BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method.

Rapid advances in genetics are linking mutations on genes to diseases at an exponential rate, yet characterizing the gene-mutation-cell-behavior relationships essential for precision medicine remains a daunting task. More than 350 mutations on small GTPase BRaf are associated with various tumors, and ∼40 mutations are associated with the neurodevelopmental disorder cardio-facio-cutaneous syndrome (CFC). We developed a fast cost-effective lentivirus-based rapid gene replacement method to interrogate the physiopathology of BRaf and ∼50 disease-linked BRaf mutants, including all CFC-linked mutants. Analysis of simultaneous multiple patch-clamp recordings from 6068 pairs of rat neurons with validation in additional mouse and human neurons and multiple learning tests from 1486 rats identified BRaf as the key missing signaling effector in the common synaptic NMDA-R-CaMKII-SynGap-Ras-BRaf-MEK-ERK transduction cascade. Moreover, the analysis creates the original big data unveiling three general features of BRaf signaling. This study establishes the first efficient procedure that permits large-scale functional analysis of human disease-linked mutations essential for precision medicine.

Initial conditioning and re-conditioning recruit different populations of 'fear neurons' in the basal amygdala of rats.

'Fear neurons' in the basal amygdala (Ba) acquire excitatory responsiveness to conditioned stimuli (CS) after fear conditioning and are believed to encode aversive valence of conditioned fear. However, it is unclear whether identical fear conditioning sessions given at different times engage the same population of 'fear neurons'. Here, we recorded electrical activity from single neurons in the Ba while the same fear conditioning paradigm was performed at two different times. Conditioned fear was monitored during CS presentation after each conditioning session in order to identify 'fear neurons'. Surprisingly, we found that initial conditioning and re-conditioning recruited different populations of 'fear neurons' in the Ba. We performed a control experiment in which conditioned fear was monitored twice after a single fear conditioning session. The majority of the 'fear neurons', which were activated during the first retrieval, were re-activated during the second retrieval, suggesting that conditioning-induced 'fear neurons' are stable. Our findings, therefore, suggest that 'fear neurons' in the Ba encode specific learned events as well as their aversive valence.

Fear renewal requires nitric oxide signaling in the lateral amygdala.

Fear renewal is defined as return of the conditioned fear responses after extinction when a conditioned stimulus (CS) is given outside of the extinction context. Previously, we have suggested that extinction induces S-nitrosylation of GluA1 in the lateral amygdala (LA), and that the extinction-induced S-nitrosylation of GluA1 lowers the threshold of GluA1 phosphorylation (at Ser 831) which is required for fear renewal. This fits nicely with the fact that fear renewal is induced by weak stimuli. However, it has not been tested whether S-nitrosylation of GluA1 in the LA is indeed required for fear renewal. In the present study, we used three different chemicals to impede protein S-nitrosylation via distinct mechanisms. Fear renewal was inhibited by microinjection of 7-Nitroindazole (nNOS inhibitor), and ZL006 (a blocker of PSD-95-nNOS interaction) before fear renewal. Furthermore, fear renewal was also attenuated by microinjection of a strong antioxidant (N-acetyl cysteine), which scavenges reactive oxygen including nitric oxide, into the LA before each extinction training. These findings suggest that protein S-nitrosylation is required for fear renewal.

Graf regulates hematopoiesis through GEEC endocytosis of EGFR.

GTPase regulator associated with focal adhesion kinase 1 (GRAF1) is an essential component of the GPI-enriched endocytic compartment (GEEC) endocytosis pathway. Mutations in the human GRAF1 gene are associated with acute myeloid leukemia, but its normal role in myeloid cell development remains unclear. We show that Graf, the Drosophila ortholog of GRAF1, is expressed and specifically localizes to GEEC endocytic membranes in macrophage-like plasmatocytes. We also find that loss of Graf impairs GEEC endocytosis, enhances EGFR signaling and induces a plasmatocyte overproliferation phenotype that requires the EGFR signaling cascade. Mechanistically, Graf-dependent GEEC endocytosis serves as a major route for EGFR internalization at high, but not low, doses of the predominant Drosophila EGFR ligand Spitz (Spi), and is indispensable for efficient EGFR degradation and signal attenuation. Finally, Graf interacts directly with EGFR in a receptor ubiquitylation-dependent manner, suggesting a mechanism by which Graf promotes GEEC endocytosis of EGFR at high Spi. Based on our findings, we propose a model in which Graf functions to downregulate EGFR signaling by facilitating Spi-induced receptor internalization through GEEC endocytosis, thereby restraining plasmatocyte proliferation.

