Anita L. Guedea

Fear-enhancing effects of septal oxytocin receptors

The nonapeptide oxytocin is considered beneficial to mental health due to its anxiolytic, prosocial and antistress effects, but evidence for anxiogenic actions of oxytocin in humans has recently emerged. Using region-specific manipulations of the mouse oxytocin receptor (Oxtr) gene (Oxtr), we identified the lateral septum as the brain region mediating fear-enhancing effects of Oxtr. These effects emerge after social defeat and require Oxtr specifically coupled to the extracellular signal–regulated protein kinase pathway.

NMDA receptors in retrosplenial cortex are necessary for retrieval of recent and remote context fear memory

Over time, memory retrieval is thought to transfer from the hippocampus to a distributed network of neocortical sites. Of these sites, the retrosplenial cortex (RSC) is robustly activated during retrieval of remotely acquired, emotionally valenced memories. It is unclear, however, whether RSC is specifically involved in memory storage or retrieval, and which neurotransmitter receptor mechanisms serve its function. We addressed these questions by inhibiting NMDARs in RSC via infusions of APV before tests for context fear in mice. Anterior cingulate cortex (ACC) and dorsal hippocampus (DH), which have been implicated in the retrieval of remote and recent memory, respectively, served as neuroanatomical controls. Surprisingly, infusion of APV only into RSC, but not ACC or DH, abolished retrieval of remote memory, as revealed by lack of freezing to the conditioning context. APV infused into RSC also impaired retrieval of recent memory, but had no effect on conditioning or memory storage. Within-subject experiments confirmed that the role of RSC in memory retrieval is not time limited. RSC-dependent context fear memory retrieval was mediated by NR2A, but not NR2B, subunit-containing NMDARs. Collectively, these data are the first demonstration that NMDARs in RSC are necessary for the retrieval of remote and recent memories of fear-evoking contexts. Dysfunction of RSC may thereby contribute significantly to the reexperiencing of traumatic memories in patients with posttraumatic stress disorder.

Segregated Populations of Hippocampal Principal CA1 Neurons Mediating Conditioning and Extinction of Contextual Fear

Learning processes mediating conditioning and extinction of contextual fear require activation of several key signaling pathways in the hippocampus. Principal hippocampal CA1 neurons respond to fear conditioning by a coordinated activation of multiple protein kinases and immediate early genes, such as cFos, enabling rapid and lasting consolidation of contextual fear memory. The extracellular signal-regulated kinase (Erk) additionally acts as a central mediator of fear extinction. It is not known however, whether these molecular events take place in overlapping or nonoverlapping neuronal populations. By using mouse models of conditioning and extinction of fear, we set out to determine the time course of cFos and Erk activity, their cellular overlap, and regulation by afferent cholinergic input from the medial septum. Analyses of cFos+ and pErk+ cells by immunofluorescence revealed predominant nuclear activation of either protein during conditioning and extinction of fear, respectively. Transgenic cFos-LacZ mice were further used to label in vivo Fos+ hippocampal cells during conditioning followed by pErk immunostaining after extinction. The results showed that these signaling molecules were activated in segregated populations of hippocampal principal neurons. Furthermore, immunotoxin-induced lesions of medial septal neurons, providing cholinergic input into the hippocampus, selectively abolished Erk activation and extinction of fear without affecting cFos responses and conditioning. These results demonstrate that extinction mechanisms based on Erk signaling involve a specific population of CA1 principal neurons distinctively regulated by afferent cholinergic input from the medial septum.

Hippocampal NMDA receptor subunits differentially regulate fear memory formation and neuronal signal propagation.

