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. Author manuscript; available in PMC: 2019 Jan 2.
Published in final edited form as: Cell Rep. 2018 May 15;23(7):2014–2025. doi: 10.1016/j.celrep.2018.04.042

Activation of EphB2 forward signaling enhances memory consolidation

Jessica M Alapin 1, Monica Dines 1, Maria Vassiliev 1, Tal Tamir 1, Alon Ram 1, Clifford Locke 2, Ji Yu 2, Raphael Lamprecht 1
PMCID: PMC6314675  NIHMSID: NIHMS1002660  PMID: 29768201

Abstract

EphB2 is involved in enhancing synaptic transmission and gene expression molecular events believed to underlie memory formation. However, it is not known whether activation of EphB2 enhances long-term memory (LTM) formation. To explore this possibility we used a novel optogenetic technique that utilizes a photoactivatable EphB2 protein (optoEphB2) to activate by light EphB2 forward signaling in pyramidal neurons in lateral amygdala (LA). Photoactivation of optoEphB2 during fear conditioning enhanced long- but not short-term auditory fear conditioning. Activation of optoEphB2 minutes after fear conditioning learning had no effect on LTM formation. Long- and short-term contextual fear conditioning were unaffected by activation of optoEphB2 in LA. Photoactivation of optoEphB2 during fear conditioning led to activation of cAMP/Ca2+ responsive element binding (CREB) protein. Shining light on a kinase-dead optoEphB2 in LA did not lead to enhancement of long-term fear conditioning memory or to activation of CREB. Long- but not short-term auditory fear conditioning memory is impaired in mice lacking EphB2 forward signaling (EphB2lacz/lacz). Activation of optoEphB2 in LA of EphB2lacz/lacz mice rescued long-term fear conditioning memory. The present findings show that the level of EphB2 forward signaling activity during learning determines the strength of long-term memory consolidation.

Introduction

EphB2 is required for normal brain development and function and its dysfunction leads to brain and behavioral impairments (Grunwald et al., 2001; Klein, 2009; Cissé et al., 2011; Attwood et al., 2011; Sheffler-Collins and Dalva, 2012; Cruz et al., 2015; Dines et al., 2015; Zhu et al., 2016). EphB receptors activation enhances glutamatergic synaptic transmission and gene expression molecular events believed to be involved in memory formation (Lamprecht and LeDoux, 2004). For example, EphB directly interacts and cooperates with NMDA receptors (NMDARs) in synapse formation and plasticity and EphB forward signaling promotes tyrosine phosphorylation of NMDAR subunit (NR2B) by Src family kinases to facilitate NMDA receptor transmission (Dalva et al., 2000; Grunwald et al., 2001; Henderson et al., 2001; Takasu et al., 2002). Moreover, activation of EphB forward signaling in conjunction with glutamate stimulation leads to an increase in Ca2+/cAMP-responsive element binding protein (CREB) phosphorylation and genes expression (Takasu et al., 2002). These observations suggest that EphB2 activation in neurons has the potential to enhance long-term memory. However, such possibility was not explored.

To investigate whether activation of EphB2 leads to memory enhancement we use in the current study a novel optogenetic approach to induce EphB2 forward signaling by light (optoEphB2) (Locke et al., 2017). This technique takes advantage of the fact that EphB2 activation is induced by receptor clustering (Schaupp et al., 2014). In optoEphB2 the EphB2 cytoplasmic domain is fused to an optimized mutant of the plant protein cryptochrome 2 (Cry2olig, residues 1–498 with E490G mutation) which is clustered by blue light (Taslimi et al., 2014; Zhang and Cui, 2015). OptoEphB2 includes also mCherry for in vivo detection and is myristoylated at the N-terminus to be directed to the membrane. Thus, application of blue light leads to EphB2 clustering and activation. This technique allows us to activate EphB2 at the highest required spatiotemporal resolution in vivo. Similar approach was used to activate TrkC in neurons (OptoTrkC; Han et al., 2016).

To examine whether EphB2 can facilitate memory formation we used auditory fear conditioning, a well-established behavioral paradigm. In this paradigm an association is formed between the auditory stimulation tone (conditioned stimulus (CS)) and an aversive mild footshock (unconditioned stimulus (US)) (Fanselow and LeDoux, 1999; LeDoux, 2000; Davis and Whalen, 2001; Sah et al, 2003; Maren, 2005). The putative site of fear conditioning memory, the lateral amygdala (LA), has been identified (Fanselow and LeDoux, 1999; Schafe et al., 2001; Rodrigues et al., 2004; Maren, 2005; Johansen et al., 2011). EphB2 is known to be expressed in LA neurons in mice (Grunwald et al., 2001). The optoEphB2 expression in this study is controlled by the CaMKII promoter to express it in pyramidal excitatory neurons in LA of mice (McDonald et al., 2002).

Using this approach we reveal that activation of EphB2 forward signaling in LA pyramidal neurons during learning, but not afterwards, enhances long- but not short-term auditory fear conditioning. Moreover, EphB2 forward signaling during fear conditioning activates CREB in LA neurons. Fear memory enhancement and CREB activation depends on optoEphB2 kinase activity as shining light on a kinase-dead optoEphB2 in LA during fear conditioning did not lead to enhancement of long-term fear conditioning memory or to activation of CREB. Moreover, EphB2 forward signaling is essential for long-term fear memory formation as mice that lack the C-terminal of EphB2 are impaired in long-term but not short-term fear memory and activation of optoEphB2 in LA of these mice during fear conditioning rescued fear LTM. Thus, EphB2 activity enhances LTM by controlling its consolidation.

Results

Activation of optoEphB2 by light leads to receptors clustering, kinase-dependent tyrosine phosphorylation and to phosphorylation of Src

