EphrinB2 in excitatory neurons and astrocytes in the basolateral amygdala controls long-term fear memory formation.

EphrinB2 regulates synaptic transmission and morphology however its role in memory formation is unknown. Here we show that deleting ephrinB2 from excitatory neurons in the basolateral amygdala (BLA) of male mice impairs long-term (LTM), but not short-term (STM), fear memory formation. Deleting ephrinB2 from astrocytes in the BLA impairs fear LTM but not STM. Removing ephrinB2 from astrocytes in the BLA reduces the level of the excitatory amino acid transporter 1 (EAAT1) in these cells. Inhibiting EAAT1 activity in the BLA during fear conditioning, by its specific inhibitor UCPH-101, impairs fear LTM showing that EAAT1 in the BLA is needed for fear LTM formation. The administration of ephrinB2 into the BLA during fear conditioning training enhances fear LTM. Moreover, ephrinB2 increases the ability of fear conditioning to activate cells in the BLA as detected by c-Fos labeling. EphrinB2 therefore determines the threshold for fear memory formation. In contrast to mature neurons, we show that ephrinB2 in neural stem cells (NSCs) is not needed for fear LTM. Our study shows that ephrinB2 in the BLA determines the strength of long-term memory consolidation.

EphB2 activation in neural stem cells in the basolateral amygdala facilitates neurogenesis and enhances long-term memory.

Many brain diseases lead to a reduction in the number of functional neurons and it would be of value to be able to increase the number of neurons in the affected brain areas. In this study, we examined whether we can promote neural stem cells to produce mature neurons and whether an increase in the mature neurons can affect cognitive performance. We detected that the EphB2 receptor is localized in immature basolateral amygdala (BLA) neurons. We therefore aimed to increase the level of EphB2 activity in neural stem cells (NSCs) in the BLA and examine the effects on the production of mature neurons and cognition. Toward that end, we utilized a photoactivatable EphB2 construct (optoEphB2) to increase EphB2 forward signaling in NSCs in the BLA. We revealed that the activation of optoEphB2 in NSCs in the BLA increased the level of immature and mature neurons in the BLA. We further found that activation of optoEphB2 in BLA NSCs enhanced auditory, but not contextual, long-term fear memory formation. Impairing EphB2 forward signaling did not affect the level of immature and mature neurons in the BLA. This study provides evidence that NSCs can be promoted to produce mature neurons by activating EphB2 to enhance specific brain functions.

What kind of phonation causes the strongest vocal fold collision? - A hemi-larynx phonation contact pressure study.

This paper presents a measurement setup which is able to measure the distribution of small scale pressure on an area of 15.2 mm × 30.4 mm with a sample rate up to 1.2 kHz. It was used to investigate the contact pressures of vocal folds during phonation. This was performed in ex vivo experiments of 11 porcine larynges. The contact pressure at the medial surface and other phonation parameters, as the glottal resistance and the closing velocity of the vocal fold, were measured at different adduction and elongation levels and air flow rates. A statistical analysis was carried out. It could be shown that the contact pressure rises, when the vocal fold is manipulated or when the flow rate is increased.

Learning-induced bidirectional enhancement of inhibitory synaptic metaplasticity.

