to noninactivated controls. Interestingly, however, lOFC inactivation deficits were characterized by increased choice of the never-reinforced stimulus and a decrease in (perseverative-like) responding at the previously rewarded stimulus. These effects are inconsistent with the historical notion of lOFC mediating response inhibition and closer to recent views of the lOFC's role in response/outcome tracking. Overall, these findings provide initial support the utility of this novel paradigm for studying cognitive flexibility and its underlying neural substrates.

Improving transparency and scientific rigor in academic publishing.

Progress in basic and clinical research is slowed when researchers fail to provide a complete and accurate report of how a study was designed, executed, and the results analyzed. Publishing rigorous scientific research involves a full description of the methods, materials, procedures, and outcomes. Investigators may fail to provide a complete description of how their study was designed and executed because they may not know how to accurately report the information or the mechanisms are not in place to facilitate transparent reporting. Here, we provide an overview of how authors can write manuscripts in a transparent and thorough manner. We introduce a set of reporting criteria that can be used for publishing, including recommendations on reporting the experimental design and statistical approaches. We also discuss how to accurately visualize the results and provide recommendations for peer reviewers to enhance rigor and transparency. Incorporating transparency practices into research manuscripts will significantly improve the reproducibility of the results by independent laboratories.

Cued fear memory generalization increases over time.

Fear memory is a highly stable and durable form of memory, even over vast (remote) time frames. Nevertheless, some elements of fear memory can be forgotten, resulting in generalization. The purpose of this study is to determine how cued fear memory generalizes over time and measure underlying patterns of cortico-amygdala synaptic plasticity. We established generalization gradients at recent (1-d) and remote (30-d) retention intervals following auditory cued fear conditioning in adult male C57BL/6 mice. Results revealed a flattening of the generalization gradient (increased generalization) that was dissociated from contextual fear generalization, indicating a specific influence of time on cued fear memory performance. This effect reversed after a brief exposure to the novel stimulus soon after learning. Measurements from cortico-amygdala imaging of the activity-regulated cytoskeletal Arc/arg 3.1 (Arc) protein using immunohistochemistry after cued fear memory retrieval revealed a stable pattern of Arc expression in the dorsolateral amygdala, but temporally dynamic expression in the cortex. Over time, increased fear memory generalization was associated with a reduction in Arc expression in the agranular insular and infralimbic cortices while discrimination learning was associated with increased Arc expression in the prelimbic cortex. These data identify the dorsolateral amygdala, medial prefrontal, and insular cortices as loci for synaptic plasticity underlying cued fear memory generalization over time.

Chronic Ethanol During Adolescence Impacts Corticolimbic Dendritic Spines and Behavior.

BACKGROUND: Risk for alcohol use disorders (AUDs) in adulthood is linked to alcohol drinking during adolescence, but understanding of the neural and behavioral consequences of alcohol exposure during adolescence remains incomplete. Here, we examined the neurobehavioral impact of adolescent chronic intermittent EtOH (CIE) vapor exposure in mice. METHODS: C57BL/6J-background Thy1-EGFP mice were CIE-exposed during adolescence or adulthood and examined, as adults, for alterations in the density and morphology of dendritic spines in infralimbic (IL) cortex, prelimbic (PL) cortex, and basolateral amygdala (BLA). In parallel, adolescent- and adult-exposed C57BL/6J mice were tested as adults for 2-bottle EtOH drinking, sensitivity to EtOH intoxication (loss of righting reflex [LORR]), blood EtOH clearance, and measures of operant responding for food reward. RESULTS: CIE during adolescence decreased IL neuronal spine density and increased the head width of relatively wide-head IL and BLA spines, whereas CIE decreased head width of relatively narrow-head BLA spines. Adolescents had higher EtOH consumption prior to CIE than adults, while CIE during adulthood, but not adolescence, increased EtOH consumption relative to pre-CIE baseline. CIE produced a tolerance-like decrease in LORR sensitivity to EtOH challenge, irrespective of the age at which mice received CIE exposure. Mice exposed to CIE during adolescence, but not adulthood, required more sessions than AIR controls to reliably respond for food reward on a fixed-ratio (FR) 1, but not subsequent FR3, reinforcement schedule. On a progressive ratio reinforcement schedule, break point responding was higher in the adolescent- than the adult-exposed mice, regardless of CIE. Finally, footshock punishment markedly suppressed responding for reward in all groups. CONCLUSIONS: Exposure to CIE during adolescence altered dendritic spine density and morphology in IL and BLA neurons, in parallel with a limited set of behavioral alterations. Together, these data add to growing evidence that key corticolimbic circuits are vulnerable to the effects of alcohol during adolescence, with lasting, potentially detrimental, consequences for behavior.