Activation of NMDA receptors (NMDAR) in the hippocampus is essential for the formation of contextual and trace memory. However, the role of individual NMDAR subunits in the molecular mechanisms contributing to these memory processes is not known. Here we demonstrate, using intrahippocampal injection of subunit‐selective compounds, that the NR2A‐preferring antagonist impaired contextual and trace fear conditioning as well as learning‐induced increase of the nuclear protein c‐Fos. The NR2B‐specific antagonist, on the other hand, selectively blocked trace fear conditioning without affecting c‐Fos levels. Studies with cultured primary hippocampal neurons, further showed that synaptic and extrasynaptic NR2A and NR2B differentially regulate the extracellular signal‐regulated kinase 1 and 2/mitogen‐ and stress‐activated protein kinase 1 (ERK1/2/MSK1)/c‐Fos pathway. Activation of the synaptic population of NMDAR induced cytosolic, cytoskeletal, and perinuclear phosphorylation of ERK1/2 (pERK1/2). The nuclear propagation of pERK1/2 signals, revealed by upregulation of the downstream nuclear targets pMSK1 and c‐Fos, was blocked by a preferential NR2A but not by a specific NR2B antagonist. Conversely, activation of total (synaptic and extrasynaptic) NMDAR engaged receptors with NR2B subunits, and resulted in membrane retention of pERK1/2 without inducing pMSK1 and c‐Fos. Stimulation of extrasynaptic NMDAR alone was consistently ineffective at activating ERK signaling. The discrete contribution of synaptic and total NR2A‐ and NR2B‐containing NMDAR to nuclear transmission vs. membrane retention of ERK signaling may underlie their specific roles in the formation of contextual and trace fear memory.

IQGAP1 Regulates NR2A Signaling, Spine Density, and Cognitive Processes

General or brain-region-specific decreases in spine number or morphology accompany major neuropsychiatric disorders. It is unclear, however, whether changes in spine density are specific for an individual mental process or disorder and, if so, which molecules confer such specificity. Here we identify the scaffolding protein IQGAP1 as a key regulator of dendritic spine number with a specific role in cognitive but not emotional or motivational processes. We show that IQGAP1 is an important component of NMDAR multiprotein complexes and functionally interacts with the NR2A subunits and the extracellular signal-regulated kinase 1 (ERK1) and ERK2 signaling pathway. Mice lacking the IQGAP1 gene exhibited significantly lower levels of surface NR2A and impaired ERK activity compared to their wild-type littermates. Accordingly, primary hippocampal cultures of IQGAP1−/− neurons exhibited reduced surface expression of NR2A and disrupted ERK signaling in response to NR2A-dependent NMDAR stimulation. These molecular changes were accompanied by region-specific reductions of dendritic spine density in key brain areas involved in cognition, emotion, and motivation. IQGAP1 knock-outs exhibited marked long-term memory deficits accompanied by impaired hippocampal long-term potentiation (LTP) in a weak cellular learning model; in contrast, LTP was unaffected when induced with stronger stimulation paradigms. Anxiety- and depression-like behavior remained intact. On the basis of these findings, we propose that a dysfunctional IQGAP1 gene contributes to the cognitive deficits in brain disorders characterized by fewer dendritic spines.

Social modeling of conditioned fear in mice by non-fearful conspecifics.

Social interactions with conspecifics markedly alter the neuroendocrine, behavioral and emotional responses to stressful events. Some of these effects involve observational learning and result in lasting changes of fear-motivated behavior. While most evidence reveals increased fearfulness after observation of fearful demonstrators (models) in a number of species, a few reports from human and non-human primates indicate that observational learning can also attenuate some forms of fear. In the present study, we set out to determine the effects of social modeling and observational learning on fear conditioning in the mouse. Observers were pre-exposed to a novel context in the presence of fearful (F group) or non-fearful (NF group) demonstrators. Mice of the F group acquired control levels of conditioned fear. On the other hand, mice of the NF group exhibited profound and persistent reduction of fear. The decrease of fear in NF observers was most likely due to context-specific impairments of fear conditioning, as revealed by selective effects on long- but not short-term contextual fear memory, and normal fear conditioning in response to a novel context or cue. The effect was lasting, but constrained by the shock intensity. Attenuation of fear conditioning resulting from interactions with non-fearful conspecifics was largely, but not entirely, mediated by vicarious learning. These findings identify an important social buffering process serving to prevent a lasting induction of fear in response to isolated, moderately intense stressful events.