Training rodents in a particularly difficult olfactory-discrimination (OD) task results in the acquisition of the ability to perform the task well, termed 'rule learning'. In addition to enhanced intrinsic excitability and synaptic excitation in piriform cortex pyramidal neurons, rule learning results in increased synaptic inhibition across the whole cortical network to the point where it precisely maintains the balance between inhibition and excitation. The mechanism underlying such precise inhibitory enhancement remains to be explored. Here, we use brain slices from transgenic mice (VGAT-ChR2-EYFP), enabling optogenetic stimulation of single GABAergic neurons and recordings of unitary synaptic events in pyramidal neurons. Quantal analysis revealed that learning-induced enhanced inhibition is mediated by increased quantal size of the evoked inhibitory events. Next, we examined the plasticity of synaptic inhibition induced by long-lasting, intrinsically evoked spike firing in post-synaptic neurons. Repetitive depolarizing current pulses from depolarized (-70 mV) or hyperpolarized (-90 mV) membrane potentials induced long-term depression (LTD) and long-term potentiation (LTP) of synaptic inhibition, respectively. We found a profound bidirectional increase in the ability to induce both LTD, mediated by L-type calcium channels, and LTP, mediated by R-type calcium channels after rule learning. Blocking the GABAB receptor reversed the effect of intrinsic stimulation at -90 mV from LTP to LTD. We suggest that learning greatly enhances the ability to modify the strength of synaptic inhibition of principal neurons in both directions. Such plasticity of synaptic plasticity allows fine-tuning of inhibition on each particular neuron, thereby stabilizing the network while maintaining the memory of the rule. KEY POINTS: Olfactory discrimination rule learning results in long-lasting enhancement of synaptic inhibition on piriform cortex pyramidal neurons. Quantal analysis of unitary inhibitory synaptic events, evoked by optogenetic minimal stimulation, revealed that enhanced synaptic inhibition is mediated by increased quantal size. Surprisingly, metaplasticity of synaptic inhibition, induced by intrinsically evoked repetitive spike firing, is increased bidirectionally. The susceptibility to both long-term depression (LTD) and long-term potentiation (LTP) of inhibition is enhanced after learning. LTD of synaptic inhibition is mediated by L-type calcium channels and LTP by R-type calcium channels. LTP is also dependent on activation of GABAB receptors. We suggest that learning-induced changes in the metaplasticity of synaptic inhibition enable the fine-tuning of inhibition on each particular neuron, thereby stabilizing the network while maintaining the memory of the rule.

Behind the Complex Interplay of Phonation: Investigating Elasticity of Vocal Folds With Pipette Aspiration Technique During Ex Vivo Phonation Experiments.

OBJECTIVES: The vibration of the vocal folds produces the primary sound for the human speech. The vibration depends mainly on the pressure, airflow of the lungs, and the material properties of the vocal folds. In order to change them, muscles in the larynx stretch the vocal folds. This interplay is rarely investigated, but can give insight in the complex process of speech production. Most material properties studies are damaging the tissue; therefore, a nondestructive one is desired. METHODS: An ex vivo phonation experiment combined with the dynamic Pipette Aspiration Technique is used to investigate 10 porcine larynges, under manipulations of different adduction and elongation levels. For each manipulation, the near surface material properties of the vocal folds are measured as well as different phonation parameters like the subglottal pressure, glottal resistance, frequency, and stiffness. Thereby, a high-speed camera was used to record the vocal fold movement. RESULTS: On most of the measured parameters, the manipulations do show an effect. Both manipulations lead to a higher phonation frequency and an increase of the stiffness of the tissue. Comparing both manipulations, the elongation results in higher elasticity values than the adduction. Different measurement parameters have been compared with each other and correlations could be found. Where the strongest correlation are found among the elasticity values of different frequencies. But it can also be seen that the elasticity values correlate with phonation parameters. CONCLUSION: It was possible to produce a data set of 560 measurements in total. To our knowledge, this is the first time Pipette Aspiration Technique was combined with ex vivo phonation measurements for combined measurements. The amount of measurement data made it possible to carry out statistic investigations. The effect of the manipulations on material properties as well as on phonation parameters could be measured and different correlations could be found. The results lead to the hypothesis that the stretch does not have a huge effect on the material properties of the lamina propria, but more on the underlying muscle.

Fear Conditioning Leads to Enduring Alterations in RNA Transcripts in Hippocampal Neuropil that are Dependent on EphB2 Forward Signaling.