EphB2 has been shown to be involved in facilitating synaptic transmission and gene expression suggested to be involved in long-term memory formation (Klein, 2009; Sheffler-Collins and Dalva, 2012). However, it is not known whether its activation can also facilitate long-term memory. Toward exploring this possibility we utilized a photoactivatable EphB2 (optoEphB2) to activate EphB2 in vivo at high spatiotemporal resolution. Figure 1A shows a time lapse experiment where optoEphB2 under the CMV promoter is activated by light in HEK293 cells. To examine optoEphB2 in membrane we used TIRF (total internal reflection fluorescence) microscopy. We observed that optoEphB2 cluster in membrane after short exposure to blue light (Figure 1B). OptoEphB2 cluster formation and dissipation was quantified in HEK cells (n=3) (Figure 1C). The results show fast induction of cluster by light that return to basal level within minutes. To test if light-induced optoEphB2 clustering resulted in its activation, we assayed for tyrosine phosphorylation in MEFs cell lysates by Western blotting. We found that mouse embryonic fibroblasts (MEFs) stably expressing optoEphB2 and illuminated with blue light for one minute produced significantly higher overall tyrosine phosphorylation compared to cells left in the dark (Figure 1D). In contrast, blue light produced no increase in tyrosine phosphorylation in cells expressing kinase-dead optoEphB2 (optoEphB2-KD), which contained a mutation (K99M in optoEphB2) in the kinase domain that prevents ATP binding (Zisch et al., 2000). Immunoblot with mCherry shows a single band at 135 KDa indicating that the optoEphB2 is not degraded (data not shown). Anti-phosphotyrosine blot analysis of immune-precipitated optoEphB2 show that optoEphB2 become phosphorylated following blue light-illumination (Figure 1E). Thus, blue light-induces optoEphB2 clustering and activation. To examine whether optoEphB2 interacts with endogenous EphB2 we co-expressed in HEK cells EphB2-V5 and optoEphB2. Pulldown was performed either against EphB2 (anti-V5; Figure 1F) or against optoEphB2 (anti-mCherry; Figure 1G). The co-ip detected no interaction in non-activated cells, no interaction in light-activated cells when pulled with anti-V5 and a very weak signal when pulled with anti-mCherry. Thus, the vast majority of the optoEphB2 do not interact with the endogenous EphB2.

Figure 1. Activation of optoEphB2 by light leads to receptor dimerization and to phosphorylation of Src.

Figure 1.

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A. Schematic description of the plasmid containing the optoEphB2 under the CMV promoter andmode of action of optoEphB2. B. Time lapse TIRF images of HEK293 cells transiently transfected with optoEphB2, showing clustering upon blue light illumination (488 nm, 250 ms pulses between frames). Time shown is in seconds. The red square is shown below as an inset. C, Quantification of optoEphB2 cluster formation and dissipation (n=3) in HEK293 cells. Cells were activated (blue bar) for 120 sec (3.5 sec per frame) and then followed with a slower time-lapse (1 min per frame) without blue light activation. D. Western blot of total phosphotyrosine in whole cell lysates of MEFs stably expressing optoEphB2 or optoEphB2-KD, following 1 minute of illumination by blue LED light (~10−2 W/cm2) or incubation in the dark. Arrow points at the molecular weight of optoEphB2. E. Anti-phosphotyrosine blot analysis of immune-precipitated optoEphB2 showing that optoEphB2 becomes phosphorylated by blue light-illumination. F. HEK cells are co-transfected with EphB2-V5 and optoEphB2. Cells are either kept in the dark or stimulated with blue light (1min) before cell lysates were collected. Lysates were immunoprecipitated with anti-V5. G. HEK cells are co-transfected with EphB2-V5 and optoEphB2. Cells are either kept in the dark or stimulated with blue light (1min) before cell lysates were collected. Lysates were immunoprecipitated with anti-mCherry.

We were interested to express optoEphB2 and optoEphB-KD in excitatory neurons in lateral amygdala (LA). We therefore produced AAV viruses expressing optoEphB2 or optoEphB-KD under the CaMKII promoter to express them in excitatory pyramidal neurons in LA (McDonald et al., 2002). Microinjection of the AAVs into LA led to optoEphB2 and optoEphB2-KD expression as seen by imaging of the mCherry in optoEphB2 in LA (Figure 2B). The expression is specific to mCherry as it omitted red but not green fluorescent light. We quantified the expression optoEphB2 and optoEphB2-KD and found that optoEphB2 (n=7) and optoEphB2KD (n=6) (mCherry expressing neurons) are expressed in 26.29+/− 1.92 and 29.099+/− 4.30 cells from all cells in LA (measured by DAPI staining), respectively (Figure 2C). There is no significant difference between the level of expression of optoEphB2 and optoEphB2-KD in LA (p>0.3). It was shown that EphB activation leads to phosphorylation of Src (Takasu et al., 2002). Shining light (same protocol of light exposure as during fear conditioning trials) in LA expressing optoEphB2 through optic fibers led to increase in Src phosphorylation 2 minutes after the end of the last light stimulation. Figure 2E shows a representative immunohistochemistry result of Src phosphorylation in LA after light stimulation. This increase in Src phosphorylation in LA is significant in animals that express optoEphB2 in LA exposed to light (n=5) when compared to LA in animals expressing optoEphB2 but not exposed to light (n=6) (p<0.01) (Figure 2E). In contrast, stimulation of optoEphB2-KD in LA of mice by light did not lead to increase in Src phosphorylation compared to LA that express optoEphB2-KD but not stimulated with light (p>0.7) (Figure 2F). The aforementioned results show that optoEphB2 can be activated by light in LA. We performed immunohistochemistry with neuronal marker (anti-NeuN) and glia markers (e.g. anti-GFAP) and found that optoEphB2 is expressed in neurons but not in glia (Figure 2H).

Figure 2. Activation of OptoEphB2 in LA by light leads to an increase in phosphorylation of Src in a kinase dependent manner.

Figure 2.

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A. Schematic description of the construct of optoEphB2 and optoEphB2 kinase dead (OptoEphB2-KD) under the CaMKII promoter that is expressed by the AAV virus. B. Expression of optoEphB2 and optoEphB2-KD, as shown by mCherry expression, in lateral amygdala one month after AAV injection. Same slides visualized under conditions for green emission. No expression detected showing the specificity of the expression. C. The percentage of optoEphB2 (n=7) and optoEphB2-KD (n=6) from cells in LA was evaluated. Similar level of expression of these constructs in LA was detected (p>0.3). D. Pattern of light stimulation of optoEphB2 (473 nm) in LA used in the pSrc experiments. E. Representative immunohistochemistry of phospho-Src measured 2 minutes after the end of optoEphB2 light stimulation in LA or of non-stimulated LA. Quantitative results of colocalization of phospho-Src with optoEphB2 in light and no light LAs. The level of co-localization is higher (p<0.01) in the light stimulated LAs (n=5) compared to non-stimulated LAs (n=6) showing that Src is phosphorylated after optoEphB2 activation. F. Representative immunohistochemistry of phospho-Src measured 2 minutes after the end of light stimulation of optoEphB2-KD in LA or of non-stimulated LA. Stimulation of optoEphB2-KD in LA of mice did not lead to increase in Src phosphorylation compared to LA that express optoEphB2-KD but not stimulated with light (p>0.7). H. Staining after injection of AAV into LA with anti-NeuN or anti-GFAP antibody.