The basolateral amygdala γ-aminobutyric acidergic system in health and disease.

The brain comprises an excitatory/inhibitory neuronal network that maintains a finely tuned balance of activity critical for normal functioning. Excitatory activity in the basolateral amygdala (BLA), a brain region that plays a central role in emotion and motivational processing, is tightly regulated by a relatively small population of γ-aminobutyric acid (GABA) inhibitory neurons. Disruption in GABAergic inhibition in the BLA can occur when there is a loss of local GABAergic interneurons, an alteration in GABAA receptor activation, or a dysregulation of mechanisms that modulate BLA GABAergic inhibition. Disruptions in GABAergic control of the BLA emerge during development, in aging populations, or after trauma, ultimately resulting in hyperexcitability. BLA hyperexcitability manifests behaviorally as an increase in anxiety, emotional dysregulation, or development of seizure activity. This Review discusses the anatomy, development, and physiology of the GABAergic system in the BLA and circuits that modulate GABAergic inhibition, including the dopaminergic, serotonergic, noradrenergic, and cholinergic systems. We highlight how alterations in various neurotransmitter receptors, including the acid-sensing ion channel 1a, cannabinoid receptor 1, and glutamate receptor subtypes, expressed on BLA interneurons, modulate GABAergic transmission and how defects of these systems affect inhibitory tonus within the BLA. Finally, we discuss alterations in the BLA GABAergic system in neurodevelopmental (autism/fragile X syndrome) and neurodegenerative (Alzheimer's disease) diseases and after the development of epilepsy, anxiety, and traumatic brain injury. A more complete understanding of the intrinsic excitatory/inhibitory circuit balance of the amygdala and how imbalances in inhibitory control contribute to excessive BLA excitability will guide the development of novel therapeutic approaches in neuropsychiatric diseases.

A dendritic organization of lateral amygdala neurons in fear susceptible and resistant mice.

Subtle differences in neuronal microanatomy may be coded in individuals with genetic susceptibility for neuropsychiatric disorders. Genetic susceptibility is a significant risk factor in the development of anxiety disorders, including post-traumatic stress disorder (PTSD). Pavlovian fear conditioning has been proposed to model key aspects of PTSD. According to this theory, PTSD begins with the formation of a traumatic memory which connects relevant environmental stimuli to significant threats to life. The lateral amygdala (LA) is considered to be a key network hub for the establishment of Pavlovian fear conditioning. Substantial research has also linked the LA to PTSD. Here we used a genetic mouse model of fear susceptibility (F-S) and resistance (F-R) to investigate the dendritic and spine structure of principal neurons located in the LA. F-S and F-R lines were bi-directionally selected based on divergent levels of contextual and cued conditioned freezing in response to fear-evoking footshocks. We examined LA principal neuron dendritic and spine morphology in the offspring of experimentally naive F-S and F-R mice. We found differences in the spatial distribution of dendritic branch points across the length of the dendrite tree, with a significant increase in branch points at more distal locations in the F-S compared with F-R line. These results suggest a genetic predisposition toward differences in fear memory strength associated with a dendritic branch point organization of principal neurons in the LA. These micro-anatomical differences in neuron structure in a genetic mouse model of fear susceptibility and resistance provide important insights into the cellular mechanisms of pathophysiology underlying genetic predispositions to anxiety and PTSD.