Alterations in mRNA transcription have been associated with changes in brain functions. We wanted to examine if fear conditioning causes long-term changes in transcriptome profiles in the basolateral amygdala (BLA) and hippocampus using RNA-Seq and laser microdissection microscopy. We further aimed to uncover whether these changes are involved in memory formation by monitoring their levels in EphB2lacZ/lacZ mice, which lack EphB2 forward signaling and can form short-term fear conditioning memory but not long-term fear conditioning memory. We found transcriptome signatures unique to each brain region that are comprise of specific cellular pathways. We also revealed that fear conditioning leads to alterations in mRNAs levels 24 h after training in hippocampal neuropil, but not in hippocampal cell layers or BLA. The two main groups of altered mRNAs encode proteins involved in neuronal transmission, neuronal morphogenesis and neuronal development and the vast majority are known to be enriched in neurons. None of these mRNAs levels were altered by fear conditioning in EphB2lacZ/lacZ mice, which were also impaired in long-term fear memory. We show here that fear conditioning leads to an enduring alteration in mRNAs levels in hippocampal neuropil that is dependent on processes mediated by EphB2 that are needed for long-term memory formation.

Rac1 GTPase activation impairs fear conditioning-induced structural changes in basolateral amygdala neurons and long-term fear memory formation.

Long-term memory formation leads to enduring alterations in synaptic efficacy and neuronal responses that may be created by changes in neuronal morphology. We show that fear conditioning leads to a long-lasting increase in the volume of the primary and secondary dendritic branches, but not of distal branches, of neurons located at the basolateral amygdala (BLA). The length of the dendritic branches is not affected by fear conditioning. Fear conditioning leads to an enduring increase in the length and volume of dendritic spines, especially in the length of the spine neck and the volume of the spine head. Fear conditioning does not affect dendritic spine density. We further reveal that activation of Rac1 in BLA during fear conditioning impairs long-term auditory, but not contextual, fear conditioning memory. Activation of Rac1 during fear conditioning prevents the enduring increase in the dendritic primary branch volume and dendritic spines length and volume. Rac1 activation per se has no effect on neuronal morphology. These results show that fear conditioning induces changes known to reduce the inhibition of signal propagation along the dendrite and the increase in synaptic efficacy whereas preventing these changes, by Rac1 activation, impairs fear memory formation.

Quasi-static ultrasound elastography of ex-vivo porcine vocal folds during passive elongation and adduction.

OBJECTIVES: The elastic properties of the vocal folds have great influence on the primary sound and thus on the entire subsequent phonation process. Muscle contractions in the larynx can alter the elastic properties of the vocal fold tissue. Quasi-static ultrasound elastography is a non-destructive examination method that can be applied to ex-vivo vocal folds. In this work, porcine vocal folds were passively elongated and adducted and the changes of the elastic properties due to that manipulations were measured. METHODS: Manipulations were performed by applying force to sewn-in sutures. Elongation was achieved by a suture attached to the thyroid cartilage, which was pulled forward by defined weights. Adduction was effected by two sutures exerting torque on the arytenoid cartilage. A series of ten specimens was examined and evaluated using a quasi-static elastography algorithm. In addition, the surface stretch was measured optically using tattooed reference points. RESULTS: This study showed that the expected stiffening of the tissue during the manipulations can be measured using quasi-static ultrasound elastography. The measured effect of elongation and adduction, both of which result in stretching of the tissue, is stiffening. However, the relative change of specific manipulations is not the same for the same load on different larynges, but is rather related to stretch caused and other uninvestigated factors. CONCLUSION: The passive elongation and adduction of vocal folds stiffen the tissue of the vocal folds and can be measured using ultrasound elastography.

Nck1 activity in lateral amygdala regulates long-term fear memory formation.

