Carolyn Graybeal

Strain Differences in Stress Responsivity Are Associated with Divergent Amygdala Gene Expression and Glutamate-Mediated Neuronal Excitability

Stress is a major risk factor for numerous neuropsychiatric diseases. However, susceptibility to stress and the qualitative nature of stress effects on behavior differ markedly among individuals. This is partly because of the moderating influence of genetic factors. Inbred mouse strains provide a relatively stable and restricted range of genetic and environmental variability that is valuable for disentangling gene–stress interactions. Here, we screened a panel of inbred strains for anxiety- and depression-related phenotypes at baseline (trait) and after exposure to repeated restraint. Two strains, DBA/2J and C57BL/6J, differed in trait and restraint-induced anxiety-related behavior (dark/light exploration, elevated plus maze). Gene expression analysis of amygdala, medial prefrontal cortex, and hippocampus revealed divergent expression in DBA/2J and C57BL/6J both at baseline and after repeated restraint. Restraint produced strain-dependent expression alterations in various genes including glutamate receptors (e.g., Grin1, Grik1). To elucidate neuronal correlates of these strain differences, we performed ex vivo analysis of glutamate excitatory neurotransmission in amygdala principal neurons. Repeated restraint augmented amygdala excitatory postsynaptic signaling and altered metaplasticity (temporal summation of NMDA receptor currents) in DBA/2J but not C57BL/6J. Furthermore, we found that the C57BL/6J-like changes in anxiety-related behavior after restraint were absent in null mutants lacking the modulatory NMDA receptor subunit Grin2a, but not the AMPA receptor subunit Gria1. Grin2a null mutants exhibited significant (∼30%) loss of dendritic spines on amygdala principal neurons under nonrestraint conditions. Collectively, our data support a model in which genetic variation in glutamatergic neuroplasticity in corticolimbic circuitry underlies phenotypic variation in responsivity to stress.

Paradoxical reversal learning enhancement by stress or prefrontal cortical damage: rescue with BDNF

Stress affects various forms of cognition. We found that moderate stress enhanced late reversal learning in a mouse touchscreen-based choice task. Ventromedial prefrontal cortex (vmPFC) lesions mimicked the effect of stress, whereas orbitofrontal and dorsolateral striatal lesions impaired reversal. Stress facilitation of reversal was prevented by BDNF infusion into the vmPFC. These findings suggest a mechanism by which stress-induced vmPFC dysfunction disinhibits learning by alternate (for example, striatal) systems.

GluN2B in corticostriatal circuits governs choice learning and choice shifting

A choice that reliably produces a preferred outcome can be automated to liberate cognitive resources for other tasks. Should an outcome become less desirable, behavior must adapt in parallel or it becomes perseverative. Corticostriatal systems are known to mediate choice learning and flexibility, but the molecular mechanisms of these processes are not well understood. We integrated mouse behavioral, immunocytochemical, in vivo electrophysiological, genetic and pharmacological approaches to study choice. We found that the dorsal striatum (DS) was increasingly activated with choice learning, whereas reversal of learned choice engaged prefrontal regions. In vivo, DS neurons showed activity associated with reward anticipation and receipt that emerged with learning and relearning. Corticostriatal or striatal deletion of Grin2b (encoding the NMDA-type glutamate receptor subunit GluN2B) or DS-restricted GluN2B antagonism impaired choice learning, whereas cortical Grin2b deletion or OFC GluN2B antagonism impaired shifting. Our convergent data demonstrate how corticostriatal GluN2B circuits govern the ability to learn and shift choice behavior.

Genetic Strain Differences in Learned Fear Inhibition Associated with Variation in Neuroendocrine, Autonomic, and Amygdala Dendritic Phenotypes

Mood and anxiety disorders develop in some but not all individuals following exposure to stress and psychological trauma. However, the factors underlying individual differences in risk and resilience for these disorders, including genetic variation, remain to be determined. Isogenic inbred mouse strains provide a valuable approach to elucidating these factors. Here, we performed a comprehensive examination of the extinction-impaired 129S1/SvImJ (S1) inbred mouse strain for multiple behavioral, autonomic, neuroendocrine, and corticolimbic neuronal morphology phenotypes. We found that S1 exhibited fear overgeneralization to ambiguous contexts and cues, impaired context extinction and impaired safety learning, relative to the (good-extinguishing) C57BL/6J (B6) strain. Fear overgeneralization and impaired extinction was rescued by treatment with the front-line anxiety medication fluoxetine. Telemetric measurement of electrocardiogram signals demonstrated autonomic disturbances in S1 including poor recovery of fear-induced suppression of heart rate variability. S1 with a history of chronic restraint stress displayed an attenuated corticosterone (CORT) response to a novel, swim stressor. Conversely, previously stress-naive S1 showed exaggerated CORT responses to acute restraint stress or extinction training, insensitivity to dexamethasone challenge, and reduced hippocampal CA3 glucocorticoid receptor mRNA, suggesting downregulation of negative feedback control of the hypothalamic–pituitary–adrenal axis. Analysis of neuronal morphology in key neural nodes within the fear and extinction circuit revealed enlarged dendritic arbors in basolateral amygdala neurons in S1, but normal infralimbic cortex and prelimbic cortex dendritic arborization. Collectively, these data provide convergent support for the utility of the S1 strain as a tractable model for elucidating the neural, molecular and genetic basis of persistent, excessive fear.

