Altered Forebrain Functional Connectivity and Neurotransmission in a Kinase-Inactive Met Mouse Model of Autism.

MET, the gene encoding the tyrosine kinase receptor for hepatocyte growth factor, is a susceptibility gene for autism spectrum disorder (ASD). Genetically altered mice with a kinase-inactive Met offer a potential model for understanding neural circuit organization changes in autism. Here, we focus on the somatosensory thalamocortical circuitry because distinct somatosensory sensitivity phenotypes accompany ASD, and this system plays a major role in sensorimotor and social behaviors in mice. We employed resting-state functional magnetic resonance imaging and in vivo high-resolution proton MR spectroscopy to examine neuronal connectivity and neurotransmission of wild-type, heterozygous Met-Emx1, and fully inactive homozygous Met-Emx1 mice. Met-Emx1 brains showed impaired maturation of large-scale somatosensory network connectivity when compared with wild-type controls. Significant sex × genotype interaction in both network features and glutamate/gamma-aminobutyric acid (GABA) balance was observed. Female Met-Emx1 brains showed significant connectivity and glutamate/GABA balance changes in the somatosensory thalamocortical system when compared with wild-type brains. The glutamate/GABA ratio in the thalamus was correlated with the connectivity between the somatosensory cortex and the thalamus in heterozygous Met-Emx1 female brains. The findings support the hypothesis that aberrant functioning of the somatosensory thalamocortical system is at the core of the conspicuous somatosensory behavioral phenotypes observed in Met-Emx1 mice.

Insulin-Independent GABAA Receptor-Mediated Response in the Barrel Cortex of Mice with Impaired Met Activity.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder caused by genetic variants, susceptibility alleles, and environmental perturbations. The autism associated geneMETtyrosine kinase has been implicated in many behavioral domains and endophenotypes of autism, including abnormal neural signaling in human sensory cortex. We investigated somatosensory thalamocortical synaptic communication in mice deficient in Met activity in cortical excitatory neurons to gain insights into aberrant somatosensation characteristic of ASD. The ratio of excitation to inhibition is dramatically increased due to decreased postsynaptic GABAAreceptor-mediated inhibition in the trigeminal thalamocortical pathway of mice lacking active Met in the cerebral cortex. Furthermore, in contrast to wild-type mice, insulin failed to increase GABAAreceptor-mediated response in the barrel cortex of mice with compromised Met signaling. Thus, lacking insulin effects may be a risk factor in ASD pathogenesis. SIGNIFICANCE STATEMENT: A proposed common cause of neurodevelopmental disorders is an imbalance in excitatory neural transmission, provided by the glutamatergic neurons, and the inhibitory signals from the GABAergic interneurons. Many genes associated with autism spectrum disorders impair synaptic transmission in the expected cell type. Previously, inactivation of the autism-associated Met tyrosine kinase receptor in GABAergic interneurons led to decreased inhibition. In thus report, decreased Met signaling in glutamatergic neurons had no effect on excitation, but decimated inhibition. Further experiments indicate that loss of Met activity downregulates GABAAreceptors on glutamatergic neurons in an insulin independent manner. These data provide a new mechanism for the loss of inhibition and subsequent abnormal excitation/inhibition balance and potential molecular candidates for treatment or prevention.

Enhancement of human neural stem cell self-renewal in 3D hypoxic culture.

The pathology of neurological disorders is associated with the loss of neuronal and glial cells that results in functional impairments. Human neural stem cells (hNSCs), due to their self-renewing and multipotent characteristics, possess enormous tissue-specific regenerative potential. However, the efficacy of clinical applications is restricted due to the lack of standardized in vitro cell production methods with the capability of generating hNSC populations with well-defined cellular compositions. At any point, a population of hNSCs may include undifferentiated stem cells, intermediate and terminally differentiated progenies, and dead cells. Due to the plasticity of hNSCs, environmental cues play crucial roles in determining the cellular composition of hNSC cultures over time. Here, we investigated the independent and synergistic effect of three important environmental factors (i.e., culture dimensionality, oxygen concentration, and growth factors) on the survival, renewal potential, and differentiation of hNSCs. Our experimental design included two dimensional (2D) versus three dimensional (3D) cultures and normoxic (21% O2 ) versus hypoxic (3% O2 ) conditions in the presence and absence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Additionally, we discuss the feasibility of mathematical models that predict hNSC growth and differentiation under these culture conditions by adopting a negative feedback regulatory term. Our results indicate that the synergistic effect of culture dimensionality and hypoxic oxygen concentration in the presence of growth factors enhances the proliferation of viable, undifferentiated hNSCs. Moreover, the same synergistic effect in the absence of growth factors promotes the differentiation of hNSCs. Biotechnol. Bioeng. 2017;114: 1096-1106. © 2016 Wiley Periodicals, Inc.