Role of oxytocin receptors in modulation of fear by social memory

RationaleOxytocin receptors (Oxtr) are important mediators of social learning and emotion, with bidirectional effects on fear and anxiety. Contrary to the anxiolytic actions of Oxtr in the amygdala, we recently showed that Oxtr in the lateral septum mediate the enhancement of fear conditioning by social defeat in mice.ObjectivesUsing positive social interactions, which impair fear conditioning, here we attempted to delineate whether the role of septal Oxtr in fear regulation depends on the valence of the social memory.MethodsPharmacological and genetic manipulations of lateral septal Oxtr were combined with the social buffering of fear paradigm, in which pre-exposure to nonfearful conspecifics reduces subsequent contextual fear conditioning, as revealed by decreased freezing behavior.ResultsAntagonism and down-regulation of Oxtr in the lateral septum abolished, while oxytocin (Oxt) administration before pre-exposure to nonfearful conspecifics facilitated the decrease of freezing behavior.ConclusionsThe septal oxytocin system enhances memory of social interactions regardless of their valence, reducing fear after positive and enhancing fear after negative social encounters. These findings explain, at least in part, the seemingly bidirectional role of Oxt in fear regulation.

GABAergic mechanisms regulated by miR-33 encode state-dependent fear

Fear-inducing memories can be state dependent, meaning that they can best be retrieved if the brain states at encoding and retrieval are similar. Restricted access to such memories can present a risk for psychiatric disorders and hamper their treatment. To better understand the mechanisms underlying state-dependent fear, we used a mouse model of contextual fear conditioning. We found that heightened activity of hippocampal extrasynaptic GABAA receptors, believed to impair fear and memory, actually enabled their state-dependent encoding and retrieval. This effect required protein kinase C-βII and was influenced by miR-33, a microRNA that regulates several GABA-related proteins. In the extended hippocampal circuit, extrasynaptic GABAA receptors promoted subcortical, but impaired cortical, activation during memory encoding of context fear. Moreover, suppression of retrosplenial cortical activity, which normally impairs retrieval, had an enhancing effect on the retrieval of state-dependent fear. These mechanisms can serve as treatment targets for managing access to state-dependent memories of stressful experiences.

Metabotropic Glutamate Receptor 5/Homer Interactions Underlie Stress Effects on Fear

BACKGROUND Glutamatergic transmission is one of the main components of the stress response; nevertheless, its role in the emotional stress sequelae is not known. Here, we investigated whether interactions between group I metabotropic glutamate receptors (metabotropic glutamate receptor 1 and metabotropic glutamate receptor 5 [mGluR5]) and Homer proteins mediate the delayed and persistent enhancement of fear induced by acute stress. METHODS Antagonists and inverse agonists of metabotropic glutamate receptor 1 and mGluR5 were injected into the hippocampus after immobilization stress and before contextual fear conditioning. Metabotropic glutamate receptor 5 was displaced from constitutive Homer scaffolds by viral transfection of Homer1a or injection of Tat decoy peptides. The effects of these manipulations on stress-enhanced fear were determined. RESULTS We show that stress induces interactions between hippocampal mGluR5 and Homer1a; causes a sustained, ligand-independent mGluR5 activity; and enhances contextual fear. Consistent with this mechanism, enhancement of fear was abolished by delayed poststress application of inverse agonists, but not antagonists, of mGluR5. The effect of stress was mimicked by virally transfected Homer1a or injection of Tat-metabotropic glutamate receptor C-tail decoy peptides into the hippocampus. CONCLUSIONS Constitutive activation of mGluR5 is identified as a principal hippocampal mechanism underlying the delayed stress effects on emotion and memory. Inverse agonists, but not antagonists, of mGluR5 are therefore proposed as a preventive treatment option for acute and posttraumatic stress disorders.

Hippocampal Erk mechanisms linking prediction error to fear extinction: Roles of shock expectancy and contextual aversive valence

Extinction of fear requires learning that anticipated aversive events no longer occur. Animal models reveal that sustained phosphorylation of the extracellular signal-regulated kinase (Erk) in hippocampal CA1 neurons plays an important role in this process. However, the key signals triggering and regulating the activity of Erk are not known. By varying the degree of expected and delivered aversive reinforcement, we demonstrate that Erk specifically responds to prediction errors of contextual aversive events. An increase of somatonuclear phospho-Erk (pErk) within principal CA1 neurons was observed only when the expectation of contextual foot shock was violated, but not when the context was consistently nonreinforced or reinforced by foot shock. The rate of error detection, Erk signaling, and fear extinction markedly depended on shock expectancy and the aversive valence of the context, as revealed by comparison of groups trained with single, continuous, or partial reinforcement. On the basis of these findings, the hippocampal Erk response to prediction errors of aversive outcome is proposed as a unique mechanism of fear extinction. Improving the detection and processing of these errors has the potential to attenuate fear responses in patients with anxiety disorders.