Aging in zebrafish is associated with reduced locomotor activity and strain dependent changes in bottom dwelling and thigmotaxis.

Aging is associated with a wide range of physiological and behavioral changes in many species. Zebrafish, like humans, rodents, and birds, exhibits gradual senescence, and thus may be a useful model organism for identifying evolutionarily conserved mechanisms related to aging. Here, we compared behavior in the novel tank test of young (6-month-old) and middle aged (12-month-old) zebrafish from two strains (TL and TU) and both sexes. We find that this modest age difference results in a reduction in locomotor activity in male fish. We also found that background strain modulated the effects of age on predator avoidance behaviors related to anxiety: older female TL fish increased bottom dwelling whereas older male TU fish decreased thigmotaxis. Although there were no consistent effects of age on either short-term (within session) or long-term (next day) habituation to the novel tank, strain affected the habituation response. TL fish tended to increase their distance from the bottom of the tank whereas TU fish had no changes in bottom distance but instead tended to increase thigmotaxis. Our findings support the use of zebrafish for the study of how age affects locomotion and how genetics interacts with age and sex to alter exploratory and emotional behaviors in response to novelty.

Individual differences in the boldness of female zebrafish are associated with alterations in serotonin function.

One of the most prevalent axes of behavioral variation in both humans and animals is risk taking, where individuals that are more willing to take risk are characterized as bold while those that are more reserved as shy. Brain monoamines (i.e., serotonin, dopamine, and norepinephrine) have been found to play a role in a variety of behaviors related to risk taking. Genetic variation related to monoamine function have also been linked to personality in both humans and animals. Using zebrafish, we investigated the relationship between monoamine function and boldness behavior during exploration of a novel tank. We found a sex-specific correlation between serotonin metabolism (5-HIAA:5-HT ratio) and boldness that was limited to female animals; there were no relationships between boldness and dopamine or norepinephrine. To probe differences in serotonergic function, we administered a serotonin reuptake inhibitor, escitalopram, to bold and shy fish, and assessed their exploratory behavior. We found that escitalopram had opposing effects on thigmotaxis in female animals with bold fish spending more time near the center of the tank and shy fish spent more time near the periphery. Taken together, our findings suggest that variation in serotonergic function makes sex-specific contributions to individual differences in risk taking behavior.

A gelatin-based feed for precise and non-invasive drug delivery to adult zebrafish.

Although the use of adult zebrafish as a model organism has increased in recent years, there is room to refine methods, such as drug delivery, to make them less invasive and more precise. Here, we describe the development of a non-invasive gelatin-based feed method that is tailored to animals based on their body mass. The feed was readily eaten by zebrafish (<1 min) with minimal leaching of compound when placed in water (<5% in 5 min). As a proof of principle, we fed fish a NMDA receptor antagonist (MK-801, 4 mg kg-1) prior to the novel tank test. We found that MK-801 caused a general decrease in predator-avoidance/anxiety-like behavior (bottom dwelling) and an increase in locomotion in male fish, but not females. Our simple, easy to prepare and individually tailored gelatin-based feed enables precisely dosed, non-invasive drug delivery to adult-stage zebrafish for the first time.

Beyond bold versus shy: Zebrafish exploratory behavior falls into several behavioral clusters and is influenced by strain and sex.

Individual differences in exploratory behavior have been found across a range of taxa and are thought to contribute to evolutionary fitness. Animals that explore more of a novel environment and visit areas of high predation risk are considered bold, whereas animals with the opposite behavioral pattern are shy. Here, we determined whether this bimodal characterization of bold versus shy adequately captures the breadth of behavioral variation in zebrafish or if there are more than these two subtypes. To identify behavioral categories, we applied unsupervised machine to three-dimensional swim traces from over 400 adult zebrafish across four strains (AB, TL, TU, and WIK) and both sexes. We found that behavior stratified into four distinct clusters: previously described bold and shy behavior and two new behavioral types we call wall-huggers and active explorers. Clusters were stable across time and influenced by strain and sex where we found that TLs were shy, female TU fish were bold, male TU fish were active explorers, and male ABs were wall-huggers. Our work suggests that zebrafish exploratory behavior has greater complexity than previously recognized and lays the groundwork for the use of zebrafish in understanding the biological basis of individual differences in behavior.

A 3D adult zebrafish brain atlas (AZBA) for the digital age.