Endogenous amyloid-ß mediates memory forgetting in the normal brain.

Amyloid beta (Aß) is known to be one of the strong candidate molecules for initiating Alzheimer's disease and has been extensively studied in the light of disease pathophysiology. However, it is still elusive what roles Aß play in the normal brain. In this study, we report that Aß is required for memory forgetting in the normal brain. We monitored object recognition memory, and in order to quench soluble Aß, we microinjected anti-Aß antibody (4G8) into the ventricles after memory acquisition. Microinjection of anti-Aß antibody prolonged the maintenance of object recognition memory. This effect appeared not to be due to modulation of memory consolidation since antibody injection after memory consolidation still had a similar effect on memory maintenance. Furthermore, the maintenance of object recognition memory was prolonged in Fcgr2b KO mice, which lacks IgG Fcγ receptor II-b (FcγRIIb), a receptor for soluble Aß oligomers. Taken together, these findings suggest that endogenous Aß is involved in memory forgetting in the normal brain.

Evidence for presynaptically silent synapses in the immature hippocampus.

Silent synapses show NMDA receptor (NMDAR)-mediated synaptic responses, but not AMPAR-mediated synaptic responses. A prevailing hypothesis states that silent synapses contain NMDARs, but not AMPARs. However, alternative presynaptic hypotheses, according to which AMPARs are present at silent synapses, have been proposed; silent synapses show slow glutamate release via a fusion pore, and glutamate spillover from the neighboring synaptic terminals. Consistent with these presynaptic hypotheses, the peak glutamate concentrations at silent synapses have been estimated to be ≪170 µM, much lower than those seen at functional synapses. Glutamate transients predicted based on the two presynaptic mechanisms have been shown to activate only high-affinity NMDARs, but not low-affinity AMPARs. Interestingly, a previous study has developed a new approach to distinguish between the two presynaptic mechanisms using dextran, an inert macromolecule that reduces the diffusivity of released glutamate: postsynaptic responses through the fusion pore mechanism, but not through the spillover mechanism, are potentiated by reduced glutamate diffusivity. Therefore, we reasoned that if the fusion pore mechanism underlies silent synapses, dextran application would reveal AMPAR-mediated synaptic responses at silent synapses. In the present study, we recorded AMPAR-mediated synaptic responses at the CA3-CA1 synapses in neonatal rats in the presence of blockers for NMDARs and GABAARs. Bath application of dextran revealed synaptic responses at silent synapses. GYKI53655, a selective AMPAR-antagonist, completely inhibited the unsilenced synaptic responses, indicating that the unsilenced synaptic responses are mediated by AMPARs. The dextran-mediated reduction in glutamate diffusivity would also lead to the activation of metabotropic glutamate receptors (mGluRs), which might induce unsilencing via the activation of unknown intracellular signaling. Hence, we determined whether mGluR-blockers alter the dextran-induced unsilencing. However, dextran application continued to produce significant synaptic unsilencing in the presence of a cocktail of the blockers for all subtypes of mGluRs. Our findings provide evidence that slowed glutamate diffusion produces synaptic unsilencing by enhancing the peak glutamate occupancy of pre-existing AMPARs, supporting the fusion pore mechanism of silent synapses.

Enhanced theta synchronization correlates with the successful retrieval of trace fear memory.