An organization of visual and auditory fear conditioning in the lateral amygdala.

Pavlovian fear conditioning is an evolutionary conserved and extensively studied form of associative learning and memory. In mammals, the lateral amygdala (LA) is an essential locus for Pavlovian fear learning and memory. Despite significant progress unraveling the cellular mechanisms responsible for fear conditioning, very little is known about the anatomical organization of neurons encoding fear conditioning in the LA. One key question is how fear conditioning to different sensory stimuli is organized in LA neuronal ensembles. Here we show that Pavlovian fear conditioning, formed through either the auditory or visual sensory modality, activates a similar density of LA neurons expressing a learning-induced phosphorylated extracellular signal-regulated kinase (p-ERK1/2). While the size of the neuron population specific to either memory was similar, the anatomical distribution differed. Several discrete sites in the LA contained a small but significant number of p-ERK1/2-expressing neurons specific to either sensory modality. The sites were anatomically localized to different levels of the longitudinal plane and were independent of both memory strength and the relative size of the activated neuronal population, suggesting some portion of the memory trace for auditory and visually cued fear conditioning is allocated differently in the LA. Presenting the visual stimulus by itself did not activate the same p-ERK1/2 neuron density or pattern, confirming the novelty of light alone cannot account for the specific pattern of activated neurons after visual fear conditioning. Together, these findings reveal an anatomical distribution of visual and auditory fear conditioning at the level of neuronal ensembles in the LA.

Traits of fear resistance and susceptibility in an advanced intercross line.

Genetic variability in the strength and precision of fear memory is hypothesised to contribute to the etiology of anxiety disorders, including post-traumatic stress disorder. We generated fear-susceptible (F-S) or fear-resistant (F-R) phenotypes from an F8 advanced intercross line (AIL) of C57BL/6J and DBA/2J inbred mice by selective breeding. We identified specific traits underlying individual variability in Pavlovian conditioned fear learning and memory. Offspring of selected lines differed in the acquisition of conditioned fear. Furthermore, F-S mice showed greater cued fear memory and generalised fear in response to a novel context than F-R mice. F-S mice showed greater basal corticosterone levels and hypothalamic corticotrophin-releasing hormone (CRH) mRNA levels than F-R mice, consistent with higher hypothalamic-pituitary-adrenal (HPA) axis drive. Hypothalamic mineralocorticoid receptor and CRH receptor 1 mRNA levels were decreased in F-S mice as compared with F-R mice. Manganese-enhanced magnetic resonance imaging (MEMRI) was used to investigate basal levels of brain activity. MEMRI identified a pattern of increased brain activity in F-S mice that was driven primarily by the hippocampus and amygdala, indicating excessive limbic circuit activity in F-S mice as compared with F-R mice. Thus, selection pressure applied to the AIL population leads to the accumulation of heritable trait-relevant characteristics within each line, whereas non-behaviorally relevant traits remain distributed. Selected lines therefore minimise false-positive associations between behavioral phenotypes and physiology. We demonstrate that intrinsic differences in HPA axis function and limbic excitability contribute to phenotypic differences in the acquisition and consolidation of associative fear memory. Identification of system-wide traits predisposing to variability in fear memory may help in the direction of more targeted and efficacious treatments for fear-related pathology.

Neurons activated during fear memory consolidation and reconsolidation are mapped to a common and new topography in the lateral amygdala.