Zebrafish have made significant contributions to our understanding of the vertebrate brain and the neural basis of behavior, earning a place as one of the most widely used model organisms in neuroscience. Their appeal arises from the marriage of low cost, early life transparency, and ease of genetic manipulation with a behavioral repertoire that becomes more sophisticated as animals transition from larvae to adults. To further enhance the use of adult zebrafish, we created the first fully segmented three-dimensional digital adult zebrafish brain atlas (AZBA). AZBA was built by combining tissue clearing, light-sheet fluorescence microscopy, and three-dimensional image registration of nuclear and antibody stains. These images were used to guide segmentation of the atlas into over 200 neuroanatomical regions comprising the entirety of the adult zebrafish brain. As an open source, online (azba.wayne.edu), updatable digital resource, AZBA will significantly enhance the use of adult zebrafish in furthering our understanding of vertebrate brain function in both health and disease.

Neural correlates of ingroup bias for prosociality in rats.

Prosocial behavior, in particular helping others in need, occurs preferentially in response to distress of one's own group members. In order to explore the neural mechanisms promoting mammalian helping behavior, a discovery-based approach was used here to identify brain-wide activity correlated with helping behavior in rats. Demonstrating social selectivity, rats helped others of their strain ('ingroup'), but not rats of an unfamiliar strain ('outgroup'), by releasing them from a restrainer. Analysis of brain-wide neural activity via quantification of the early-immediate gene c-Fos identified a shared network, including frontal and insular cortices, that was active in the helping test irrespective of group membership. In contrast, the striatum was selectively active for ingroup members, and activity in the nucleus accumbens, a central network hub, correlated with helping. In vivo calcium imaging showed accumbens activity when rats approached a trapped ingroup member, and retrograde tracing identified a subpopulation of accumbens-projecting cells that was correlated with helping. These findings demonstrate that motivation and reward networks are associated with helping an ingroup member and provide the first description of neural correlates of ingroup bias in rodents.

Recovery of "Lost" Infant Memories in Mice.

Hippocampus-dependent, event-related memories formed in early infancy in human and non-human animals are rapidly forgotten. Recently we found that high levels of hippocampal neurogenesis contribute to accelerated rates of forgetting during infancy. Here, we ask whether these memories formed in infancy are permanently erased (i.e., storage failure) or become progressively inaccessible with time (i.e., retrieval failure). To do this, we developed an optogenetic strategy that allowed us to permanently express channelrhodopsin-2 (ChR2) in neuronal ensembles that were activated during contextual fear encoding in infant mice. We then asked whether reactivation of ChR2-tagged ensembles in the dentate gyrus was sufficient for memory recovery in adulthood. We found that optogenetic stimulation of tagged dentate gyrus neurons recovered "lost" infant memories up to 3 months following training and that memory recovery was associated with broader reactivation of tagged hippocampal and cortical neuronal ensembles.

Eukaryotic elongation factor 2 kinase upregulates the expression of proteins implicated in cell migration and cancer cell metastasis.

Eukaryotic elongation factor 2 kinase (eEF2K) negatively regulates the elongation phase of mRNA translation and hence protein synthesis. Increasing evidence indicates that eEF2K plays an important role in the survival and migration of cancer cells and in tumor progression. As demonstrated by two-dimensional wound-healing and three-dimensional transwell invasion assays, knocking down or inhibiting eEF2K in cancer cells impairs migration and invasion of cancer cells. Conversely, exogenous expression of eEF2K or knocking down eEF2 (the substrate of eEF2K) accelerates wound healing and invasion. Importantly, using LC-HDMSE analysis, we identify 150 proteins whose expression is decreased and 73 proteins which are increased upon knocking down eEF2K in human lung carcinoma cells. Of interest, 34 downregulated proteins are integrins and other proteins implicated in cell migration, suggesting that inhibiting eEF2K may help prevent cancer cell mobility and metastasis. Interestingly, eEF2K promotes the association of integrin mRNAs with polysomes, providing a mechanism by which eEF2K may enhance their cellular levels. Consistent with this, genetic knock down or pharmacological inhibition of eEF2K reduces the protein expression levels of integrins. Notably, pharmacological or genetic inhibition of eEF2K almost completely blocked tumor growth and effectively prevented the spread of tumor cells in vivo. High levels of eEF2K expression were associated with invasive carcinoma and metastatic tumors. These data provide the evidence that eEF2K is a new potential therapeutic target for preventing tumor metastasis.

Contextual fear conditioning in zebrafish.

Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development of a contextual fear conditioning paradigm using an electric shock as the aversive stimulus. We find that contextual fear conditioning is modulated by shock intensity, prevented by an established amnestic agent (MK-801), lasts at least 14 d, and exhibits extinction. Furthermore, fish of various background strains (AB, Tu, and TL) are able to acquire fear conditioning, but differ in fear extinction rates. Taken together, we find that contextual fear conditioning in zebrafish shares many similarities with the widely used contextual fear conditioning paradigm in rodents. Combined with the amenability of genetic manipulation in zebrafish, we anticipate that our paradigm will prove to be a useful complementary system in which to examine the molecular basis of vertebrate learning and memory.

Chemogenetic Interrogation of a Brain-wide Fear Memory Network in Mice.

Behavior depends on coordinated activity across multiple brain regions. Within such networks, highly connected hub regions are assumed to disproportionately influence behavioral output, although this hypothesis has not been systematically evaluated. Previously, by mapping brain-wide expression of the activity-regulated gene c-fos, we identified a network of brain regions co-activated by fear memory. To test the hypothesis that hub regions are more important for network function, here, we simulated node deletion in silico in this behaviorally defined functional network. Removal of high degree nodes produced the greatest network disruption (e.g., reduction in global efficiency). To test these predictions in vivo, we examined the impact of post-training chemogenetic silencing of different network nodes on fear memory consolidation. In a series of independent experiments encompassing 25% of network nodes (i.e., 21/84 brain regions), we found that node degree accurately predicted observed deficits in memory consolidation, with silencing of highly connected hubs producing the largest impairments.

Functional Connectivity of Multiple Brain Regions Required for the Consolidation of Social Recognition Memory.

Social recognition memory is an essential and basic component of social behavior that is used to discriminate familiar and novel animals/humans. Previous studies have shown the importance of several brain regions for social recognition memories; however, the mechanisms underlying the consolidation of social recognition memory at the molecular and anatomic levels remain unknown. Here, we show a brain network necessary for the generation of social recognition memory in mice. A mouse genetic study showed that cAMP-responsive element-binding protein (CREB)-mediated transcription is required for the formation of social recognition memory. Importantly, significant inductions of the CREB target immediate-early genes c-fos and Arc were observed in the hippocampus (CA1 and CA3 regions), medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), and amygdala (basolateral region) when social recognition memory was generated. Pharmacological experiments using a microinfusion of the protein synthesis inhibitor anisomycin showed that protein synthesis in these brain regions is required for the consolidation of social recognition memory. These findings suggested that social recognition memory is consolidated through the activation of CREB-mediated gene expression in the hippocampus/mPFC/ACC/amygdala. Network analyses suggested that these four brain regions show functional connectivity with other brain regions and, more importantly, that the hippocampus functions as a hub to integrate brain networks and generate social recognition memory, whereas the ACC and amygdala are important for coordinating brain activity when social interaction is initiated by connecting with other brain regions. We have found that a brain network composed of the hippocampus/mPFC/ACC/amygdala is required for the consolidation of social recognition memory.SIGNIFICANCE STATEMENT Here, we identify brain networks composed of multiple brain regions for the consolidation of social recognition memory. We found that social recognition memory is consolidated through CREB-meditated gene expression in the hippocampus, medial prefrontal cortex, anterior cingulate cortex (ACC), and amygdala. Importantly, network analyses based on c-fos expression suggest that functional connectivity of these four brain regions with other brain regions is increased with time spent in social investigation toward the generation of brain networks to consolidate social recognition memory. Furthermore, our findings suggest that hippocampus functions as a hub to integrate brain networks and generate social recognition memory, whereas ACC and amygdala are important for coordinating brain activity when social interaction is initiated by connecting with other brain regions.

Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions.