Mechanisms underlying delay fear conditioning in which conditioned stimuli (CS) are paired and co-terminated with unconditioned stimuli (US), have been extensively characterized, thus expanding knowledge concerning learning and memory. However, trace fear conditioning in which CS and US are separated by trace interval periods, has received much less attention though it involves cognitive processes including timing and working memories. Various brain regions including the hippocampus are known to play an important role in memory acquisition and/or retrieval of trace fear conditioning. However, neural correlates, which are specific for the discrete steps in trace fear conditioning, have not been characterized thoroughly. Here, we investigated the network activities between the dorsal and ventral hippocampi at different stages of memory processing after trace fear conditioning. When fear memory was retrieved successfully, theta synchronization between the two regions was enhanced relative to preconditioning levels. The enhancement in theta synchronization was observed only during the trace interval period but not during CS presentation or after the trace interval period. Thus, the enhanced theta synchronization between the dorsal and ventral hippocampi may underlie a cognitive process associated with the trace interval period when fear memory is retrieved successfully.

AMPA receptor exchange underlies transient memory destabilization on retrieval.

A consolidated memory can be transiently destabilized by memory retrieval, after which memories are reconsolidated within a few hours; however, the molecular substrates underlying this destabilization process remain essentially unknown. Here we show that at lateral amygdala synapses, fear memory consolidation correlates with increased surface expression of calcium-impermeable AMPA receptors (CI-AMPARs), which are known to be more stable at the synapse, whereas memory retrieval induces an abrupt exchange of CI-AMPARs to calcium-permeable AMPARs (CP-AMPARs), which are known to be less stable at the synapse. We found that blockade of either CI-AMPAR endocytosis or NMDA receptor activity during memory retrieval, both of which blocked the exchange to CP-AMPARs, prevented memory destabilization, indicating that this transient exchange of AMPARs may underlie the transformation of a stable memory into an unstable memory. These newly inserted CP-AMPARs gradually exchanged back to CI-AMPARs within hours, which coincided with the course of reconsolidation. Furthermore, blocking the activity of these newly inserted CP-AMPARs after retrieval impaired reconsolidation, suggesting that they serve as synaptic "tags" that support synapse-specific reconsolidation. Taken together, our results reveal unexpected physiological roles of CI-AMPARs and CP-AMPARs in transforming a consolidated memory into an unstable memory and subsequently guiding reconsolidation.

Delayed escape behavior requires claustral activity.

Animals are known to exhibit innate and learned forms of defensive behaviors, but it is unclear whether animals can escape through methods other than these forms. In this study, we develop the delayed escape task, in which male rats temporarily hold the information required for future escape, and we demonstrate that this task, in which the subject extrapolates from past experience without direct experience of its behavioral outcome, does not fall into either of the two forms of behavior. During the holding period, a subset of neurons in the rostral-to-striatum claustrum (rsCla), only when pooled together, sustain enhanced population activity without ongoing sensory stimuli. Transient inhibition of rsCla neurons during the initial part of the holding period produces prolonged inhibition of the enhanced activity. The transient inhibition also attenuates the delayed escape behavior. Our data suggest that the rsCla activity bridges escape-inducing stimuli to the delayed onset of escape.

Long-term neural correlates of reversible fear learning in the lateral amygdala.

Fear conditioning and extinction are behavioral models that reflect the association and dissociation of environmental cues to aversive outcomes, both known to involve the lateral amygdala (LA). Accordingly, responses of LA neurons to conditioned stimuli (CS) increase after fear conditioning and decrease partially during extinction. However, the long-term effects of repeated fear conditioning and extinction on LA neuronal firing have not been explored. Here we show, using stable, high signal-to-noise ratio single-unit recordings, that the ensemble activity of all recorded LA neurons correlates tightly with conditioned fear responses of rats in a conditioning/extinction/reconditioning paradigm spanning 3 d. This CS-evoked ensemble activity increased after conditioning, decreased after extinction, and was repotentiated after reconditioning. Cell-by-cell analysis revealed that among the LA neurons that displayed potentiated responses after initial fear conditioning, some exhibited weakened CS responses after extinction (extinction-susceptible), whereas others remained potentiated (extinction-resistant). The majority of extinction-susceptible neurons exhibited strong potentiation after reconditioning, suggesting that this distinct subpopulation (reversible fear neurons) encodes updated CS-unconditioned stimulus (US) association strength. Interestingly, these reversible fear neurons displayed larger, more rapid potentiation during reconditioning compared with the initial conditioning, providing a neural correlate of savings after extinction. In contrast, the extinction-resistant fear neurons did not show further increases after reconditioning, suggesting that this subpopulation encodes persistent fear memory representing the original CS-US association. This longitudinal report on LA neuronal activity during reversible fear learning suggests the existence of distinct populations encoding various facets of fear memory and provides insight into the neuronal mechanisms of fear memory modulation.