A key question in neuroscience is how memory is selectively allocated to neural networks in the brain. This question remains a significant research challenge, in both rodent models and humans alike, because of the inherent difficulty in tracking and deciphering large, highly dimensional neuronal ensembles that support memory (i.e., the engram). In a previous study we showed that consolidation of a new fear memory is allocated to a common topography of amygdala neurons. When a consolidated memory is retrieved, it may enter a labile state, requiring reconsolidation for it to persist. What is not known is whether the original spatial allocation of a consolidated memory changes during reconsolidation. Knowledge about the spatial allocation of a memory, during consolidation and reconsolidation, provides fundamental insight into its core physical structure (i.e., the engram). Using design-based stereology, we operationally define reconsolidation by showing a nearly identical quantity of neurons in the dorsolateral amygdala (LAd) that expressed a plasticity-related protein, phosphorylated mitogen-activated protein kinase, following both memory acquisition and retrieval. Next, we confirm that Pavlovian fear conditioning recruits a stable, topographically organized population of activated neurons in the LAd. When the stored fear memory was briefly reactivated in the presence of the relevant conditioned stimulus, a similar topography of activated neurons was uncovered. In addition, we found evidence for activated neurons allocated to new regions of the LAd. These findings provide the first insight into the spatial allocation of a fear engram in the LAd, during its consolidation and reconsolidation phase.

Chronic nicotine exposure produces lateralized, age-dependent dendritic remodeling in the rodent basolateral amygdala.

This study investigated the dendritic morphology of neurons located in the right and left basolateral amygdala (BLA) and infralimbic (IL) cortex following chronic nicotine exposure during adolescence or adulthood. Sprague-Dawley rats were administered subcutaneous injections of nicotine (0.5 mg/kg; free base) or saline three times per week for 2 weeks (six total injections). The dose period began on either postnatal day (P) 32 (adolescent) or P61 (adult). Twenty days following the end of dosing, brains were processed for Golgi-Cox staining, and dendrites from principal neurons in the BLA and pyramidal neurons in the IL were digitally reconstructed in three dimensions. Morphometric analysis revealed a contrasting pattern of BLA dendritic morphology between the adolescent and adult pretreatment groups. In the adult control group, basilar dendritic length did not differ with respect to hemisphere. Nicotine induced robust hemispheric asymmetry by increasing dendritic length in the right hemisphere only. In contrast, adolescent nicotine exposure did not produce significant alteration of basilar dendritic morphology. There was, however, an indication that nicotine eliminated a naturally existing hemispheric asymmetry in the younger cohort. At both ages, nicotine produced a reduction in complexity of the apical tree of principal neurons. Chronic nicotine did not affect the dendritic morphology of pyramidal neurons from the IL in either age group, indicating another dimension of anatomical specificity. Collectively, these data implicate the BLA as a target for lasting neuroplasticity associated with chronic nicotine exposure. Further, hemispheric differences in dendritic morphology were uncovered that depended on the age of nicotine exposure, a finding that underscores the importance of considering laterality when investigating neurodevelopmental effects of drug exposure.

Assaying Fear Memory Discrimination and Generalization: Methods and Concepts.

Generalization describes the transfer of conditioned responding to stimuli that perceptually differ from the original conditioned stimulus. One arena in which discriminant and generalized responding is of particular relevance is when stimuli signal the potential for harm. Aversive (fear) conditioning is a leading behavioral model for studying associative learning and memory processes related to threatening stimuli. This article describes a step-by-step protocol for studying discrimination and generalization using cued fear conditioning in rodents. Alternate conditioning paradigms, including context generalization, differential generalization, discrimination training, and safety learning, are also described. The protocol contains instructions for constructing a cued fear memory generalization gradient and methods for isolating discrete cued-from-context cued conditioned responses (i.e., "the baseline issue"). The preclinical study of generalization is highly pertinent in the context of fear learning and memory because a lack of fear discrimination (overgeneralization) likely contributes to the etiology of anxiety-related disorders and post-traumatic stress disorder. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Tone cued fear generalization gradient Basic Protocol 2: Quantification of freezing Support Protocol: Alternate conditioning paradigms.

Know safety, no fear.