Fear conditioning leads to long-term fear memory formation and is a model for studying fear-related psychopathological conditions such as phobias and post-traumatic stress disorder. Long-term fear memory formation is believed to involve alterations of synaptic efficacy mediated by changes in synaptic transmission and morphology in lateral amygdala (LA). Nck1 is a key neuronal adaptor protein involved in the regulation of the actin cytoskeleton and the neuronal processes believed to be involved in memory formation. However, the role of Nck1 in memory formation is not known. Here we explored the role of Nck1 in fear memory formation in lateral amygdala (LA). Reduction of Nck1 in excitatory neurons in LA enhanced long-term, but not short-term, auditory fear conditioning memory. Activation of Nck1, by using a photoactivatable Nck1 (PA-Nck1), during auditory fear conditioning in excitatory neurons in LA impaired long-term, but not short-term, fear memory. Activation of Nck1 immediately or a day after fear conditioning did not affect fear memory. The hippocampal-mediated contextual fear memory was not affected by the reduction or activation of Nck1 in LA. We show that Nck1 is localized to the presynapses in LA. Nck1 activation in LA excitatory neurons decreased the frequency of AMPA receptors-mediated miniature excitatory synaptic currents (mEPSCs). Nck1 activation did not affect GABA receptor-mediated inhibitory synaptic currents (mIPSCs). These results show that Nck1 activity in excitatory neurons in LA regulates glutamate release and sets the threshold for fear memory formation. Moreover, our research shows that Nck1 may serve as a target for pharmacological treatment of fear and anxiety disorders.

EphrinA4 mimetic peptide impairs fear conditioning memory reconsolidation in lateral amygdala.

Fear memory may undergo a process after memory reactivation called reconsolidation. To examine the roles of ephrinA4 in fear memory reconsolidation an inhibitory ephrinA4 mimetic peptide (pep-ephrinA4), that targets the EphA binding site and inhibits EphA activation, was used. Pep-ephrinA4 was microinjected into the lateral amygdala (LA) of fear-conditioned rats 24 h after training and 30 min before tone CS memory retrieval. Memory retrieval was unaffected by pep-ephrinA4. However, the animals were impaired in fear memory tested 1 h or 24 h afterward when compared to controls. Fear-conditioned animals injected with pep-ephrinA4 into LA immediately after long-term memory retrieval were unaffected when tested 24 h afterward. Microinjection into LA of a peptide originated from an ephrinA4 site that does not interact with EphA did not affect fear memory reconsolidation. Rats that were administrated with pep-ephrinA4 systemically 24 h after fear conditioning and 30 min before CS memory retrieval were impaired in long-term fear conditioning memory tested 24 h afterward when compared to the control peptide. These results show that ephrinA4 binding sites are needed for long-term fear memory reconsolidation in LA and may serve as a target for the treatment of fear-related disorders by blocking reconsolidation.

Actin Cytoskeleton Role in the Maintenance of Neuronal Morphology and Long-Term Memory.

Evidence indicates that long-term memory formation creates long-lasting changes in neuronal morphology within a specific neuronal network that forms the memory trace. Dendritic spines, which include most of the excitatory synapses in excitatory neurons, are formed or eliminated by learning. These changes may be long-lasting and correlate with memory strength. Moreover, learning-induced changes in the morphology of existing spines can also contribute to the formation of the neuronal network that underlies memory. Altering spines morphology after memory consolidation can erase memory. These observations strongly suggest that learning-induced spines modifications can constitute the changes in synaptic connectivity within the neuronal network that form memory and that stabilization of this network maintains long-term memory. The formation and elimination of spines and other finer morphological changes in spines are mediated by the actin cytoskeleton. The actin cytoskeleton forms networks within the spine that support its structure. Therefore, it is believed that the actin cytoskeleton mediates spine morphogenesis induced by learning. Any long-lasting changes in the spine morphology induced by learning require the preservation of the spine actin cytoskeleton network to support and stabilize the spine new structure. However, the actin cytoskeleton is highly dynamic, and the turnover of actin and its regulatory proteins that determine and support the actin cytoskeleton network structure is relatively fast. Molecular models, suggested here, describe ways to overcome the dynamic nature of the actin cytoskeleton and the fast protein turnover and to support an enduring actin cytoskeleton network within the spines, spines stability and long-term memory. These models are based on long-lasting changes in actin regulatory proteins concentrations within the spine or the formation of a long-lasting scaffold and the ability for its recurring rebuilding within the spine. The persistence of the actin cytoskeleton network within the spine is suggested to support long-lasting spine structure and the maintenance of long-term memory.