Vanishing white matter (VWM) is a leukodystrophy with predominantly early-childhood onset. Affected children display various neurological signs, including ataxia and spasticity, and die early. VWM patients have bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (eIF2B). eIF2B regulates protein synthesis rates under basal and cellular stress conditions. The underlying molecular mechanism of how mutations in eIF2B result in VWM is unknown. Previous studies suggest that brain white matter astrocytes are primarily affected in VWM. We hypothesized that the translation rate of certain astrocytic mRNAs is affected by the mutations, resulting in astrocytic dysfunction. Here we subjected primary astrocyte cultures of wild type (wt) and VWM (2b5ho ) mice to pulsed labeling proteomics based on stable isotope labeling with amino acids in cell culture (SILAC) with an L-azidohomoalanine (AHA) pulse to select newly synthesized proteins. AHA was incorporated into newly synthesized proteins in wt and 2b5ho astrocytes with similar efficiency, without affecting cell viability. We quantified proteins synthesized in astrocytes of wt and 2b5ho mice. This proteomic profiling identified a total of 80 proteins that were regulated by the eIF2B mutation. We confirmed increased expression of PROS1 in 2b5ho astrocytes and brain. A DAVID enrichment analysis showed that approximately 50% of the eIF2B-regulated proteins used the secretory pathway. A small-scale metabolic screen further highlighted a significant change in the metabolite 6-phospho-gluconate, indicative of an altered flux through the pentose phosphate pathway (PPP). Some of the proteins migrating through the secretory pathway undergo oxidative folding reactions in the endoplasmic reticulum (ER), which produces reactive oxygen species (ROS). The PPP produces NADPH to remove ROS. The proteomic and metabolomics data together suggest a deregulation of ER function in 2b5ho mouse astrocytes.

Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling.

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory properties, so this finding indicates it may be involved in metabolic remodeling and also serve as a novel candidate biomarker. Levels of translation factor eEF1 also increased during TAC, likely contributing to faster cell mass accumulation. Interestingly those two candidates were not up-regulated in pregnancy or exercise induced CH, indicating PKM2 and eEF1 were pathological CH specific markers. We anticipate that the methodologies described here will be valuable for other researchers studying protein synthesis in primary cells.

c-Jun-N-terminal kinase 1 is necessary for nicotine-induced enhancement of contextual fear conditioning.

Acute nicotine enhances hippocampus-dependent learning. Identifying how acute nicotine improves learning will aid in understanding how nicotine facilitates the development of maladaptive memories that contribute to drug-seeking behaviors, help development of medications to treat disorders associated with cognitive decline, and advance understanding of the neurobiology of learning and memory. The effects of nicotine on learning may involve recruitment of signaling through the c-Jun N-terminal kinase family (JNK 1-3). Learning in the presence of acute nicotine increases the transcription of mitogen-activated protein kinase 8 (MAPK8, also known as JNK1), likely through a CREB-dependent mechanism. The functional significance of JNK1 in the effects of acute nicotine on learning, however, is unknown. The current studies undertook a backward genetic approach to determine the functional contribution JNK1 protein makes to nicotine-enhanced contextual fear conditioning. JNK1 wildtype (WT) and knockout (KO) mice were administered acute nicotine prior to contextual and cued fear conditioning. 24h later, mice were evaluated for hippocampus-dependent (contextual fear conditioning) and hippocampus-independent (cued fear conditioning) memory. Nicotine selectively enhanced contextual conditioning in WT mice, but not in KO mice. Nicotine had no effect on hippocampus-independent learning in either genotype. JNK1 KO and WT mice given saline showed similar levels of learning. These data suggest that JNK1 may be recruited by nicotine and is functionally necessary for the acute effects of nicotine on learning and memory.

Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons.

Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia.

Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.

Dynamics of elongation factor 2 kinase regulation in cortical neurons in response to synaptic activity.

The rapid regulation of cell signaling in response to calcium in neurons is essential for real-time processing of large amounts of information in the brain. A vital regulatory component, and one of the most energy-intensive biochemical processes in cells, is the elongation phase of mRNA translation, which is controlled by the Ca(2+)/CaM-dependent elongation factor 2 kinase (eEF2K). However, little is known about the dynamics of eEF2K regulation in neurons despite its established role in learning and synaptic plasticity. To explore eEF2K dynamics in depth, we stimulated synaptic activity in mouse primary cortical neurons. We find that synaptic activity results in a rapid, but transient, increase in eEF2K activity that is regulated by a combination of AMPA and NMDA-type glutamate receptors and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin complex 1 (mTORC1) pathways. We then used computational modeling to test the hypothesis that considering Ca(2+)-coordinated MEK/ERK, mTORC1, and eEF2k activation is sufficient to describe the observed eEF2K dynamics. Although such a model could partially fit the empirical findings, it also suggested that a crucial positive regulator of eEF2K was also necessary. Through additional modeling and empirical evidence, we demonstrate that AMP kinase (AMPK) is also an important regulator of synaptic activity-driven eEF2K dynamics in neurons. Our combined modeling and experimental findings provide the first evidence that it is necessary to consider the combined interactions of Ca(2+) with MEK/ERK, mTORC1, and AMPK to adequately explain eEF2K regulation in neurons.