EphB2 activation in lateral amygdala during fear conditioning learning trials enhances long-term auditory fear memory

We were interested to explore the possibility that EphB2 activity in LA may affect fear long-term memory (LTM) formation. Toward that end, we activated optoEphB2 in LA, bilaterally, using optic fibers, during the CS-US presentations in fear conditioning training (Figure 3A). Control group expressed optoEphB2 in LA and was subjected to fear conditioning training but not exposed to light stimulation through the optic fibers. Freezing during training was not different between the groups (F(1,25)= 0.21, p>0.8) there is no treatment × tone trial interaction (F(2,50)= 1.06, p>0.3) (Figure 2B) indicating that EphB2 activation does not affect freezing per se, foot shock sensitivity and CS and US processing in the LA. Long-term auditory fear conditioning memory formation in animals subjected to light (n=10) was enhanced compared to animals not subjected to light (n=17) (F(1,25)= 5.65, p<0.026) (Figure 3D). There is no treatment × tone trial interaction (F(4,100)=0.221, p>0.9) indicating that the rate of changes in fear responses along the test trials was similar in all groups. These results show that activation of EphB2 during auditory fear conditioning trials leads to enhancement of long-term auditory fear memory.

Figure 3. EphB2 activation in lateral amygdala during fear conditioning learning trials enhances long-term, but not short-term, auditory fear memory.

Figure 3.

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A. Schematic description of protocol of behavior and light stimulation for the LTM experiment. B. Freezing during training in response to the tone CS is not different between light (n=10) and no light (n=17) animals (F(1,25)= 0.21, p>0.8). C. Contextual fear conditioning tested 24 hours after fear conditioning is not different between the light and no light groups (p>0.6). D. Auditory fear conditioning tested 48 hours after fear conditioning is enhanced in animals subjected to light activation of optoEphB2 compared to no light mice (F(1,25)= 5.65, p<0.026). E. Schematic description of protocol of behavior and light stimulation for the STM experiment. F. Freezing during training in response to the tone CS is not different between light (n=9) and no light (n=7) animals (F(1,14)= 0.011, p>0.9). G. Contextual fear conditioning tested 1 hour after fear conditioning is not different between the light and no light groups (p>0.6). H. Auditory fear conditioning tested 2 hours after fear conditioning is not different between animals subjected to light activation of optoEphB2 compared to no light mice (F(1,14)=0.043, p>0.8). I. Schematic description of protocol of behavior and light stimulation for the post-training activation of optoEphB2 experiment. J. Freezing during training in response to the tone CS is not different between light (n=10) and no light (n=8) animals (F(1,16)=0.312, p>0.5). K. Contextual fear conditioning tested 24 hours after fear conditioning is not different between the light and no light groups (p>0.9). L. Auditory fear conditioning tested 48 hours after fear conditioning is not different between animals subjected to light activation of optoEphB2 compared to no light mice (F(1,14)= 0.251, p>0.6).

EphB2 activation in lateral amygdala during fear conditioning learning trials has no effect on long-term contextual fear memory

The aforementioned observations show that activation of EphB2 during learning in lateral amygdala leads to enhancement of long-term auditory fear memory. We were interested to explore whether such activation affects also contextual fear conditioning known to be mediated by the hippocampus (Kim and Fanselow, 1992; Phillips and Ledoux, 1992). Activation of EphB2 in LA (n=10) during fear conditioning has no effect on long-term contextual fear memory when compared to animals where optoEphB2 was not activated (n=17) (p>0.6) (Figure 3C). These results show that activation of EphB2 in lateral amygdala during fear conditioning trials has no effect on long-term contextual fear memory.

Activation of EphB2 during training has no effect on short-term fear memory formation

We were interested to explore the possibility that EphB2 activity in LA may affect fear short-term memory (STM) formation. Toward that end, we activated optoEphB2 in LA, using optic fibers, during the CS-US presentations in fear conditioning training (Figure 3E). Control group expressed optoEphB2 in LA and was subjected to fear conditioning training but not exposed to light stimulation through the optic fibers. Fear conditioning memory was tested 2 hrs after training. Freezing during training was not different between the groups (F(1,14)= 0.011, p>0.9) there is no treatment × tone trial interaction (F(1.176,16.463)= 0.098, p>0.7) (Figure 3F). There was no significant difference between the light (n=9) and no light (n=7) groups when tested for fear STM (F(1,14)=0.043, p>0.8) (Figure 3H). There is no treatment × tone trial interaction (F(2.292, 32.092)= 0.597, p>0.5). These results show that EphB2 activation in LA has no effect on fear STM formation.

EphB2 activation in lateral amygdala during fear conditioning learning trials has no effect on short-term contextual fear memory

We were interested to explore whether optoEphB2 activation affects short-term contextual fear conditioning. Fear conditioning memory was tested 1 hr after training. Activation of EphB2 in LA (n=9) during fear conditioning has no effect on short-term contextual fear memory when compared to animals where optoEphB2 was not activated (n=7) (p>0.6) (Figure 3G). These results show that activation of EphB2 during fear conditioning trials has no effect on short-term contextual fear memory.

Activation of EphB2 after fear conditioning training has no effect on long-term fear memory formation

EphB activation that leads to enhancement of neuronal functions such as gene expression is effective only in conjunction with synaptic transmission (Takasu et al., 2002). We were therefore interested to explore whether activation of optoEphB2 is effective specifically during fear conditioning training (as shown above) or whether activation of optoEphB2 after fear conditioning training will also have an effect on long-term memory. Toward that end we trained the animals, that expressed optoEphB2 in LA, for fear conditioning and shined light through the optic fibers onto LA 6 minutes after the last CS (3 pulses of 20 seconds light similar to light stimulation protocol done during training) (Figure 3I). Freezing during training was not different between the groups (F(1,16)=0.312, p>0.5) there is no treatment × tone trial interaction (F(2,32)=0.351, p>0.7) (Figure 3J). Animals were tested for long-term fear memory formation 24 hrs after training. As seen in figure 3 post-training light stimulation (n=10) had no significant effect on long-term contextual (p>0.9; Figure 3K) and auditory (F(1,14)= 0.251, p>0.6; with no interaction F(4,64)= 1.094, p>0.3; Figure 2L) fear memory formation when compared to non-stimulated animals (n=8). The results together with the aforementioned observations show that EphB2 facilitates long-term memory when activated during training but not afterwards.

Light application to a kinase dead optoEphB2 in lateral amygdala has no effect on long-term fear conditioning memory formation

We were interested to examine whether the enhancement of fear memory by activation of optoEphB2 depends on its kinase activity. Toward that end we utilized a kinase dead mutant of optoEphB2 (optoEphB2-KD). We applied light into LA of mice that express optoEphB2-KD during fear conditioning and compared the effects to mice that express optoEphB2-KD in LA but were not subjected to light (Figure 4A). Freezing during training was not different between the groups (F(1,12)=0.993, p>0.3) there is no treatment × tone trial interaction (F(2,24)=0.111, p>0.8) (Figure 4B). There was no significant difference between the light (n=7) and no light (n=7) groups when tested for long-term contextual fear conditioning (p>0.8) (Figure 4C). There was no significant difference between the light (n=7) and no light (n=7) groups when tested for auditory fear conditioning LTM (F(1,12)=0.054, p>0.8) (Figure 4D). There is no treatment × tone trial interaction (F(1.681, 20167)=0.166, p>0.9). These results show that the enhancement of fear memory formation by optoEphB2 depends on its kinase activity. Moreover, it shows that application of light per se has no effect on LTM.