Quantitative proteomics of auditory fear conditioning.

Auditory fear conditioning is a well-characterized rodent learning model where a neutral auditory cue is paired with an aversive outcome to induce associative fear memory. The storage of long-term auditory fear memory requires long-term potentiation (LTP) in the lateral amygdala and de novo protein synthesis. Although many studies focused on individual proteins have shown their contribution to LTP and fear conditioning, non-biased genome-wide studies have only recently been possible with microarrays, which nevertheless fall short of measuring changes at the level of proteins. Here we employed quantitative proteomics to examine the expression of hundreds of proteins in the lateral amygdala in response to auditory fear conditioning. We found that various proteins previously implicated in LTP, learning and axon/dendrite growth were regulated by fear conditioning. A substantial number of proteins that were regulated by fear conditioning have not yet been studied specifically in learning or synaptic plasticity.

Impairment of fear memory consolidation in maternally stressed male mouse offspring: evidence for nongenomic glucocorticoid action on the amygdala.

The environment in early life elicits profound effects on fetal brain development that can extend into adulthood. However, the long-lasting impact of maternal stress on emotional learning remains largely unknown. Here, we focus on amygdala-related learning processes in maternally stressed mice. In these mice, fear memory consolidation and certain related signaling cascades were significantly impaired, though innate fear, fear memory acquisition, and synaptic NMDA receptor expression in the amygdala were unaltered. In accordance with these findings, maintenance of long-term potentiation (LTP) at amygdala synapses, but not its induction, was significantly impaired in the maternally stressed animals. Interestingly, amygdala glucocorticoid receptor expression was reduced in the maternally stressed mice, and administration of glucocorticoids (GCs) immediately after fear conditioning and LTP induction restored memory consolidation and LTP maintenance, respectively, suggesting that a weakening of GC signaling was responsible for the observed impairment. Furthermore, microinfusion of a membrane-impermeable form of GC (BSA-conjugated GC) into the amygdala mimicked the restorative effects of GC, indicating that a nongenomic activity of GC mediates the restorative effect. Together, these findings suggest that prenatal stress induces long-term dysregulation of nongenomic GC action in the amygdala of adult offspring, resulting in the impairment of fear memory consolidation. Since modulation of amygdala activity is known to alter the consolidation of emotionally influenced memories allocated in other brain regions, the nongenomic action of GC on the amygdala shown herein may also participate in the amygdala-dependent modulation of memory consolidation.

Preso regulation of dendritic outgrowth through PI(4,5)P2-dependent PDZ interaction with ßPix.

In neuronal development, dendritic outgrowth and arborization are important for the establishment of neural circuit formation. A previous study reported that PSD-95-interacting regulator of spine morphogenesis (Preso) formed a complex with PAK-interacting exchange factor-beta (ßPix) via PSD-95/Dlg/ZO-1 (PDZ) interaction. Here, we report that Preso and its binding protein, ßPix, are localized in dendritic growth cones. Knockdown and dominant-negative inhibition of Preso in cultured neurons markedly reduced the dendritic outgrowth but not branching, and led to a decrease in the intensity of ßPix and F-actin in neuronal dendritic tips. Moreover, phosphatidylinositol 4,5-bisphosphate (PIP(2) ) induced a conformational change in Preso toward the open PDZ domain and enhanced the interaction with ßPix. In addition, the Preso band 4.1 protein, ezrin, radixin and moesin (FERM) domain mutant is unable to interact with PIP(2) and it did not rescue the Preso-knockdown effect. These results indicate that PIP(2) is a key signalling molecule that regulates dendritic outgrowth through activation of small GTPase signalling via interaction between Preso and ßPix.

PDZ-scaffold protein, Tamalin promotes dendritic outgrowth and arborization in rat hippocampal neuron.