Astrocyte-mediated hepatocyte growth factor/scatter factor supplementation restores GABAergic interneurons and corrects reversal learning deficits in mice.

Many psychiatric and neurological disorders present persistent neuroanatomical abnormalities in multiple brain regions that may reflect a common origin for a developmental disturbance. In mammals, many of the local GABAergic inhibitory interneurons arise from a single subcortical source. Perturbations in the ontogeny of the GABAergic interneurons may be reflected in the adult by interneuron deficits in both frontal cerebral cortical and striatal regions. Disrupted GABAergic circuitry has been reported in patients with schizophrenia and frontal lobe epilepsy and may contribute to their associated impairments in behavioral flexibility. The present study demonstrates that one type of behavioral flexibility, reversal learning, is dependent upon proper numbers of GABAergic interneurons. Mice with abnormal interneuron ontogeny have reduced numbers of parvalbumin-expressing GABAergic local interneurons in the orbitofrontal cortical and striatal regions and impaired reversal leaning. Using a genetic approach, both the anatomical and functional deficiencies are restored with exogenous postnatal growth factor supplementation. These results show that GABAergic local circuitry is critical for modulating behavioral flexibility and that birth defects can be corrected by replenishing crucial growth factors.

Double dissociation of the effects of medial and orbital prefrontal cortical lesions on attentional and affective shifts in mice.

Many neuropsychiatric diseases are associated with cognitive rigidity linked to prefrontal dysfunction. For example, schizophrenia and Parkinson's disease are associated with performance deficits on the Wisconsin Card Sorting Test, which evaluates attentional set shifting. Although the genetic underpinnings of these disorders can be reproduced in mice, there are few models for testing the functional consequences. Here, we demonstrate that an analog of the Wisconsin Card Sorting Test, developed in marmosets and recently adapted to rats, is a behavioral model of prefrontal function in mice. Systematic analysis demonstrated that formation of the attentional set in mice is dependent on the number of problem sets. We found that mice, like rats and primates, exhibit both affective and attentional sets, and these functions are disrupted by neurotoxic damage to orbitofrontal and medial prefrontal cortical areas, respectively. These data are identical to studies in rats and similar to the deficits reported after prefrontal damage in a comparable task in marmosets. These results provide a behavioral model to assess prefrontal function in mice.

Loss of skills and onset patterns in neurodevelopmental disorders: Understanding the neurobiological mechanisms.

Patterns of onset in Autism Spectrum Disorder, including a pattern that includes loss of previously acquired skills, have been identified since the first reports of the disorder. However, attempts to study such "regression" have been limited to clinical studies, that until recently mostly involved retrospective reports. The current report reflects discussion that occurred at an NIMH convened meeting in 2016 with the purpose of bridging clinical autism research with basic and translational work in this area. This summary describes the state of the field with respect to clinical studies, describing gaps in knowledge based on limited methods and prospective data collected. Biological mechanisms that have been shown to account for regression early in development in specific conditions are discussed, as well as potential mechanisms that have not yet been explored. Suggestions include use of model systems during the developmental period and cutting-edge methods, including non-invasive imaging that may afford opportunities for a better understanding of the neurobiological pathways that result in loss of previously-attained skills. Autism Res 2018, 11: 212-222. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Loss of previously acquired skills, or regression, has been reported in Autism Spectrum Disorder since Kanner's reports in the 1950's. The current report reflects discussion from an NIMH convened meeting in 2016 with the purpose of bridging clinical autism research with basic and translational work in this area. This summary describes the state of the field regarding clinical studies and suggests use of model systems during the developmental period and cutting-edge methods, for a better understanding of the neurobiological pathways that result in loss of previously-attained skills.

Dominant-Negative DISC1 Alters the Dopaminergic Modulation of Inhibitory Interneurons in the Mouse Prefrontal Cortex.