Figure 4. Light application to a kinase dead optoEphB2 in lateral amygdala has no effect on long-term fear conditioning memory formation.

Figure 4.

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A. Schematic description of protocol of behavior and light stimulation. B. Freezing during training in response to the tone CS is not different between light (n=7) and no light (n=7) animals (F(1,12)=0.993, p>0.3). C. Contextual fear conditioning tested 24 hours after fear conditioning is not different between the light and no light groups (p>0.8). D. Auditory fear conditioning tested 48 hours after fear conditioning is not different between animals subjected to light activation of optoEphB2 compared to no light mice (F(1,12)=0.054, p>0.8).

Activation of EphB2 during fear conditioning leads to phosphorylation of CREB

Activation of EphB leads to an increase in Src-dependent phosphorylation of CREB at Ser133 (Takasu et al., 2002). We show above (Fig 2) that photoactivation of optoEphB2 leads to Src phosphorylation. Overexpression of CREB in LA facilitates fear memory formation in a Ser133-phosphorylation dependent manner (Josselyn et al., 2001). We were therefore interested to explore the possibility the photoactivation of EphB2 during fear conditioning leads to an increase in phosphorylation of CREB at Ser133. As shown in figures 5B and 5C optoEphB2 activation by light during fear conditioning led to a significant increase in CREB phosphorylation at Ser133 in LA neurons that express optoEphB2 when compared to optoEphB2 expressing neurons in LA of animals that were subjected to fear conditioning but not subjected to light (p<0.031) indicating that activation of optoEphB2 in LA during fear conditioning led to a significant increase in CREB phosphorylation at Ser133 in LA. Similar results are shown using Western blot (Figure 5D) exhibiting an increase in CREB phosphorylation in LA of fear conditioned animals that express optoEphB2 in LA subjected to light during training compared to LA of fear conditioned mice that express optoEphB2 but not subjected to light (p<0.033). Application of light to the optoEphB2 kinase dead (optoEphB2-KD) in LA of fear conditioned animals during training (n=5) did not induce CREB phosphorylation in optoEphB2-KD expressing neurons compared to neurons in LA of fear conditioned mice that express optoEphB2-KD but not subjected to light during training (n=4) (p>0.8) indicating that the increased in CREB phosphorylation depends on EphB2 kinase activity (Figure 5E). Thus, these results together with previous observations infer that EphB2 enhances fear memory formation in LA through activation of CREB.

Figure 5. Activation of EphB2 during fear conditioning leads to phosphorylation of CREB.

Figure 5.

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A. Description of protocol used for CREB phosphorylation. B. Representative immunohistochemistry of phospho-CREB in LA of light stimulated or of non-stimulated LA of optoEphB2 expressing mouse. Arrows are examples of neurons where phospho-CREB and optoEphB2 colocalize. C. Quantified results of colocalized phospho-CREB and optoEphB2 in LA of mice. OptoEphB2 activation during fear conditioning led to a significant increase in CREB phosphorylation at Ser133 in LA neurons that express optoEphB2 when compared to optoEphB2 expressing neurons in LA of animals that were subjected to fear conditioning but were not subjected to light (p<0.031). D. Western blot analysis shows an increase in CREB phosphorylation in fear conditioned animals that express optoEphB2 in LA that are subjected to light in LA compared to LA in fear conditioned mice that express optoEphB2 but not subjected to light (p<0.033). E. Application of light to the optoEphB2 kinase dead (optoEphB2-KD) during fear conditioned (n=5) did not induce CREB phosphorylation in optoEphB2-KD expressing neurons compared to neurons is LA of fear conditioned mice that express optoEphB2-KD but not subjected to light (n=4) (p>0.8)

Mice lacking the C-terminal domain of EphB2 are impaired in long-term auditory fear memory formation

The observations above show that activation of EphB2 trough dimerization of its C-terminal part in LA facilitated auditory long-term fear memory formation. We therefore were interested to explore whether removing the C-terminal of EphB2 will have an opposite effect on the ability to form long-term fear memory. Toward that end, we studied fear memory formation in knockin mice where a EphB2-lacZ (Nuk-LacZ) mutant allele, designed to encode a fusion protein, comprised of the extracellular, transmembrane and juxtamembrane domains but lacks the entire tyrosine kinase catalytic and C-terminal domains of EphB2, was introduced into mice (EphB2lacz/lacz) by homologues recombination (Henkemeyer et al., 1996; Figure 4A). EphB2-Lacz is expressed in lateral amygdala (Figure 6B). We trained the EphB2lacz/lacz mice (n=9) for fear conditioning and compared their freezing during the long-term auditory fear memory test to freezing of wt mice (n=10). EphB2lacz/lacz mice froze significantly less than the wt mice (F(1,17)=17.198, p<0.002) (Fig. 6D). There is no significant group X tone interaction (F(4,68)= 0.691, p>0.6). These results indicate that EphB2 forward signaling mediated by the tyrosine kinase catalytic and C-terminal domains is essential for auditory fear LTM formation.

Figure 6. Mice lacking the C-terminal domain of EphB2 are impaired in long- but not short-term auditory fear memory formation.

Figure 6.

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A. Schematic description of the C-terminal truncated EphB2 expressed in knockin mice. B. The truncated EphB2 is expressed in lateral amygdala (B2) as detected by the β-Galactosidase assay. Truncated EphB2 is not expressed in wild type animals (B1). C. Short-term auditory fear memory in EphB2lacz/lacz mice (n=11) tested for fear memory formation 2 hrs after fear conditioning training is not significantly different from fear STM in wt mice (n=8) (F(1,17)=0.023, p>0.8). D. Long-term auditory fear memory in EphB2lacz/lacz mice (n=9) 24 hrs after fear conditioning training is significantly impaired when compared to wt mice (n=10) (F(1,17)=17.198, p<0.002).

