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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.

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 up-regulation 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 versus membrane retention of ERK signaling may underlie their specific roles in the formation of contextual and trace fear memory.

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 ascFos, 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. TransgeniccFos-LacZmice were further used to labelin vivoFos+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.

The hippocampus plays an important role in processing surprising events, such as new stimuli or known stimuli gaining new behavioral significance (Ploghaus et al. 2000; Lisman and Grace 2005). Accordingly, fear conditioning formed after delivery of unexpected reinforcement, and fear extinction occurring after omission of expected aversive reinforcement, depend on hippocampal activity (Knight et al. 2004; Lattal et al. 2006). Whereas fear conditioning is rapidly and robustly acquired after a single exposure to a novel context paired with foot shock, extinction typically requires multiple nonreinforced contextual exposures (Vianna et al. 2001; Fischer et al. 2004; Chen et al. 2005). These differences may be caused by several factors. First, extinction learning may depend on distinctive molecular mechanisms optimally requiring repeated stimulus presentations. Second, extinction could be slower because it involves new learning as well as retrieval (Ouyang and Thomas 2005), transient destabilization (Lee et al. 2008), and depotentiation (Kim et al. 2007) of the conditioning memory. Third, the expectancy of the aversive event acquired during conditioning needs to be modified for extinction to occur (Lovibond 2004). Fourth, if the aversive valence of the unconditioned stimulus (UCS) is transferred to the conditioned stimulus (CS) during conditioning, so that the CS not only serves as a predictor of UCS (expectancy learning) but also gains its aversive motivational properties (evaluative learning), extinction of fear may be markedly delayed (Baeyens et al. 1990; Vansteenwegen et al. 2006; Blechert et al. 2008). Despite the multitude of learning processes involved in conditioning and subsequent extinction of fear, the specific mechanisms by which each process individually contributes to fear regulation are not known. At a molecular level, the extracellular signal-regulated kinase (Erk) has recently been identified as an important mediator of fear extinction (Szapiro et al. 2003; Chen et al. 2005; Tronson et al. 2008). Erk shows rapid and sustained phosphorylation shortly preceding and during fear extinction, which subsides after fear has stably extinguished (Fischer et al. 2007; Ryu et al. 2008). This activation pattern suggests that Erk is most likely triggered by some of the stimuli uniquely involved in extinction. Here, we show that prediction error of aversive events, but not memory retrieval, lack of reinforcement, or fear response, is the most likely signal activating Erk in hippocampal neurons. The shock expectancy and contextual aversive valence encoded during conditioning significantly accelerated and delayed, respectively, the rate of error detection, Erk signaling, and fear extinction.