Every day we are bombarded by stimuli that must be assessed for their potential for harm or benefit. Once a stimulus is learned to predict harm, it can elicit fear responses. Such learning can last a lifetime but is not always beneficial for an organism. For an organism to thrive in its environment, it must know when to engage in defensive, avoidance behaviors and when to engage in non-defensive, approach behaviors. Fear should be suppressed in situations that are not dangerous: when a novel, innocuous stimulus resembles a feared stimulus, when a feared stimulus no longer predicts harm, or when there is an option to avoid harm. A cardinal feature of anxiety disorders is the inability to suppress fear adaptively. In PTSD, for instance, learned fear is expressed inappropriately in safe situations and is resistant to extinction. In this review, we discuss mechanisms of suppressing fear responses during stimulus discrimination, fear extinction, and active avoidance, focusing on the well-studied tripartite circuit consisting of the amygdala, medial prefrontal cortex and hippocampus.

Improving transparency and scientific rigor in academic publishing.

Progress in basic and clinical research is slowed when researchers fail to provide a complete and accurate report of how a study was designed, executed, and the results analyzed. Publishing rigorous scientific research involves a full description of the methods, materials, procedures, and outcomes. Investigators may fail to provide a complete description of how their study was designed and executed because they may not know how to accurately report the information or the mechanisms are not in place to facilitate transparent reporting. Here, we provide an overview of how authors can write manuscripts in a transparent and thorough manner. We introduce a set of reporting criteria that can be used for publishing, including recommendations on reporting the experimental design and statistical approaches. We also discuss how to accurately visualize the results and provide recommendations for peer reviewers to enhance rigor and transparency. Incorporating transparency practices into research manuscripts will significantly improve the reproducibility of the results by independent laboratories. SIGNIFICANCE: Failure to replicate research findings often arises from errors in the experimental design and statistical approaches. By providing a full account of the experimental design, procedures, and statistical approaches, researchers can address the reproducibility crisis and improve the sustainability of research outcomes. In this piece, we discuss the key issues leading to irreproducibility and provide general approaches to improving transparency and rigor in reporting, which could assist in making research more reproducible.

Improving transparency and scientific rigor in academic publishing.

Progress in basic and clinical research is slowed when researchers fail to provide a complete and accurate report of how a study was designed, executed, and the results analyzed. Publishing rigorous scientific research involves a full description of the methods, materials, procedures, and outcomes. Investigators may fail to provide a complete description of how their study was designed and executed because they may not know how to accurately report the information or the mechanisms are not in place to facilitate transparent reporting. Here, we provide an overview of how authors can write manuscripts in a transparent and thorough manner. We introduce a set of reporting criteria that can be used for publishing, including recommendations on reporting the experimental design and statistical approaches. We also discuss how to accurately visualize the results and provide recommendations for peer reviewers to enhance rigor and transparency. Incorporating transparency practices into research manuscripts will significantly improve the reproducibility of the results by independent laboratories.

Functional Neuronal Topography: A Statistical Approach to Micro Mapping Neuronal Location.

In order to understand the relationship between neuronal organization and behavior, precise methods that identify and quantify functional cellular ensembles are required. This is especially true in the quest to understand the mechanisms of memory. Brain structures involved in memory formation and storage, as well as the molecular determinates of memory are well-known, however, the microanatomy of functional neuronal networks remain largely unidentified. We developed a novel approach to statistically map molecular markers in neuronal networks through quantitative topographic measurement. Brain nuclei and their subdivisions are well-defined - our approach allows for the identification of new functional micro-regions within established subdivisions. A set of analytic methods relevant for measurement of discrete neuronal data across a diverse range of brain subdivisions are presented. We provide a methodology for the measurement and quantitative comparison of functional micro-neural network activity based on immunohistochemical markers matched across individual brains using micro-binning and heat mapping within brain sub-nuclei. These techniques were applied to the measurement of different memory traces, allowing for greater understanding of the functional encoding within sub-nuclei and its behavior mediated change. These approaches can be used to understand other functional and behavioral questions, including sub-circuit organization, normal memory function and the complexities of pathology. Precise micro-mapping of functional neuronal topography provides essential data to decode network activity underlying behavior.