Short-term auditory fear memory formation is intact in C-terminal truncated EphB2 mice

We were interested to explore whether short-term memory is affected in mice that lack the C-terminal domains of EphB2. Toward that end, we tested fear memory formation 2 hrs after fear conditioning training in EphB2lacz/lacz mice (n=11) and compared it to fear memory formation in wt mice (n=8). There is no significant change in freezing during STM memory formation between the groups (F(1,17)=0.023, p>0.8) (Fig. 6C). There is no group X tone interaction (F(4,68)=0.428, p>0.7). These results show that EphB2 forward signaling is not essential for auditory fear conditioning STM formation. In addition, it also shows that freezing per se, foot shock sensitivity and CS and US processing in the EphB2lacz/lacz mice are not impaired.

Activation of EphB2 forward signaling in lateral amygdala of C-terminal truncated EphB2 mice enhances long-term fear conditioning memory

We were interested to explore whether optoEphB2 activation can rescue long-term fear memory in mice that lack the C-terminal domains of EphB2 (EphB2lacz/lacz). We therefore expressed optoEphB2 in LA of EphB2lacz/lacz mice and activated it by light during fear conditioning training and compared it to fear conditioning in EphB2lacz/lacz mice that express optoEphB2 in LA but were not subjected to light. As can be seen in figure 7B the mice groups were not different in freezing responses during training (F(1,7)=0.354, p>0.5) there is no treatment × tone trial interaction (F(2,14)=0.612, p>0.5). Activation of optoEphB2 by light in LA enhanced the formation of long-term contextual fear conditioning memory when compared to controls with no light (p<0.035) (Figure 7C). Activation of optoEphB2 by light in LA enhanced the formation of long-term auditory fear conditioning memory when compared to controls with no light (F(1,7)=7.386, p<0.031) (Figure 7D). There is no group X tone interaction (F(4,28)=1.029, p>0.4). These results show that optoEphB2 can enhance long-term auditory fear memory formation in EphB2lacz/lacz mice showing the necessity of forward EphB2 signaling in LA for memory formation and enhancement. Moreover, it shows that optoEphB2 forward signaling can replace the function of endogenous EphB2 forward signaling for fear memory formation and enhancement.

Figure 7. Activation of EphB2 forward signaling in lateral amygdala of C-terminal truncated EphB2 mice enhances long-term fear conditioning memory.

Figure 7.

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A. Schematic description of protocol of behavior and light stimulation. B. Freezing during training in response to the tone CS is not different between light (n=5) and no light (n=4) animals (F(1,7)=0.354, p>0.5). C. Contextual fear conditioning tested 24 hours after fear conditioning is enhanced in the light group compared to no light group (p<0.035). D. Auditory fear conditioning tested 48 hours after fear conditioning is enhanced in animals subjected to light activation of optoEphB2 compared to no light mice (F(1,7)=7.386, p<0.031).

Discussion

EphB2 is involved in enhancing synaptic transmission and gene expression believed to underlie memory formation. In this study we examined whether activation of EphB2 in lateral amygdala (LA) enhances memory formation. Toward that end, we utilized a novel optogenetic technique that allows activation of EphB2 by light (optoEphB2) (Locke et al., 2017). We revealed that activation of optoEphB2 by blue light in LA during fear conditioning led to enhancement of long-term, but not short-term, auditory fear conditioning memory. Activation of optoEphB2 minutes after fear conditioning had no effect on long-term fear memory formation. Long- and short-term contextual fear conditioning memories, mediated by the hippocampus, were unaffected by activation of optoEphB2 in LA. We next examined the downstream effectors that are activated by optoEphB2 and revealed that CREB is activated following optoEphB2 photoactivation during fear conditioning leading to an increase in number of CREB-active neurons in LA. We also show that enhancement of fear conditioning and CREB activation in LA depend on optoEphB2 kinase activity. Finally, we revealed that EphB2 forward signaling is essential for fear LTM as long-term, but not short-term, fear memory is impaired in mice that lack the C-terminal portion of EphB2 and activation of optoEphB2 in LA of these mice during training enhance fear LTM.

Using photoactivatable EphB2 we revealed that activation of EphB2 forward signaling in LA enhances long-term but not short-term auditory fear memory formation. These observations show that increase of EphB2 activity during training does not enhance fear memory acquisition but rather its consolidation. Consistent with this conclusion are our observations that EphB2 forward signaling is necessary for the consolidation, but not acquisition, of fear memory as auditory long-term, but not short-term, fear conditioning is abolished in mice with C-terminal-truncated EphB2. Thus, not only that EphB2 forward signaling enhances memory consolidation its absence abolishes it.

Results from the current study show that fear memory formation can be enhanced by forward signaling of EphB2 in LA and does not require reverse signaling through ephrinBs. EphrinBs reverse signaling is involved in neuronal morphogenesis such as spine and synapse formation (e.g. Segura et al., 2007; Klein, 2009) and synaptic plasticity (e.g. Armstrong et al., 2006; Bouzioukh et al., 2007) cellular events believed to be involved in long-term memory formation (Lamprecht and LeDoux, 2004). However, our results show that such events mediated by ephrin reverse signaling are not necessary for the enhancement of fear long-term memory in LA. Thus, increase in EphB2 forward signaling enhances memory formation in LA independent of EphB2 reverse signaling.

EphB2 forward signaling activation leads to CREB phosphorylation and gene expression. Treatment of neurons that express EphB with ephrinB2-Fc, followed by glutamate stimulation, increased CREB Ser133 phosphorylation and downstream reporter-gene transcription. This effect was dependent on NMDA receptor and Src activation. Moreover, CREB phosphorylation is abolished in neurons expressing an EphB2 construct containing a deletion of the EphB2 cytoplasmic domain (Takasu et al., 2002). CREB plays a key role in the formation of fear memories in lateral amygdala. Overexpressing CREB in LA facilitates fear memory formation (Josselyn et al., 2001). Such enhancement effects of CREB depend on phosphorylation at Ser133, because similar overexpression of mCREB, a mutated version of CREB that cannot be phosphorylated at the Ser133 residue, does not facilitate LTM (Josselyn et al., 2001). Moreover, LA neurons with increased CREB are preferentially activated by fear memory expression, suggesting that they are selectively recruited into the memory trace (Han et al., 2007). Selectively deleting these neurons overexpressing CREB after learning blocked expression of that fear memory (Han et al., 2009). We examined whether photoactivation of optoEphB2 during fear conditioning activates CREB in LA. We revealed that activation of optoEphB2 increased the level of phospho-CREB in LA compared to animals that underwent fear conditioning but the optoEphB2 was not activated. Taken together, our results imply that optoEphB2 enhances long-term fear memory by activating CREB in LA pyramidal neurons.

We show that activation of optoEphB2 after fear conditioning has no effect on long-term fear memory formation. This observation indicates that activation of EphB2 is useful only during training in conjunction with synaptic transmission in LA. This result is consistent with the study showing that ephrinB2-Fc stimulation of EphB leads to CREB phosphorylation and enhancement of CREB-dependent transcription only if it is subjected together with glutamate application to neurons (Takasu et al., 2002). Glutamate or ephrinB2-Fc alone had no effect on CREB phosphorylation. Glutamate is released during fear conditioning and glutamate receptors are essential in amygdala during fear conditioning for fear memory formation (e.g. Rodrigues et al., 2001).