A truncated disrupted in schizophrenia 1 (Disc1) gene increases the risk of psychiatric disorders, probably affecting cortical interneurons. Here, we sought to determine whether this cell population is affected in mice carrying a truncated (Disc1) allele (DN-DISC1). We utilized whole cell recordings to assess electrophysiological properties and modulation by dopamine (DA) in two classes of interneurons: fast-spiking (FS) and low threshold-spiking (LTS) interneurons in wild-type and DN-DISC1 mice. In DN-DISC1 mice, FS interneurons, but not LTS interneurons, exhibited altered action potentials. Further, the perineuronal nets that surround FS interneurons exhibited abnormal morphology in DN-DISC1 mice, and the DA modulation of this cell type was altered in DN-DISC1 mice. We conclude that early-life manipulation of a gene associated with risk of psychiatric disease can result in dysfunction, but not loss, of specific GABAergic interneurons. The resulting alteration of excitatory-inhibitory balance is a critical element in DISC1 pathophysiology.

Regulation of neocortical interneuron development and the implications for neurodevelopmental disorders.

Neurodevelopmental disorders typically have complex endophenotypes, which can include abnormalities in neuronal excitability, processing of complex information, as well as behaviors such as anxiety and social interactions. Converging experimental and clinical evidence suggests that altered interneuron development may underlie part of the pathophysiological process of such disorders. Consistent with this, mice with abnormal hepatocyte growth factor signaling exhibit disturbances in the development of specific interneuron subclasses that are paralleled by seizure activity and a complex behavioral phenotype. Mutations in molecules that regulate different aspects of interneuron development could provide the heterogeneity in genetic susceptibility that, when combined with environmental disturbances, results in a phenotypic spectrum that serves as the hallmark pathophysiology for autism, mental retardation, schizophrenia and other neurodevelopmental disorders.

Three-Dimensional Environment Sustains Morphological Heterogeneity and Promotes Phenotypic Progression During Astrocyte Development.

Astrocytes are critical for coordinating normal brain function by regulating brain metabolic homeostasis, synaptogenesis and neurotransmission, and blood-brain barrier permeability and maintenance. Dysregulation of normal astrocyte ontogeny contributes to neurodevelopmental and neurodegenerative disorders, epilepsies, and adverse responses to injury. To achieve these multiple essential roles, astrocyte phenotypes are regionally, morphologically, and functionally heterogeneous. Therefore, the best regenerative medicine strategies may require selective production of distinct astrocyte subpopulations at defined maturation levels. However, little is known about the mechanisms that direct astrocyte diversity or whether heterogeneity is represented in biomaterials. In vitro studies report lack of normal morphologies and overrepresentation of the glial scar type of reactive astrocyte morphology and expression of markers, questioning how well the in vitro astrocytes represent glia in vivo and whether in vitro tissue engineering methods are suitable for regenerative medicine applications. Our previous work with neurons suggests that the three-dimensional (3D) environment, when compared with standard two-dimensional (2D) substrate, yields cellular and molecular behaviors that more closely approximately normal ontogeny. To specifically study the effects of dimensionality, we used purified glial fibrillary acidic protein (GFAP)-expressing primary cerebral cortical astrocyte cultures from single pups and characterized the cellular maturation profiles in 2D and 3D milieu. We identified four morphological groups in vitro: round, bipolar, stellate, and putative perivascular. In the 3D hydrogel culture environment, postnatal astrocytes transitioned from a population of nearly all round cells and very few bipolar cells toward a population with significant fractions of round, stellate, and putative perivascular cells within a few days, following the in vivo ontogeny. In 2D, however, the population shift from round and bipolar to stellate and perivascular was rarely observed. The transition to distinct cellular morphologies in 3D corresponded to the in vivo expression of phenotypic markers, supporting the generation of mature heterogeneous glial populations in vitro. This study presents quantitative data supporting that 3D culture is critical for sustaining the heterogeneity of astrocytes in vitro and for generating a representation of the in vivo portfolio of heterogeneous populations of astrocytes required for therapeutic interventions in neurodevelopmental disorders, epilepsy, and brain injury.

Genetic disruption of cortical interneuron development causes region- and GABA cell type-specific deficits, epilepsy, and behavioral dysfunction.