Does gene deletion of AMPA GluA1 phenocopy features of schizoaffective disorder

Glutamatergic dysfunction is strongly implicated in schizophrenia and mood disorders. GluA1 knockout (KO) mice display schizophrenia- and depression-related abnormalities. Here, we asked whether GluA1 KO show mania-related abnormalities. KO were tested for behavior in approach/avoid conflict tests, responses to repeated forced swim exposure, and locomotor responses under stress and after psychostimulant treatment. The effects of rapid dopamine depletion and treatment with lithium or GSK-3β inhibitor on KO locomotor hyperactivity were tested. Results showed that KO exhibited novelty- and stress-induced locomotor hyperactivity, reduced forced swim immobility and alterations in approach/avoid conflict tests. Psychostimulant treatment and dopamine depletion exacerbated KO locomotor hyperactivity. Lithium, but not GSK-3β inhibitor, treatment normalized KO anxiety-related behavior and partially reversed hyperlocomotor behavior, and also reversed elevated prefrontal cortex levels of phospho-MARCKS and phospho-neuromodulin. Collectively, these findings demonstrate mania-related abnormalities in GluA1 KO and, combined with previous findings, suggest this mutant may provide a novel model of features of schizoaffective disorder.

Do GluA1 knockout mice exhibit behavioral abnormalities relevant to the negative or cognitive symptoms of schizophrenia and schizoaffective disorder

The glutamate system has been strongly implicated in the pathophysiology of psychotic illnesses, including schizophrenia and schizoaffective disorder. We recently found that knockout (KO) mice lacking the AMPA GluA1 subunit displayed behavioral abnormalities relevant to some of the positive symptoms of these disorders. Here we phenotyped GluA1 KO mice for behavioral phenotypes pertinent to negative and cognitive/executive symptoms. GluA1 KO mice were tested for conspecific social interactions, the acquisition and extinction of an operant response for food-reward, operant-based pairwise visual discrimination and reversal learning, and impulsive choice in a delay-based cost/benefit decision-making T-maze task. Results showed that GluA1 KO mice engaged in less social interaction than wildtype (WT) controls when tested in a non-habituated, novel environment, but, conversely, displayed more social interaction in a well habituated, familiar environment. GluA1 KO mice were faster to acquire an operant stimulus-response for food reward than WT and were subsequently slower to extinguish the response. Genotypes showed similar pairwise discrimination learning and reversal, although GluA1 KO mice made fewer errors during early reversal. GluA1 KO mice also displayed increased impulsive choice, being less inclined to choose a delayed, larger reward when given a choice between this and a smaller, immediate reward, compared to WT mice. Finally, sucrose preference did not differ between genotypes. Collectively, these data add to the growing evidence that GluA1 KO mice display at least some phenotypic abnormalities mimicking those found in schizophrenia/schizoaffective disorder. Although these mice, like any other single mutant line, are unlikely to model the entire disease, they may nevertheless provide a useful tool for studying the role of GluA1 in certain aspects of the pathophysiology of major psychotic illness.

Prefrontal single-unit firing associated with deficient extinction in mice

The neural circuitry mediating fear extinction has been increasingly well studied and delineated. The rodent infralimbic subregion (IL) of the ventromedial prefrontal cortex (vmPFC) has been found to promote extinction, whereas the prelimbic cortex (PL) demonstrates an opposing, pro-fear, function. Studies employing in vivo electrophysiological recordings have observed that while increased IL single-unit firing and bursting predicts robust extinction retrieval, increased PL firing can correlate with sustained fear and poor extinction. These relationships between single-unit firing and extinction do not hold under all experimental conditions, however. In the current study, we further investigated the relationship between vmPFC and PL single-unit firing and extinction using inbred mouse models of intact (C57BL/6J, B6) and deficient (129S1/SvImJ, S1) extinction strains. Simultaneous single-unit recordings were made in the PL and vmPFC (encompassing IL) as B6 and S1 mice performed extinction training and retrieval. Impaired extinction retrieval in S1 mice was associated with elevated PL single-unit firing, as compared to firing in extinguishing B6 mice, consistent with the hypothesized pro-fear contribution of PL. Analysis of local field potentials also revealed significantly higher gamma power in the PL of S1 than B6 mice during extinction training and retrieval. In the vmPFC, impaired extinction in S1 mice was also associated with exaggerated single-unit firing, relative to B6 mice. This is in apparent contradiction to evidence that IL activity promotes extinction, but could reflect a (failed) compensatory effort by the vmPFC to mitigate fear-promoting activity in other regions, such as the PL or amygdala. In support of this hypothesis, augmenting IL activity via direct infusion of the GABAA receptor antagonist picrotoxin rescued impaired extinction retrieval in S1 mice. Chronic fluoxetine treatment produced modest reductions in fear during extinction retrieval and increased the number of Zif268-labeled cells in layer II of IL, but failed to increase vmPFC single-unit firing. Collectively, these findings further support the important contribution these cortical regions play in determining the balance between robust extinction on the one hand, and sustained fear on the other. Elucidating the precise nature of these roles could help inform understanding of the pathophysiology of fear-related anxiety disorders.