In conclusion, our study shows for the first time that the activation of EphB2 forward signaling during associative learning leads to CREB phosphorylation and to enhancement of long-term memory. Lack of EphB2 forward signaling abolishes long-term memory formation. EphB2 activation may be involved in enhanced pathological fear responses in fear-related diseases. Using the optoEphB2 technique may be used to improve memory in memory-related disorders.

Methods

Animals

Adult (8 weeks of age) male C57BL/6J mice weighing 22–28 g (ordered from Harlan laboratories) were used for behavioral and optogenetics studies. The generation of EphB2lacz/lacz has been described previously (Henkemeyer et al., 1996). Mutant mice were maintained in a heterozygous state on a 129XC57BI/6 background. Colonies founder mice were obtained from Prof. Ruediger Klein (Max Planck Institute of Neurobiology, Martinsried, Germany) and were bred at University of Haifa. Mice were housed at 22°C in a 12h light/dark cycle, with free access to food and water. Behavioral experiments were approved by the University of Haifa Institutional Committee for animal experiments in accordance with National Institutes of Health guidelines.

Live-cell imaging

NIH3T3 cells were maintained in Dulbecco’s Modified Eagle Medium (Gibco, Grand Island, NY or Lonza, Basel, Switzerland) with fetal bovine serum (FBS, BioWest, Kansas City, MO) and penicillin/streptomycin (Gibco). Transient transfection of optoEphB2 containing plasmid was carried out using Lipofectamine 2000 (Invitrogen, Grand Island, NY) following the manufacturer’s protocol. Images were taken in TIRF on an Olympus IX81 inverted microscope. A 561-nm laser was used to excite mCherry on optoEphB and 250-ms pulses of 488-nm light from an argon ion laser were delivered in between frames to photoactivate Cry2.

Western blots analysis of MEFs

Mouse embryonic fibroblasts (MEFs) expressing optoEphB2 grown in a 6-cm dish were illuminated with a blue light (~10–2 W/cm2) LED array for 1 minute. Control cells were kept in the dark. MEFs were lysed in modified kinase lysis buffer (KLB) as previously described (Ditlev et al., 2012), with 0.1% SDS added to aid in solubilizing large optoEphB2 or KD-optoEphB2 clusters. Proteins were separated by gel electrophoresis on 4–15% gradient polyacrylamide gels (BioRad, Hercules, CA), transferred to nitrocellulose membranes (General Electric Healthcare, Pittsburgh, PA), and blotted with anti-phosphotyrosine (Cell Signaling Technologies, Danvers, MA). Blots were visualized on an Odyssey IR scanner (LI-COR) using secondary antibodies labeled with IRDye 800. For co-IP experiments antibodies that were used are: anti-mCherry (thermos fisher PA5–34974), anti-V5 (thermos fisher R96025), rabbit anti-actin (CST 8457; Cell signaling Technologies) and mouse anti-actin (CST 3700; Cell signaling Technologies).

Western blot analysis for pCREB in mice

After decapitation LA tissue was dissected. Samples were homogenized in lysis buffer (10% glycerol, 1% triton X-100, 1 mM EDTA, 50 mM HEPES, 150 mM NaCl, phosphatase inhibitor 1:1000) and centrifuged for 5 min at 10,000 rpm. Afterwards, SDS sample buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2.3% sodium dodecyl sulfate (SDS) and 5% b-mercaptoethanol) was added to the supernatant. Samples were heated at 80°C for 5 min and stored at −20°C until use. Samples were subjected to SDS polyacrylamide gel electrophoresis (SDS–PAGE) followed by Western blot analysis. Blots were blocked in Tris-buffered saline solution containing 0.1% Tween 20 (TBST) and 5% BSA for 1 h at room temperature. The blots were incubated with anti-Phospho-CREB (Ser133, 1:1000; Cell signaling Laboratories) or anti-mCherry (1:1000; Abcam) overnight at 4°C. Blots were washed thrice with TBST and incubated for 1 h at room temperature with peroxidase anti-rabbit secondary antibody (1:10,000; Jackson ImmunoResearch Laboratories). The blots were then washed thrice in TBST and exposed to enhanced chemiluminescence with the 20-500-120 EZ ECL kit (Biological Industries, Kibbutz Beit-Haemek, Israel). Blots were exposed in ChemiDox XRS (Bio-Rad), and analyzed by Quantity-One 4.5.0 software (Bio-Rad).

AAV production and microinjection

High titer AAV viruses (>1X10^13 VG/ml) (SignaGen Laboratories, MD, USA) that express optoEphB2 under short CaMKII promoter were used. OptoEphB2-KD under short CaMKII promoter expressing AAVs at high titer (1.5X10^13) was produced by ELSC Vector Core Facility (Hebrew University of Jerusalem, Israel). Animals were anesthetized with Medetomidine (Domitor) 1mg/ml and Ketamine 100mg/ml cocktail, diluted in sterile isotonic saline (administered doses: Ketamine 50mg/kg; Domitor 0.5mg/kg; 100μl/10gm of animal body weight). Dipyrone (50%) was injected for analgesia before surgery and consecutive 3 days after surgery. AAV particles were injected (0.5μl/hemisphere, 0.1μl/min) aimed to LA/BLA. After virus injection, intracranial optic fibers (Thor labs, Fiber Optic Cannula, ∅1.25 mm Stainless Ferrule, ∅200 μm Core, 0.39 NA) were implanted 0.5 mm above the virus injection sites. Animals were allowed to recuperate for 4 weeks before behavioral experiments (e.g. Dana et al., 2014). After behavioral or histological procedures the animals were perfused and localization of AAVs was examined. Only mice with expression of mCherry within the borders of LA/BLA were included in the data analysis.

Fear conditioning

Animals were placed in the conditioning chamber and two minutes afterwards subjected to 3 pairs of tone (Conditioned stimulus (CS) - 20 secs, 2.8 kHz, 85 dB) that co-terminated with a foot shock (Unconditioned stimulus (US) - 2 secs, 0.8 mA). The inter-trial interval was 120 secs. Mice were tested for short-term (STM) or for long-term memory (LTM). For contextual fear conditioning the mice were tested in the same context (1 hr (STM) or 24hrs (LTM) after training) followed by auditory fear conditioning in a different context (2hrs (STM) or 48 hrs (LTM)). Freezing was scored automatically by the machine and the threshold during testing was identical to all animals.