A Quasi-Static Quantitative Ultrasound Elastography Algorithm Using Optical Flow.

Ultrasound elastography is a constantly developing imaging technique which is capable of displaying the elastic properties of tissue. The measured characteristics could help to refine physiological tissue models, but also indicate pathological changes. Therefore, elastography data give valuable insights into tissue properties. This paper presents an algorithm that measures the spatially resolved Young's modulus of inhomogeneous gelatin phantoms using a CINE sequence of a quasi-static compression and a load cell measuring the compressing force. An optical flow algorithm evaluates the resulting images, the stresses and strains are computed, and, conclusively, the Young's modulus and the Poisson's ratio are calculated. The whole algorithm and its results are evaluated by a performance descriptor, which determines the subsequent calculation and gives the user a trustability index of the modulus estimation. The algorithm shows a good match between the mechanically measured modulus and the elastography result-more precisely, the relative error of the Young's modulus estimation with a maximum error 35%. Therefore, this study presents a new algorithm that is capable of measuring the elastic properties of gelatin specimens in a quantitative way using only the image data. Further, the computation is monitored and evaluated by a performance descriptor, which measures the trustability of the results.

Dynamic Biomechanical Analysis of Vocal Folds Using Pipette Aspiration Technique.

The voice producing process is a complex interplay between glottal pressure, vocal folds, their elasticity and tension. The material properties of vocal folds are still insufficiently studied, because the determination of material properties in soft tissues is often difficult and connected to extensive experimental setups. To shed light on this less researched area, in this work, a dynamic pipette aspiration technique is utilized to measure the elasticity in a frequency range of 100-1000 Hz. The complex elasticity could be assessed with the phase shift between exciting pressure and tissue movement. The dynamic pipette aspiration setup has been miniaturized with regard to a future in-vivo application. The techniques were applied on 3 different porcine larynges 4 h and 1 d postmortem, in order to investigate the deterioration of the tissue over time and analyze correlation in elasticity values between vocal fold pairs. It was found that vocal fold pairs do have different absolute elasticity values but similar trends. This leads to the assumption that those trends are more important for phonation than having same absolute values.

The role of p21-activated kinase in maintaining the fear learning-induced modulation of excitation/inhibition ratio in lateral amygdala.

We study the relations between different learning paradigms and enduring changes in excitatory synaptic transmission. Here we show that auditory fear conditioning (AFC), but not olfactory fear conditioning (OFC) training, led to enduring enhancement in AMPA-mediated miniature EPSCs (mEPSCs). Moreover, olfactory unpaired training led to a stable significant reduction in excitatory synaptic transmission. However, olfactory discrimination learning (OD) did not modulate postsynaptic AMPA-mediated mEPSCs in LA. The p21-activated kinase (PAK) activity, previously shown to have a key role in maintaining persistent long-lasting enhancement in synaptic inhibition after OFC, has an opposing effect on excitatory synaptic transmission. PAK maintained the level of excitatory synaptic transmission in the amygdala in all experimental groups, except in neurons in the OFC trained rats. PAK also maintained excitatory synaptic transmission in all neurons of auditory fear conditioning and naïve training groups except in neurons of the auditory safety learning. Safety learning was previously shown in our study to enhance synaptic inhibition. We thus suggest that PAK maintains inhibitory synaptic transmission in a learning-dependent manner and on the other hand affects excitatory synaptic transmission only in groups where learning has not affected inhibitory transmission. Thus, PAK controls learning-induced changes in the excitation/inhibition balance.

The Role of Rac GTPase in Dendritic Spine Morphogenesis and Memory.