The generation of properly functioning circuits during brain development requires precise timing of cell migration and differentiation. Disruptions in the developmental plan may lead to neurological and psychiatric disorders. Neocortical circuits rely on inhibitory GABAergic interneurons, the majority of which migrate from subcortical sources. We have shown that the pleiotropic molecule hepatocyte growth factor/scatter factor (HGF/SF) mediates interneuron migration. Mice with a targeted mutation of the gene encoding urokinase plasminogen activator receptor (uPAR), a key component in HGF/SF activation and function, have decreased levels of HGF/SF and a 50% reduction in neocortical GABAergic interneurons at embryonic and perinatal ages. Disruption of interneuron development leads to early lethality in most models. Thus, the long-term consequences of such perturbations are unknown. Mice of the uPAR-/- strain survive until adulthood, and behavior testing demonstrates that they have an increased anxiety state. The uPAR-/- strain also exhibits spontaneous seizure activity and higher susceptibility to pharmacologically induced convulsions. The neocortex of the adult uPAR-/- mouse exhibits a dramatic region- and subtype-specific decrease in GABA-immunoreactive interneurons. Anterior cingulate and parietal cortical areas contain 50% fewer GABAergic interneurons compared with wild-type littermates. However, interneuron numbers in piriform and visual cortical areas do not differ from those of normal mice. Characterization of interneuron subpopulations reveals a near complete loss of the parvalbumin subtype, with other subclasses remaining intact. These data demonstrate that a single gene mutation can selectively alter the development of cortical interneurons in a region- and cell subtype-specific manner, with deficits leading to long-lasting changes in circuit organization and behavior.

Interneurons are necessary for coordinated activity during reversal learning in orbitofrontal cortex.

BACKGROUND: Cerebral cortical gamma-aminobutyric acidergic interneuron dysfunction is hypothesized to lead to cognitive deficits comorbid with human neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. We have previously shown that mice that harbor mutations in the Plaur gene, which is associated with schizophrenia, have deficits in frontal cortical parvalbumin-expressing interneurons. Plaur mice have impaired reversal learning, similar to deficits observed in patients with schizophrenia. METHODS: We examined the role of parvalbumin interneurons in orbitofrontal cortex during reversal learning by recording single unit activity from 180 control and 224 Plaur mouse neurons during a serial reversal task. Neural activity was analyzed during correct and incorrect decision choices and reward receipt. RESULTS: Neurons in control mice exhibited strong phasic responses both during discrimination and reversal learning to decisions and rewards, and the strength of the response was correlated with behavioral performance. Although baseline firing was significantly enhanced in Plaur mice, neural selectivity for correct or erroneous decisions was diminished and not correlated with behavior, and reward encoding was downscaled. In addition, Plaur mice showed a significant reduction in the number of neurons that encoded expected outcomes across task phases during the decision period. CONCLUSIONS: These data indicate that parvalbumin interneurons are necessary for the representation of outcomes in orbitofrontal cortex. Deficits in inhibition blunt selective neural firing during key decisions, contributing to behavioral inflexibility. These data provide a potential explanation for disorders of cognitive control that accompany the loss of these gamma-aminobutyric acidergic interneurons in human neuropsychiatric disorders, such as autism, epilepsy, and schizophrenia.

Growth and developmental body composition of the cebus monkey (Cebus albifrons).

The carcasses of 37 Cebus albifrons, Colombia (19 male, 17 female, 1 unknown) with ages ranging from premature stillborn to 8 yr, were analyzed for body composition. The absolute content of water, protein, fat, and ash were determined by standard techniques and were analyzed as functions of carcass weight and age. The weight of the carcass was directly proportional to the whole body weight over the entire range of weights studied. All parameters but fat were linearly related to carcass weight; the relationships of protein and water to age were best described by exponential equations, whereas that of ash to age was linear. Variability in the fat content of the carcass precluded the fitting of predictive equations on the basis of either weight or age. Analysis of the relative (percent) composition of both the whole and fat-free carcass provided exponential equations to describe the pattern of development of protein, water, and their ratio. Using these mathematical models, it was calculated that chemical maturity, with regard to water and protein, probably occurred by 8 wk of age. Percent ash composition of whole carcass and fat-free carcass was described by linear equations. Longitudinal growth data from 89 male and 76 female C. albifrons, born and reared in the departmental breeding colony, were obtained over 12 yr. An exponential equation relating body weight to age (R2 = 0.999) described the patern of growth for the first 2 yr of life; thereafter, the pattern was more varied as the animals approached and reached sexual maturity.

Prefrontal cognitive deficits in mice with altered cerebral cortical GABAergic interneurons.