Laser stimulation

Optic fibers were connected to a 473-nm blue laser diode (Shanghai Dreamlasers) via FC/PC adaptors. The light intensity ~15 mW/mm2 was measured at the tip of the fiber. A control group of animals got equal amount of viruses microinjected into LA/BLA along with the optic fiber implantation but did not receive light stimulation.

Immunohistochemistry

Immunohistochemistry experiments were performed one day after memory test. The animals were subjected to light in one side and the other side served as non-stimulated control. The animals were anesthetized immediately after the last light stimulation, for phospho-Src, or 7 min after fear conditioning and light stimulation, for phospho-CREB, by inhalation of isofluorane. Then mice were perfused intracardially with PBS followed by 4% paraformaldehyde in PBS using digital peristaltic pump (MU-D01, Major Science). After perfusion, mice were decapitated and their brains removed and placed in 30% sucrose, 1% paraformaldehyde, in PBS, 48 hours at 4°C for postfixation. Brains were frozen and sliced using a cooled cryostat (Leica, CM1900) at a thickness of 45μm. Slices from each animal were first treated with blocking solution (10% NGS, 3% BSA and 3% Triton in PBS 1x) and incubated for 1 hr at room temperature on a shaker at medium speed. Then the slices were subjected to anti-phospho-Src (Tyr-416) antibody (Cell Signaling, 1:300) or anti-phospho-CREB (Ser-133) antibody (Cell Signaling, 1:400) and were incubated overnight at 4°C on a shaker at medium speed. On the second day the slices were washed 3 times with PBS and subjected to secondary antibody Alexa 488 anti-rabbit (Molecular probes, 1:1000). For the NeuN and GFAP staining the slices were subjected to mouse anti-GFAP antibody (1:3000; Sigma-Aldrich) or rabbit-anti NeuN antibody (1:500; MBL) and were incubated overnight at 4°C on a shaker at medium speed. On the second day the slices were washed 3 times with PBS 1x and incubated with secondary Alexa 488 anti-rabbit or Alexa 488 anti-mouse antibody was added (1:1000; Molecular Probes) for 2 hrs at RT. Photographs of the brain slides were taken using a Nikon confocal microscope and quantification was done using Image J. For pSrc cell bodies were quantified and for pCREB stained nuclei were quantified. Before monitoring for viral expression the slides with animal numbers were mixed and examined blindly. The neurons are quantified from a single LA slice from each animal that was picked randomly.

β-Galactosidase assay

Wild type mice and EphB2lacZ/lacZ mice were scarified by decapitation. Brains were excised and immediately frozen in liquid nitrogen. After freezing, brains were covered with aluminum foil and kept at −80 °C until sectioning. Fifty μm brain sections were sliced with a cooled cryostat (Leica, CM1900) and mounted on Super Frost-coated slides. The slides with the slices were washed twice for 10 min with 0.01 M PBS, and then were fixated for 10 min at room temperature (RT) with the fixation solution (2% formaldehyde and 0.2% glutaraldehyde in PBS). After fixation the slides were washed again with PBS and incubated with the X-Gal staining solution (SigmaAldrich) at 37°C for at least 2 h. The tissue was removed from the staining solution and the slides were washed for 30 min with PBS. After washing, Slow Fade antifade medium (Invitrogen) was added to each dry slice. Slides were kept in the dark at 4°C until image acquisition.

Statistics

Data were analyzed with repeated measures ANOVA for auditory fear conditioning and Mann-Whitney U test for the contextual fear conditioning, immunohistochemistry and Western blot studies with an α level of 0.05 using the PASW statistics 20.

Acknowledgments

The study was funded by The German-Israeli Foundation for Scientific Research and Development (GIF) the Israel Science Foundation grant for R.L.