Thyroid receptor ß involvement in the effects of acute nicotine on hippocampus-dependent memory.

Cigarette smoking is common despite adverse health effects. Nicotine's effects on learning may contribute to addiction by enhancing drug-context associations. Effects of nicotine on learning could be direct or could occur by altering systems that modulate cognition. Because thyroid signaling can alter cognition and nicotine/smoking may change thyroid function, nicotine could affect learning through changes in thyroid signaling. These studies investigate the functional contributions of thyroid receptor (TR) subtypes ß and α1 to nicotine-enhanced learning and characterize the effects of acute nicotine and learning on thyroid hormone levels. We conducted a high throughput screen of transcription factor activity to identify novel targets that may contribute to the effects of nicotine on learning. Based on these results, which showed that combined nicotine and learning uniquely acted to increase TR activation, we identified TRs as potential targets of nicotine. Further analyses were conducted to determine the individual and combined effects of nicotine and learning on thyroid hormone levels, but no changes were seen. Next, to determine the role of TRß and TRα1 in the effects of nicotine on learning, mice lacking the TRß or TRα1 gene and wildtype littermates were administered acute nicotine prior to fear conditioning. Nicotine enhanced contextual fear conditioning in TRα1 knockout mice and wildtypes from both lines but TRß knockout mice did not show nicotine-enhanced learning. This finding supports involvement of TRß signaling in the effect of acute nicotine on hippocampus-dependent memory. Acute nicotine enhances learning and these effects may involve processes regulated by the transcription factor TRß.

BDNF stimulation of protein synthesis in cortical neurons requires the MAP kinase-interacting kinase MNK1.

Although the MAP kinase-interacting kinases (MNKs) have been known for >15 years, their roles in the regulation of protein synthesis have remained obscure. Here, we explore the involvement of the MNKs in brain-derived neurotrophic factor (BDNF)-stimulated protein synthesis in cortical neurons from mice. Using a combination of pharmacological and genetic approaches, we show that BDNF-induced upregulation of protein synthesis requires MEK/ERK signaling and the downstream kinase, MNK1, which phosphorylates eukaryotic initiation factor (eIF) 4E. Translation initiation is mediated by the interaction of eIF4E with the m(7)GTP cap of mRNA and with eIF4G. The latter interaction is inhibited by the interactions of eIF4E with partner proteins, such as CYFIP1, which acts as a translational repressor. We find that BDNF induces the release of CYFIP1 from eIF4E, and that this depends on MNK1. Finally, using a novel combination of BONCAT and SILAC, we identify a subset of proteins whose synthesis is upregulated by BDNF signaling via MNK1 in neurons. Interestingly, this subset of MNK1-sensitive proteins is enriched for functions involved in neurotransmission and synaptic plasticity. Additionally, we find significant overlap between our subset of proteins whose synthesis is regulated by MNK1 and those encoded by known FMRP-binding mRNAs. Together, our data implicate MNK1 as a key component of BDNF-mediated translational regulation in neurons.

Stronger learning recruits additional cell-signaling cascades: c-Jun-N-terminal kinase 1 (JNK1) is necessary for expression of stronger contextual fear conditioning.

Increased training often results in stronger memories but the neural changes responsible for these stronger memories are poorly understood. It is proposed here that higher levels of training that result in stronger memories recruit additional cell signaling cascades. This study specifically examined if c-Jun N-terminal kinase 1 (JNK1) is involved in the formation of stronger fear conditioning memories. Wildtype (WT), JNK1 heterozygous (Het), and JNK1 knockout (KO) mice were fear conditioned with 1 trial, 2 trials, or 4 trials. All mice learned both contextual (hippocampus-dependent) and cued (hippocampus-independent) fear conditioning but for contextual fear conditioning only, the JNK1 KO mice did not show higher levels of learning with increased trials. That is, WT mice showed a significant linear increase in contextual fear conditioning as training trials increased from 1 to 2 to 4 trials whereas KO mice showed the same level of contextual fear conditioning as WT mice for 1 trial training but did not have increased levels of contextual fear conditioning with additional trials. These data suggest that JNK1 may not be critical for learning but when higher levels of hippocampus-dependent learning occur, JNK1 signaling is recruited and is necessary for stronger hippocampus-dependent memory formation.