Alterations of inhibitory GABAergic neurons are implicated in multiple psychiatric and neurological disorders, including schizophrenia, autism and epilepsy. In particular, interneuron deficits in prefrontal areas, along with presumed decreased inhibition, have been reported in several human patients. The majority of forebrain GABAergic interneurons arise from a single subcortical source before migrating to their final regional destination. Factors that govern the interneuron populations have been identified, demonstrating that a single gene mutation may globally affect forebrain structures or a single area. In particular, mice lacking the urokinase plasminogen activator receptor (Plaur) gene have decreased GABAergic interneurons in frontal and parietal, but not caudal, cortical regions. Plaur assists in the activation of hepatocyte growth factor/scatter factor (HGF/SF), and several of the interneuron deficits are correlated with decreased levels of HGF/SF. In some cortical regions, the interneuron deficit can be remediated by endogenous overexpression of HGF/SF. In this study, we demonstrate decreased parvalbumin-expressing interneurons in the medial frontal cortex, but not in the hippocampus or basal lateral amygdala in the Plaur null mouse. The Plaur null mouse demonstrates impaired medial frontal cortical function in extinction of cued fear conditioning and the inability to form attentional sets. Endogenous HGF/SF overexpression increased the number of PV-expressing cells in medial frontal cortical areas to levels greater than found in wildtype mice, but did not remediate the behavioral deficits. These data suggest that proper medial frontal cortical function is dependent upon optimum levels of inhibition and that a deficit or excess of interneuron numbers impairs normal cognition.

Neural ECM and epilepsy.

Currently, there are about 20 antiepileptic drugs on market. Still, seizures in about 30% of patients with epilepsy are not adequately controlled, or the drugs cause quality-of-life-compromising adverse events. Importantly, there are no treatments to combat epileptogenesis, a process that leads to the development of epilepsy and its progression. To fill the gaps in the treatment of epilepsy, there is an urgent need for identification of novel treatment targets. Data emerging over the recent years have shown that different components of the extracellular matrix (ECM) contribute to many components of tissue reorganization during epileptogenesis and the ECM is also a major regulator of synaptic excitability. Here, we review the role of urokinase-type plasminogen activator receptor interactome, matrix metalloproteinases, tenascin-R, and LGI1 in epileptogenesis and ictogenesis. Moreover, the role of the ECM in epilepsy-related comorbidities is reviewed. As there is active development of new imaging methods, we also summarize the data available on imaging of the ECM in epilepsy.

Interneuron development and epilepsy: early genetic defects cause long-term consequences in seizures and susceptibility.

Errors in the generation of the inhibitory GABAergic interneurons of the cerebral cortex and hippocampus have variable consequences. Studies of the molecular pathways of interneuron development reveal genes that are associated with human epilepsies. Animal models of gene variants exhibit seizures and abnormal electroencephalographic activity, providing unique models for discovering better treatments for individual forms of epilepsy.

Neural structures underlying set-shifting: roles of medial prefrontal cortex and anterior cingulate cortex.

Impaired attentional set-shifting and inflexible decision-making are problems frequently observed during normal aging and in several psychiatric disorders. To understand the neuropathophysiology of underlying inflexible behavior, animal models of attentional set-shifting have been developed to mimic tasks such as the Wisconsin Card Sorting Task (WCST), which tap into a number of cognitive functions including stimulus-response encoding, working memory, attention, error detection, and conflict resolution. Here, we review many of these tasks in several different species and speculate on how prefrontal cortex and anterior cingulate cortex might contribute to normal performance during set-shifting.

Reversal learning and attentional set-shifting in mice.

Schizophrenia is a complex developmental disorder that presents challenges to modern neuroscience in terms of discovering etiology and aiding in effective treatment of afflicted humans. One approach is to divide the constellation of symptoms of human neuropsychiatric disorders into discrete units for study. Multiple animal models are used to study brain ontogeny, response to psychoactive compounds, substrates of defined behaviors. Frontal cortical areas have been found to have abnormal anatomy and neurotransmitter levels in postmortem brains from schizophrenic patients. The mouse model has the advantage of rather straightforward genetic manipulation and offers numerous genetic variations within the same species. However, until recently, the behavioral analyses in the mice lagged behind the primate and rat, especially with respect to testing of frontal cortical regions. Current reports of mouse prefrontal anatomy and function advocate the mouse as a feasible animal model to study prefrontal cortical function. This review highlights the most recent developments from behavioral paradigms for testing orbital and medial prefrontal cortical function in pharmacological and genetic models of human schizophrenia.