The ability to form memories in the brain is needed for daily functions, and its impairment is associated with human mental disorders. Evidence indicates that long-term memory (LTM)-related processes such as its consolidation, extinction and forgetting involve changes of synaptic efficacy produced by alterations in neural transmission and morphology. Modulation of the morphology and number of dendritic spines has been proposed to contribute to changes in neuronal transmission mediating such LTM-related processes. Rac GTPase activity is regulated by synaptic activation and it can affect spine morphology by controlling actin-regulatory proteins. Recent evidence shows that changes in Rac GTPase activity affect memory consolidation, extinction, erasure and forgetting and can affect spine morphology in brain areas that mediate these behaviors. Altered Rac GTPase activity is associated with abnormal spine morphology and brain disorders. By affecting Rac GTPase activity we can further understand the roles of spine morphogenesis in memory. Moreover, manipulation of Rac GTPase activity may serve as a therapeutic tool for the treatment of memory-related brain diseases.

A Cellular Mechanism of Learning-Induced Enhancement of Synaptic Inhibition: PKC-Dependent Upregulation of KCC2 Activation.

Long-term memory of complex olfactory learning is expressed by wide spread enhancement in excitatory and inhibitory synaptic transmission onto piriform cortex pyramidal neurons. A particularly interesting modification in synaptic inhibition is the hyperpolarization of the reversal potential of the fast post synaptic inhibitory potential (fIPSP). Here we study the mechanism underlying the maintenance of such a shift in the fIPSP. Blocking of the neuronal specific K+-Cl- co-transporter (KCC2) in neurons of trained rats significantly depolarized the averaged fIPSP reversal potential of the spontaneous miniature inhibitory post synaptic currents (mIPSCs), to the averaged pre-training level. A similar effect was obtained by blocking PKC, which was previously shown to upregulate KCC2. Accordingly, the level of PKC-dependent phosphorylation of KCC2, at the serine 940 site, was significantly increased after learning. In contrast, blocking two other key second messenger systems CaMKII and PKA, which have no phosphorylation sites on KCC2, had no effect on the fIPSP reversal potential. Importantly, the PKC inhibitor also reduced the averaged amplitude of the spontaneous miniature excitatory synaptic currents (mEPSCs) in neurons of trained rats only, to the pre-training level. We conclude that learning-induced hyper-polarization of the fIPSP reversal potential is mediated by PKC-dependent increase of KCC2 phosphorylation.

Learning-induced enduring changes in inhibitory synaptic transmission in lateral amygdala are mediated by p21-activated kinase.

In this study we explored whether learning leads to enduring changes in inhibitory synaptic transmission in lateral amygdala (LA). We revealed that olfactory discrimination (OD) learning in rats led to a long-lasting increase in postsynaptic GABAA channel-mediated miniature inhibitory postsynaptic currents (mIPSCs) in LA. Olfactory fear conditioning, but not auditory fear conditioning, also led to enduring enhancement in GABAA-mediated mIPSCs. Auditory fear conditioning, but not olfactory fear conditioning or OD learning, induced an enduring reduction in the frequency but not the current of mIPSC events. We found that p21-activated kinase (PAK) activity is needed to maintain OD and olfactory fear conditioning learning-induced enduring enhancement of mIPSCs. Further analysis revealed that OD led to an increase in GABAA channel conductance whereas olfactory fear conditioning increased the number of GABAA channels. These alterations in GABAA channels conductance and level are controlled by PAK activity. Our study shows that the learning-induced increase in postsynaptic inhibitory transmission in LA is specific to the sensory modality. However, the mechanism that mediates the increase in inhibitory transmission, namely the increase in the conductance or in the level of GABAA channel, is learning specific.NEW & NOTEWORTHY Here we studied whether learning leads to long-lasting alterations in inhibitory synaptic transmission in lateral amygdala (LA). We revealed that learning led to enduring changes in inhibitory synaptic transmission in LA that are affected by the sensory modality (auditory or olfaction) used during learning. However, the mechanism that mediated the changes in inhibitory transmission (alterations in GABAA channel level or conductance) depended on the type of learning. These long-lasting alterations are maintained by p21-activated kinase.