Tamalin is a scaffold protein known to regulate membrane trafficking through its interaction with cytohesin-2/ARNO, guanine nucleotide exchange factor (GEF) on ADP-ribosylation factor (Arf) 1/6, and induces actin reorganization. However, the neuronal function of Tamalin is not well understood. Here, we report that Tamalin participates in neurite development through the association with exchange factor for Arf6 (EFA6A)/Arf6 signaling. In immature hippocampal neuron, Tamalin knockdown markedly reduced the dendritic outgrowth, the number of dendritic tips and the levels of filamentous actin (F-actin) and microtubule-associated protein 2 (MAP2) in dendrites. In addition, Tamalin colocalized with EFA6A and Arf6 in the dendritic shaft. Tamalin knockdown reduced the number, size, and intensity of endogenous EFA6A cluster, whereas overexpression of Tamalin showed opposite effects compared with those of knockdown. These results suggest that Tamalin is responsible for neuronal dendritic development via regulation of EFA6A/Arf6-mediated cytoskeleton dynamics.

Reactivation of fear memory renders consolidated amygdala synapses labile.

It is believed that memory reactivation transiently renders consolidated memory labile and that this labile or deconsolidated memory is reconsolidated in a protein synthesis-dependent manner. The synaptic correlate of memory deconsolidation upon reactivation, however, has not been fully characterized. Here, we show that 3,5-dihydroxyphenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRI), induces synaptic depotentiation only at thalamic input synapses onto the lateral amygdala (T-LA synapses) where synaptic potentiation is consolidated, but not at synapses where synaptic potentiation is not consolidated. Using this mGluRI-induced synaptic depotentiation (mGluRI-depotentiation) as a marker of consolidated synapses, we found that mGluRI-depotentiation correlated well with the state of memory deconsolidation and reconsolidation in a predictable manner. DHPG failed to induce mGluRI-depotentiation in slices prepared immediately after reactivation when the reactivated memory was deconsolidated. DHPG induced mGluRI-depotentiation 1 h after reactivation when the reactivated memory was reconsolidated, but it failed to do so when reconsolidation was blocked by a protein synthesis inhibitor. To test the memory-specificity of mGluRI-depotentiation, conditioned fear was acquired twice using two discriminative tones (2.8 and 20 kHz). Under this condition, mGluRI-depotentiation was fully impaired in slices prepared immediately after reactivation with both tones, whereas mGluRI-depotentiation was partially impaired immediately after reactivation with the 20 kHz tone. Consistently, microinjection of DHPG into the LA 1 h after reactivation reduced fear memory retention, whereas DHPG injection immediately after reactivation failed to do so. Our findings suggest that, upon memory reactivation, consolidated T-LA synapses enter a temporary labile state, displaying insensitivity to mGluRI-depotentiation.

Modulation of fear memory by retrieval and extinction: a clue for memory deconsolidation.

Memories are fragile and easily forgotten at first, but after a consolidation period of hours to weeks, are inscribed in our brains as stable traces, no longer vulnerable to conventional amnesic treatments. Retrieval of a memory renders it labile, akin to the early stages of consolidation. This phenomenon has been explored as memory reactivation, in the sense that the memory is temporarily 'deconsolidated', allowing a short time window for amnesic intervention. This window closes again after reconsolidation, which restores the stability of the memory. In contrast to this 'transient deconsolidation' and the short-spanned amnesic effects of consolidation blockers, some specific treatments can disrupt even consolidated memory, leading to apparent amnesia. We propose the term 'amnesic deconsolidation' to describe such processes that lead to disruption of consolidated memory and/or consolidated memory traces. We review studies of these 'amnesic deconsolidation' treatments that enhance memory extinction, alleviate relapse, and reverse learning-induced plasticity. The transient deconsolidation that memory retrieval induces and the amnesic deconsolidation that these regimes induce both seem to dislodge a component that stabilizes consolidated memory. Characterizing this component, at both molecular and network levels, will provide a key to developing clinical treatments for memory-related disorders and to defining the consolidated memory trace.

In vitro synaptic reconsolidation in amygdala slices prepared from rat brains.