Dorsolateral Striatum Engagement Interferes with Early Discrimination Learning.

In current models, learning the relationship between environmental stimuli and the outcomes of actions involves both stimulus-driven and goal-directed systems, mediated in part by the DLS and DMS, respectively. However, though these models emphasize the importance of the DLS in governing actions after extensive experience has accumulated, there is growing evidence of DLS engagement from the onset of training. Here, we used in vivo photosilencing to reveal that DLS recruitment interferes with early touchscreen discrimination learning. We also show that the direct output pathway of the DLS is preferentially recruited and causally involved in early learning and find that silencing the normal contribution of the DLS produces plasticity-related alterations in a PL-DMS circuit. These data provide further evidence suggesting that the DLS is recruited in the construction of stimulus-elicited actions that ultimately automate behavior and liberate cognitive resources for other demands, but with a cost to performance at the outset of learning.

Nicotine-induced and D1-receptor-dependent dendritic remodeling in a subset of dorsolateral striatum medium spiny neurons.

Nicotine is one of the most addictive substances known, targeting multiple memory systems, including the ventral and dorsal striatum. One form of neuroplasticity commonly associated with nicotine is dendrite remodeling. Nicotine-induced dendritic remodeling of ventral striatal medium spiny neurons (MSNs) is well-documented. Whether MSN dendrites in the dorsal striatum undergo a similar pattern of nicotine-induced structural remodeling is unknown. A morphometric analysis of Golgi-stained MSNs in rat revealed a natural asymmetry in dendritic morphology across the mediolateral axis, with larger, more complex MSNs found in the dorsolateral striatum (DLS). Chronic nicotine produced a lasting (at least 21day) expansion in the dendritic complexity of MSNs in the DLS, but not dorsomedial striatum (DMS). Given prior evidence that MSN subtypes can be distinguished based on dendritic morphology, MSNs were segregated into morphological subpopulations based on the number of primary dendrites. Analysis of these subpopulations revealed that DLS MSNs with more primary dendrites were selectively remodeled by chronic nicotine exposure and remodeling was specific to the distal-most portions of the dendritic arbor. Co-administration of the dopamine D1 receptor (D1R) antagonist SCH23390 completely reversed the selective effects of nicotine on DLS MSN dendrite morphology, supporting a causal role for dopamine signaling at D1 receptors in nicotine-induced dendrite restructuring. Considering the functional importance of the DLS in shaping and expressing habitual behavior, these data support a model in which nicotine induces persistent and selective changes in the circuit connectivity of the DLS that may promote and sustain addiction-related behavior.

Alcohol exposure during adolescence impairs auditory fear conditioning in adult Long-Evans rats.

Few studies have examined long-term effects of ethanol on auditory fear conditioning, and fewer still have examined whether adolescence represents a unique period of vulnerability. We investigated the impact of ethanol consumption during adolescence and adulthood on fear conditioning, following an extended abstinence period. Male and female Long-Evans rats (N = 80) consumed 10% ethanol or water (control) in a limited-access drinking paradigm (1 h) between postnatal (P) days 28-45 (adolescent) and P80-97 (adult). After the abstinence period (30 days), ethanol and control groups were assessed on the auditory fear-conditioning task. Alcohol consumption impaired tone conditioning in the male and female adolescent group. There were no persisting effects of adult dosing. In addition, adolescent rats consumed more ethanol than adults. These data provide evidence that ethanol consumption during adolescence produces enduring effects on auditory fear conditioning. The age-specific effect of ethanol may be attributable to an interplay of higher ethanol intake and the unique neurobiological characteristics of adolescents.