Age dependent forebrain structural changes in mice deficient in the autism associated gene Met tyrosine kinase.

The MET tyrosine kinase has been identified as a susceptibility gene in patients with autism spectrum disorders. MET is expressed in the forebrain during prenatal and postnatal development. After birth, MET participates in dendritic outgrowth and circuit formation. Alterations in neuronal development, particularly in the cerebral cortex, may contribute to the pathology of developmental disorders, including autism. Patients with autism can exhibit abnormal cortical volumes and head circumferences. We tested the hypothesis that impaired Met signaling during development alters forebrain structure. We have utilized a conditional mutant mouse line which expresses a kinase-dead Met restricted to the cerebral cortex and hippocampal structures. In these mice, we have used magnetic resonance imaging (MRI) to analyze the structure of the cerebral cortex and related structures across postnatal development. We found that the rostral cortex, caudal hippocampus, dorsal striatum, thalamus, and corpus callosum were all larger in adult, but not juvenile, mutant mice relative to control mice. The specificity of the changes suggests that aberrant expansion of the forebrain is consistent with continued axonal and dendritic growth, potentially leading to improper circuit formation and maintenance.

Substrate three-dimensionality induces elemental morphological transformation of sensory neurons on a physiologic timescale.

The natural environment of a neuron is the three-dimensional (3D) tissue. In vivo, embryonic sensory neurons transiently express a bipolar morphology with two opposing neurites before undergoing cytoplasmic and cytoskeletal rearrangement to a more mature pseudo-unipolar axonal arbor before birth. The unipolar morphology is crucial in the adult for correct information transmission from the periphery to the central nervous system. On two-dimensional (2D) substrates this transformation is delayed significantly or absent. We report that a 3D culture platform can invoke the characteristic transformation to the unipolar axonal arbor within a time frame similar to in vivo, overcoming the loss of this essential milestone in 2D substrates. Additionally, 3D substrates alone provided an environment that promoted axonal branching features that reflect morphological patterns observed in vivo. We have also analyzed the involvement of soluble cues in these morphogenic processes by culturing the neurons in the presence and absence of nerve growth factor (NGF), a molecule that plays distinct roles in the development of the peripheral and central nervous systems. Without NGF, both 2D and 3D cultures had significant decreases in the relative population of unipolar neurons as well as shorter neurite lengths and fewer branch points compared to cultures with NGF. Interestingly, branching features of neurons cultured in 3D without NGF resemble those of neurons cultured in 2D with NGF. Therefore, neurons cultured in 3D without NGF lost the ability to differentiate into unipolar neurons, suggesting that this morphological hallmark requires not only presentation of soluble cues like NGF, but also the surrounding 3D presentation of adhesive ligands to allow for realization of the innate morphogenic program. We propose that in a 3D environment, various matrix and soluble cues are presented toward all surfaces of the cell; this optimized milieu allows neurons to elaborate their genuine phenotype and follow programmed instructions that are intrinsic to the neuron, but disrupted when cells were dissected from the embryo. Thus, this study presents quantitative data supporting that 3D substrates are critical for sustaining the in vivo ontogeny of neurons and deciphering signaling mechanisms necessary for designing biomaterial scaffolds for nerve generation and repair.

Examining the genetic and neural components of cognitive flexibility using mice.

This commentary summarizes the research presented during the symposium "Examining the genetic and neural components of cognitive flexibility using mice" at the annual meeting of the International Behavioral Neuroscience Society 2011. Research presented includes examining: 1) Corticostriatal networks underlying reversal learning using GluN2B knockout mice, cFos expression, and in vivo electrophysiological recording; 2) Cerebellar contribution to reversal learning using mutants with Purkinje cell loss and in vivo electrochemical recording; 3) Parvalbumin contribution to reversal learning and set-shifting using PLAUR mutants and in vitro recording to examine fast-spiking interneurones; and 4) Alpha 7 nAChR contribution to reversal learning, set-shifting, motivation, and the 'eureka moment' of rule acquisition. It is proposed that these studies revealed more about the neurobiology underlying these behaviors than could be discovered using pharmacological techniques alone. Together, the research presented stressed the importance of exploring the genetic contribution to neuropsychiatric disease and the important role that the mouse, coupled with robust behavioral measures, can play in understanding neurobiology underlying cognitive flexibility.