The retrieval of consolidated fear memory causes it to be labile or deconsolidated, and the deconsolidated fear memory is reconsolidated over time in a protein synthesis-dependent manner. We have recently developed an ex vivo model where during fear memory deconsolidation and reconsolidation the synaptic state can be monitored at thalamic input synapses onto the lateral amygdala (T-LA synapses), a storage site for auditory fear memory. In this ex vivo model, the deconsolidation and reconsolidation processes of auditory fear memory in the intact brain were prevented following brain slicing; therefore, we could monitor the synaptic state for memory deconsolidation and reconsolidation at the time of brain slicing. However, why the synaptic reconsolidation process stopped after brain slicing in the ex vivo model is not known. One possibility is that brain slicing severs neuromodulatory innervations, which are required for memory reconsolidation, from other brain regions (e.g., noradrenergic innervation). In the present study, we supplemented amygdala slices with exogenous norepinephrine as a substitute for the severed noradrenergic innervations. DHPG (a group I metabotropic glutamate receptor agonist)-induced depotentiation (mGluRI-depotentiation), a marker for consolidated synapses, was observed following norepinephrine application to slices prepared immediately after tone presentation (fear memory retrieval) to rats that had been pre-conditioned to a tone paired with a shock. These results suggest that noradrenergic activation initiates synaptic reconsolidation. In contrast, mGluRI-depotentiation was absent following norepinephrine application to slices that were prepared immediately after the tone presentation (no fear memory retrieval) to rats when a tone and a shock were unpaired, ruling out the possibility that noradrenergic activation somehow facilitates a subsequent synaptic depression induced by DHPG irrespective of synaptic reconsolidation. Furthermore, the restored mGluRI-depotentiation following application of exogenous norepinephrine was dependent on de novo protein synthesis, as is memory reconsolidation. Thus, our findings suggest that T-LA synapses from acute slice preparations can undergo a reconsolidation process, thereby providing an optimal preparation to study a fear memory reconsolidation process in vitro.

Adrenal peripheral clock controls the autonomous circadian rhythm of glucocorticoid by causing rhythmic steroid production.

Glucocorticoid (GC) is an adrenal steroid with diverse physiological effects. It undergoes a robust daily oscillation, which has been thought to be driven by the master circadian clock in the suprachiasmatic nucleus of the hypothalamus via the hypothalamus-pituitary-adrenal axis. However, we show that the adrenal gland has its own clock and that the peripheral clockwork is tightly linked to steroidogenesis by the steroidogenic acute regulatory protein. Examination of mice with adrenal-specific knockdown of the canonical clock protein BMAL1 reveals that the adrenal clock machinery is required for circadian GC production. Furthermore, behavioral rhythmicity is drastically affected in these animals, together with altered expression of Period1, but not Period2, in several peripheral organs. We conclude that the adrenal peripheral clock plays an essential role in harmonizing the mammalian circadian timing system by generating a robust circadian GC rhythm.

Longitudinal recordings of single units in the basal amygdala during fear conditioning and extinction.

The balance between activities of fear neurons and extinction neurons in the basolateral nucleus of the basal amygdala (BAL) has been hypothesized to encode fear states after extinction. However, it remains unclear whether these neurons are solely responsible for encoding fear states. In this study, we stably recorded single-unit activities in the BAL during fear conditioning and extinction for 3 days, providing a comprehensive view on how different BAL neurons respond during fear learning. We found BAL neurons that showed excitatory responses to the conditioned stimulus (CS) after fear conditioning ('conditioning-potentiated neurons') and another population that showed excitatory responses to the CS after extinction ('extinction-potentiated neurons'). Interestingly, we also found BAL neurons that developed inhibitory responses to the CS after fear conditioning ('conditioning-inhibited neurons') or after extinction ('extinction-inhibited neurons'). BAL neurons that showed excitatory responses to the CS displayed various functional connectivity with each other, whereas less connectivity was observed among neurons with inhibitory responses to the CS. Intriguingly, we found correlative neuronal activities between conditioning-potentiated neurons and neurons with inhibitory responses to the CS. Our findings suggest that distinct BAL neurons, which are responsive to the CS with excitation or inhibition, encode various facets of fear conditioning and extinction.