Durable fear memories require PSD-95.

Traumatic fear memories are highly durable but also dynamic, undergoing repeated reactivation and rehearsal over time. Although overly persistent fear memories underlie anxiety disorders, such as posttraumatic stress disorder, the key neural and molecular mechanisms underlying fear memory durability remain unclear. Postsynaptic density 95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability and certain types of memory. Using a loss-of-function mutant mouse lacking the guanylate kinase domain of PSD-95 (PSD-95GK), we analyzed the contribution of PSD-95 to fear memory formation and retrieval, and sought to identify the neural basis of PSD-95-mediated memory maintenance using ex vivo immediate-early gene mapping, in vivo neuronal recordings and viral-mediated knockdown (KD) approaches. We show that PSD-95 is dispensable for the formation and expression of recent fear memories, but essential for the formation of precise and flexible fear memories and for the maintenance of memories at remote time points. The failure of PSD-95GK mice to retrieve remote cued fear memory was associated with hypoactivation of the infralimbic (IL) cortex (but not the anterior cingulate cortex (ACC) or prelimbic cortex), reduced IL single-unit firing and bursting, and attenuated IL gamma and theta oscillations. Adeno-associated virus-mediated PSD-95 KD in the IL, but not the ACC, was sufficient to impair recent fear extinction and remote fear memory, and remodel IL dendritic spines. Collectively, these data identify PSD-95 in the IL as a critical mechanism supporting the durability of fear memories over time. These preclinical findings have implications for developing novel approaches to treating trauma-based anxiety disorders that target the weakening of overly persistent fear memories.

Predictably Irrational: Assaying Cognitive Inflexibility in Mouse Models of Schizophrenia

The development of sophisticated, translatable mouse-based assays modeling the behavioral manifestations of neuropsychiatric diseases, such as schizophrenia, has lagged the advances in molecular and genomic techniques. Our laboratory has made efforts to fill this gap by investing in the development of novel assays, including adapting a touchscreen-based method for measuring cognitive and executive functions for use in mice. As part of these efforts, a recent study by Brigman et al. (2009) investigated the effects of subchronic phencyclidine treatment on mouse touchscreen-based pairwise visual discrimination and reversal learning. Here, we summarize the results of that study, and place them in the larger context of ongoing efforts to develop valid mouse “models” of schizophrenia, with a focus on reversal learning and other measures of cognitive flexibility. Touchscreen-based systems could provide a tractable platform for fully utilizing the mouse to elucidate the pathophysiology of cognitive inflexibility in schizophrenia and other neuropsychiatric disorders.

Fear memory impairing effects of systemic treatment with the NMDA NR2B subunit antagonist, Ro 25-6981, in mice: Attenuation with ageing

N-methyl-D-aspartate receptors (NMDARs) are mediators of synaptic plasticity and learning and are implicated in the pathophysiology of neuropsychiatric disease and age-related cognitive dysfunction. NMDARs are heteromers, but the relative contribution of specific subunits to NMDAR-mediated learning is not fully understood. We characterized pre-conditioning systemic treatment of the NR2B subunit-selective antagonist Ro 25-6981 for effects on multi-trial, one-trial and low-shock Pavlovian fear conditioning in C57BL/6J mice. Ro 25-6981 was also profiled for effects on novel open field exploration, elevated plus-maze anxiety-like behavior, startle reactivity, prepulse inhibition of startle, and nociception. Three-month (adult) and 12-month old C57BL/6Tac mice were compared for Ro 25-6981 effects on multi-trial fear conditioning, and corticolimbic NR2B protein levels. Ro 25-6981 moderately impaired fear learning in the multi-trial and one-trial (but not low-shock) conditioning paradigms, but did not affect exploratory or anxiety-related behaviors or sensory functions. Memory impairing effects of Ro 25-6981 were absent in 12-month old mice, although NR2B protein levels were not significantly altered. Present data provide further evidence of the memory impairing effects of selective blockade of NR2B-containing NMDARs, and show loss of these effects with ageing. This work could ultimately have implications for elucidating the pathophysiology of learning dysfunction in neuropsychiatric disorders and ageing.