References

  1. Armstrong JN, Saganich MJ, Xu NJ, Henkemeyer M, Heinemann SF, Contractor A. (2006). B-ephrin reverse signaling is required for NMDA-independent long-term potentiation of mossy fibers in the hippocampus. J Neurosci. 26, 3474–3481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Attwood BK, Bourgognon JM, Patel S, Mucha M, Schiavon E, Skrzypiec AE, Young KW, Shiosaka S, Korostynski M, Piechota M, Przewlocki R, Pawlak R. (2011). Neuropsin cleaves EphB2 in the amygdala to control anxiety. Nature. 473, 372–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bouzioukh F1, Wilkinson GA, Adelmann G, Frotscher M, Stein V, Klein R. (2007). Tyrosine phosphorylation sites in ephrinB2 are required for hippocampal long-term potentiation but not long-term depression. J Neurosci. 27, 11279–11288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cissé M, Halabisky B, Harris J, Devidze N, Dubal DB, Sun B, Orr A, Lotz G, Kim DH, Hamto P, Ho K, Yu GQ, Mucke L. (2011). Reversing EphB2 depletion rescues cognitive functions in Alzheimer model. Nature. 469, 47–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Contractor A, Rogers C, Maron C, Henkemeyer M, Swanson GT, & Heinemann SF (2002). Trans-synaptic Eph receptor-ephrin signaling in hippocampal mossy fiber LTP. Science, 296, 1864–1869. [DOI] [PubMed] [Google Scholar]
  6. Cruz E, Soler-Cedeño O, Negrón G, Criado-Marrero M, Chompré G, Porter JT (2015). Infralimbic EphB2 Modulates Fear Extinction in Adolescent Rats. J Neurosci. 35(36):12394–403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dalva MB, Takasu MA, Lin MZ, Shamah SM, Hu L, Gale NW, & Greenberg ME (2000). EphB receptors interact with NMDA receptors and regulate excitatory synapse formation.Cell 103, 945–956. [DOI] [PubMed] [Google Scholar]
  8. Dana H, Chen TW, Hu A, Shields BC, Guo C, Looger LL, Kim DS, Svoboda K (2014). Thy1-GCaMP6 transgenic mice for neuronal population imaging in vivo.PLoS One 9, e108697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davis M, & Whalen PJ (2001). The amygdala: vigilance and emotion. Molecular Psychiatry, 6,13–34. [DOI] [PubMed] [Google Scholar]
  10. Dines M, Grinberg S, Vassiliev M, Ram A, Tamir T, Lamprecht R (2015). The roles of Eph receptors in contextual fear conditioning memory formation. Neurobiol Learn Mem. 124, 62–70. [DOI] [PubMed] [Google Scholar]
  11. Ditlev JA, Michalski PJ, Huber G, Rivera GM, Mohler WA, Loew LM, and Mayer BJ (2012). Stoichiometry of Nck-dependent actin polymerization in living cells. J. Cell Biol. 197, 643–658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fanselow MS, & LeDoux JE (1999). Why we think plasticity underlying Pavlovian fear conditioning occurs in the basolateral amygdala. Neuron, 23, 229 – 232. [DOI] [PubMed] [Google Scholar]
  13. Grunwald IC, Korte M, Wolfer D, Wilkinson GA, Unsicker K, Lipp HP, Bonhoeffer T, & Klein R (2001). Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity. Neuron 32, 1027–1040. [DOI] [PubMed] [Google Scholar]
  14. Han JH, Kushner SA, Yiu AP, Cole CJ, Matynia A, Brown RA, Neve RL, Guzowski JF, Silva AJ, Josselyn SA. (2007). Neuronal competition and selection during memory formation. Science. 316, 457–460. [DOI] [PubMed] [Google Scholar]
  15. Han JH, Kushner SA, Yiu AP, Hsiang HL, Buch T, Waisman A, Bontempi B, Neve RL, Frankland PW, Josselyn SA (2009). Selective erasure of a fear memory. Science. 323, 1492–6. [DOI] [PubMed] [Google Scholar]
  16. Han KA, Woo D, Kim S, Choii G, Jeon S, Won SY, Kim HM, Heo WD, Um JW, Ko J (2016). Neurotrophin-3 Regulates Synapse Development by Modulating TrkC-PTPσ Synaptic Adhesion and Intracellular Signaling Pathways. J Neurosci. 36, 4816–4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Henderson JT, Georgiou J, Jia Z, Robertson J, Elowe S, Roder JC, & Pawson T (2001). The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function. Neuron 32, 1041–1056. [DOI] [PubMed] [Google Scholar]
  18. Henkemeyer M, Orioli D, Henderson JT, Saxton TM, Roder J, Pawson T, & Klein R (1996). Nuk controls pathfinding of commissural axons in the mammalian central nervous system. Cell 86, 35–46. [DOI] [PubMed] [Google Scholar]
  19. Johansen JP, Cain CK, Ostroff LE, & LeDoux JE (2011). Molecular mechanisms of fear learning and memory. Cell 147, 509–524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Josselyn SA, Shi C, Carlezon WA Jr, Neve RL, Nestler EJ, Davis M (2001). Long-term memory is facilitated by cAMP response element-binding protein overexpression in the amygdala. J Neurosci. 21, 2404–2412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kim JJ and Fanselow MS (1992). Modality-specific retrograde amnesia of fear.Science. 256, 675–677. [DOI] [PubMed] [Google Scholar]
  22. Klein R (2009). Bidirectional modulation of synaptic functions by Eph/ephrin signaling. Nature Neuroscience 12, 15–20. [DOI] [PubMed] [Google Scholar]
  23. Lamprecht R, & LeDoux J (2004). Structural plasticity and memory. Nature Reviews Neuroscience 5, 45–54. [DOI] [PubMed] [Google Scholar]
  24. LeDoux JE (2000). Emotion circuits in the brain. Annual Review in Neuroscience 23,155–184. [DOI] [PubMed] [Google Scholar]
  25. Locke C, Machida K, Wu Y, Yu J (2017). Optogenetic activation of EphB2 receptor in dendrites induced actin polymerization by activating Arg kinase.Biol Open. 6, 1820–1830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Maren S (2005). Synaptic mechanisms of associative memory in the amygdala. Neuron 47, 783–786. [DOI] [PubMed] [Google Scholar]
  27. McDonald AJ, Muller JF, Mascagni F (2002). GABAergic innervation of alpha type II calcium/calmodulin-dependent protein kinase immunoreactive pyramidal neurons in the rat basolateral amygdala. J Comp Neurol 446, 199–218. [DOI] [PubMed] [Google Scholar]
  28. Phillips RG and LeDoux JE. (1992), Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav Neurosci. 106, 274–285. [DOI] [PubMed] [Google Scholar]
  29. Rodrigues SM, Schafe GE, & LeDoux JE (2001). Intra-amygdala blockade of the NR2B subunit of the NMDA receptor disrupts the acquisition but not the expression of fear conditioning. J Neurosci. 21, 6889–6896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rodrigues SM, Schafe GE, & LeDoux JE (2004). Molecular mechanisms underlying emotional learning and memory in the lateral amygdala. Neuron 44, 75–91. [DOI] [PubMed] [Google Scholar]
  31. Sah P, Faber ES, Lopez De Armentia M, & Power J (2003). The amygdaloid complex: anatomy and physiology. Physiological Reviews, 83, 803–834. [DOI] [PubMed] [Google Scholar]
  32. Schafe GE, Nader K, Blair HT, & LeDoux JE (2001). Memory consolidation of Pavlovian fear conditioning: a cellular and molecular perspective. Trends in Neuroscience 24, 540–546. [DOI] [PubMed] [Google Scholar]
  33. Schaupp A, Sabet O, Dudanova I, Ponserre M, Bastiaens P, Klein R (2014). The composition of EphB2 clusters determines the strength in the cellular repulsion response. J Cell Biol. 204, 409–422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Segura I, Essmann CL, Weinges S, Acker-Palmer A. (2007). Grb4 and GIT1 transduce ephrinB reverse signals modulating spine morphogenesis and synapse formation. Nat Neurosci. 10, 301–310. [DOI] [PubMed] [Google Scholar]
  35. Sheffler-Collins SI, & Dalva MB (2012). EphBs: an integral link between synaptic function and synaptopathies.Trends in Neuroscience, 35, 293–304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Takasu MA, Dalva MB, Zigmond RE, & Greenberg ME (2002). Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors. Science 295, 491–495. [DOI] [PubMed] [Google Scholar]
  37. Taslimi A, Vrana JD, Chen D, Borinskaya S, Mayer BJ, Kennedy MJ, Tucker CL (2014). An optimized optogenetic clustering tool for probing protein interaction and function. Nat Commun. 5, 4925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tolias KF, Bikoff JB, Burette A, Paradis S, Harrar D, Tavazoie S, Weinberg RJ, & Greenberg ME (2005). The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines.Neuron 45, 525–538. [DOI] [PubMed] [Google Scholar]
  39. Zhang K & Cui B (2015). Optogenetic control of intracellular signaling pathways. Trends Biotechnol. 33, 92–100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhu XN, Liu XD, Zhuang H, Henkemeyer M, Yang JY, Xu NJ (2016). Amygdala EphB2 Signaling Regulates Glutamatergic Neuron Maturation and Innate Fear. J Neurosci. 36, 10151–10162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zisch AH, Pazzagli C, Freeman AL, Schneller M, Hadman M, Smith JW, Ruoslahti E, and Pasquale EB (2000). Replacing two conserved tyrosines of the EphB2 receptor with glutamic acid prevents binding of SH2 domains without abrogating kinase activity and biological responses. Oncogene 19, 177–187. [DOI] [PubMed] [Google Scholar]

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