Long-term memory is maintained by continuous activity of Arp2/3 in lateral amygdala.

Evidence indicates that long-term memory formation involves alterations in synaptic efficacy produced by modifications in neural transmission and morphology. However, it is not clear how such changes induced by learning, that encode memory, are maintained over long period of time to preserve long-term memory. It has been shown that the actin nucleating protein Arp2/3 is essential for supporting neuronal morphology and synaptic transmission. We therefore hypothesized that continuous Arp2/3 activity is needed to maintain long-term memory over time. To test this hypothesis we microinjected into lateral amygdala (LA) of rats CK-666, a specific inhibitor of Arp2/3, two days after fear conditioning and tested the effect on long-term fear memory maintenance a day afterward. We found that injection of CK-666 two days after training abolished fear conditioning memory. Fear conditioning could be formed when a control compound CK-689 was applied two days after training. Microinjection of CK-666 a day before fear conditioning training had no effect on fear conditioning learning and long-term memory formation. We revealed that Arp2/3 is also needed to maintain long-term conditioned taste aversion (CTA) memory in LA. Microinjection of CK-666 two days after CTA training impaired long-term memory tested a day afterwards. We conclude that continuous activity of Arp2/3 in LA is essential for the maintenance of long-term memory.

EphB2 receptor forward signaling is needed for normal long-term memory formation in aged mice.

The molecular mechanisms underpinning age-related changes in the ability to form long-term memory need to be clarified. EphB2 receptors and their ephrin ligands are involved in key cellular functions such as neuronal morphogenesis and synaptic transmission believed to be involved in long-term memory formation. We were therefore interested to explore whether EphB2 is involved in the alterations in memory formation abilities observed in old age. Toward that end, we examined the ability to form long-term memory in mice that lack EphB2 (EphB2-/-). A previous study has shown that the ability to form long-term conditioned taste aversion (CTA) memory in young EphB2-/- mice remains intact. In the present study, we report that long-term CTA memory formation is improved in old wild-type mice but not in age-matched old EphB2-/- mice. To further explore EphB2 mechanisms responsible for this difference in memory formation ability, we examined CTA memory in EphB2lacZ/lacZ mice devoid of EphB2 forward signaling. We found that the ability to create CTA long-term memory is unaffected in young EphB2lacZ/lacZ mice. However, the ability to form an increased long-term CTA memory shown in old wild-type mice is impaired in old EphB2lacZ/lacZ mice. The inability to form enhanced CTA long-term memory in EphB2-/- and EphB2lacZ/lacZ old mice was not caused by differences in taste perception or ability to consume fluids. Thus, our observations show that the absence of EphB2 forward signaling in old mice impairs the ability to form enhanced long-term CTA memory and indicate that EphB2 forward signaling is needed for normal memory formation in aged mice.

Regulation of signaling proteins in the brain by light.

In order to study the role of signaling proteins, such as kinases and GTPases, in brain functions it is necessary to control their activity at the appropriate spatiotemporal resolution and to examine the cellular and behavioral effects of such changes in activity. Reduced spatiotemporal resolution in the regulation of these proteins activity will impede the ability to understand the proteins normal functions as longer modification of their activity in non-normal locations could lead to effects different from their natural functions. To control intracellular signaling proteins at the highest temporal resolution recent innovative optogenetic approaches were developed to allow the control of photoactivable signaling proteins activity by light. These photoactivatable proteins can be activated in selected cell population in brain and in specific subcellular compartments. Minimal-invasive tools are being developed to photoactivate these proteins for study and therapy. Together these techniques afford an unprecedented spatiotemporal control of signaling proteins activity to unveil the function of brain proteins with high accuracy in behaving animals. As dysfunctional signaling proteins are involved in brain diseases, the optogenetic technique has also the potential to be used as a tool to treat brain diseases.