Molecule to Cognition unit

Bong-Kiun Kaang, Dept of Biological Sciences, SNU HP

2010

* Detection of TrkB Receptors Distributed in Cultured Hippocampal Neurons through Bioconjugation between Highly Luminescent (Quantum Dot-Neutravidin) and (Biotinylated Anti-TrkB Antibody) on Neurons by Combined Atomic Force Microscope and Confocal Laser Scanning Microscope.
[Pubmed] [PDF]

We developed highly luminescent and cost-effective quantum dot (QD)-neutravidin (NTV) bioconjugates to detect the tyrosine kinase B (TrkB) receptors distributed in the cultured hippocampus neurons. Hippocampal neurons were incubated with biotinylated anti-TrkB antibody, followed by further incubation with QD-NTV bioconjugates. QD-NTV biomarkers on the extracellular domain of TrkB receptors were imaged by the combined atomic force microscope and confocal laser scanning microscope (AFM-CLSM) providing resolved (nanometer-scale) structural and fluorescent images. We found that TrkB receptors were distributed over the neuronal cell bodies (soma) and neurites. TrkB receptors in the somata looked more concentrated, but those in the neurites appeared punctate. Thus, our QD-based immunocytochemistry technique combined with an AFM-CLSM can be used for threedimensional morphology of neurons on nanometer-scale structural resolution and their fluorescence images with QDs. Furthermore, this technique can be applied for real-time fluorescence imaging or long-term study of live neurons.

* Neuronal RNA granule contains ApCPEB1, a novel Aplysia CPEB, in Aplysia sensory neuron.
[Pubmed] [PDF]

The cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) binds to CPE containing mRNAs on their 3' untranslated regions (3'UTRs). This RNA binding protein comes out many important tasks, especially in learning and memory, by modifying the translational efficiency of target mRNAs via poly (A) tailing. Overexpressed CPEB has been reported to induce the formation of stress granules (SGs), a sort of RNA granule in mammalian cell lines. RNA granule is considered to be a potentially important factor in learning and memory. However, there is no study about RNA granule in Aplysia. To examine whether an Aplysia CPEB, ApCPEB1, forms RNA granules, we overexpressed ApCPEB1-EGFP in Aplysia sensory neurons. Consistent with the localization of mammalian CPEB, overexpressed ApCPEB1 formed granular structures, and was colocalized with RNAs and another RNA binding protein, ApCPEB, showing that ApCPEB1 positive granules are RNA-protein complexes. In addition, ApCPEB1 has a high turnover rate in RNA granules which were mobile structures. Thus, our results indicate that overexpressed ApCPEB1 is incorporated into RNA granule which is a dynamic structure in Aplysia sensory neuron. We propose that ApCPEB1 granule might modulate translation, as other RNA granules do, and furthermore, influence memory.

* Protein synthesis and degradation are required for the incorporation of modified information into the pre-existing object-location memory.
[Pubmed] [PDF]

Although some reports indicate that protein synthesis dependent process may be induced by updating information, the role of protein synthesis and degradation in changing the content of pre-existing memory is yet unclear. In this study, we utilized an object rearrangement task, in which partial information related to a pre-existing memory is changed, promoting memory modification. Inhibitors of both protein synthesis and protein degradation impaired adequate incorporation of the altered information, each in a distinctive way. These results indicate that protein synthesis and degradation play key roles in memory modification.

2009

* Social Isolation Selectively Increases Anxiety in Mice without Affecting Depression-like Behavior.
[Pubmed] [PDF]

It is hypothesized that a number of environmental factors affect animals’ behavior. Without controlling these variables, it is very hard for researchers to get not only reliable, but replicable data from various behavioral experiments testing animals’ cognitive as well as emotional functions. For example, laboratory mice which had restricted environment showed different synaptic potentiation properties with wild mice (Zhao MG et al., 2009). While performing behavioral experiments, however, it is sometimes inevitable that the researcher changes the animals’ environments, as by switching the cages in which experimental animals are housed and separating animals raised together into small experimental groups. In this study, we investigated the effect of environmental changes on mice’s emotional behaviors by socially isolating them or reducing the size of their cage. We found that social isolation selectively increases the animals' levels of anxiety, while leaving depression-like behaviors unchanged. On the other hand, alteration of the housing dimensions affected neither their anxiety levels nor their depression-like behaviors. These results suggest that environmental variables may have a prominent impact on experimental animals’ emotional behaviors and possibly their psychological states, leading to bias in the behavioral data produced from experiments. Key Words: Anxiety level, Social isolation, Housing environment, Experimental animals, Open field test, Tail suspension test, Forced swim test

* Effects of Protease Treatment and Animal Behavior on the Dissociative Culture of Aplysia Neurons.
[Pubmed] [PDF]

The dissociative culture technique of Aplysia neuron is one of the key methods that have been used for studies of cellular and molecular mechanisms of neuronal functioning. However, despite the advantages this method offers as an experimental model, its technical efficiency has had room for improvement. In this study, we examined certain putative factors that might affect the culture quality. The effects of neuronal damage induced by physical injuries, heat shock, and surface protein degradation were evaluated along with the correlation between the culture quality and animal behavior. As a result, we found that physical injury can be a critical factor that affects culture quality, whereas the heat shock and surface protein degradation had negligible effect on it. In addition, we discovered that siphon retraction time was not a good measurement for healthy neurons. Based on these findings, we suggest here an improved method in which the degree of physical injury is reduced by means of multiple protease treatment.

* Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia.
[Pubmed] [PDF]

Serotonin (5-HT) plays a critical role in modulating synaptic plasticity in the marine mollusc Aplysia and in the mammalian nervous system. In Aplysia sensory neurons, 5-HT can activate several signal cascades, including PKA and PKC, presumably via distinct types of G proteincoupled receptors. However, the molecular identities of these receptors have not yet been identified. We here report the cloning and functional characterization of a 5-HT receptor that is positively coupled to adenylyl cyclase in Aplysia neurons. The cloned receptor, 5-HTapAC1, stimulates the production of cAMP in HEK293T cells and in Xenopus oocytes. Moreover, the knockdown of 5-HTapAC1 expression by RNA interference blocked 5-HT-induced cAMP production in Aplysia sensory neurons and blocked synaptic facilitation in nondepressed or partially depressed sensory-to-motor neuron synapses. These data implicate 5-HTapAC1 as a major modulator of learning related synaptic facilitation in the direct sensory to motor neuron pathway of the gill withdrawal reflex.

* Transcriptome and protein domain analyses in Aplysia nervous system with evolutionary implications.
[Pubmed] [PDF]

The sea hare Aplysia is a powerful model organism for studying the structure and function of the nervous system. Recently, the genomic characterization of Aplysia has been facilitated: A large scale EST sequences was acquired by sequencing cDNA libraries from A. californica and a parallel EST database of the closely related species A. kurodai was reported. These EST databases provide useful tools for both molecular biology and bioinformatics. In our previous report, we demonstrated the utility of the database by screening the candidate genes for the synaptic plasticity and the behavioral sensitization using the microarray containing A. kurodai ESTs. In this addendum, we have expanded our study to show that the protein domain repertoire and the abundance of regulatory genes displayed a linear relationship with the evolution of the complex brains in different lineages. This distinct set of protein domains may play critical roles in evolution of the nervous systems.

* Induction of neuronal vascular endothelial growth factor expression by cAMP in the dentate gyrus of the hippocampus is required for antidepressant-like behaviors.
[Pubmed] [PDF]

The cAMP cascade and vascular endothelial growth factor (VEGF) are critical modulators of depression. Here we have tested whether the antidepressive effect of the cAMP cascade is mediated by VEGF in the adult hippocampus.Weused a conditional genetic system in which the Aplysia octopamine receptor (Ap oa1), a Gs-coupled receptor, is transgenically expressed in the forebrain neurons of mice. Chronic activation of the heterologous Ap oa1 by its natural ligand evoked antidepressant-like behaviors, accompanied by enhanced phosphorylation of cAMP response element-binding protein and transcription of VEGF in hippocampal dentate gyrus (DG) neurons. Selective knockdown of VEGF in these cells during the period of cAMP elevation inhibited the antidepressant-like behaviors. These findings reveal a molecular interaction between the cAMP cascade and VEGF expression, and the pronounced behavioral consequences of this interaction shed light on the mechanism underlying neuronal VEGF functions in antidepression.

* Two major gate-keepers in the self-renewal of neural stem cells: Erk1/2 and PLCgamma1 in FGFR signaling.
[Pubmed] [PDF]

An accumulating body of evidence shows that reactivated long-term memory undergoes a dynamic process called reconsolidation, in which de novo protein synthesis is required to maintain the memory. These findings open up a new dimension in the field of memory research. However, few studies have shown how once-consolidated memory becomes labile. The authors’ recent findings have demonstrated that preexisting long-term memory becomes unstable via the ubiquitin/ proteasome-dependent protein degradation pathway and that this labile state is required for the reorganization of fear memory. Here, the authors review this finding and focus on the labile state that is critical for the reorganization

* Synaptic Protein Degradation as a Mechanism in Memory Reorganization.
[Pubmed] [PDF]

Neural stem cells are undifferentiated precursor cells that proliferate, self-renew, and give rise to neuronal and glial lineages. Understanding the molecular mechanisms underlying their self-renewal is an important aspect in neural stem cell biology. The regulation mechanisms governing self-renewal of neural stem cells and the signaling pathways responsible for the proliferation and maintenance of adult stem cells remain largely unknown. In this issue of Molecular Brain [Ma DK et al. Molecular genetic analysis of FGFR1 signaling reveals distinct roles of MAPK and PLC1 activation for self-renewal of adult neural stem cells. Molecular Brain 2009, 2:16], characterized the different roles of MAPK and PLC1 in FGFR1 signaling in the self-renewal of neural stem cells. These novel findings provide insights into basic neural stem cell biology and clinical applications of potential stem-cell-based therapy.

* The role of lipid binding for the targeting of synaptic proteins into synaptic vesicles.
[Pubmed] [PDF]

An accumulating body of evidence shows that reactivated long-term memory undergoes a dynamic process called reconsolidation, in which de novo protein synthesis is required to maintain the memory. These findings open up a new dimension in the field of memory research. However, few studies have shown how once-consolidated memory becomes labile. The authors’ recent findings have demonstrated that preexisting long-term memory becomes unstable via the ubiquitin/ proteasome-dependent protein degradation pathway and that this labile state is required for the reorganization of fear memory. Here, the authors review this finding and focus on the labile state that is critical for the reorganization

* Effect of ablated hippocampal neurogenesis on the formation and extinction of contextual fear memory.
[Pubmed] [PDF]

Newborn neurons in the subgranular zone (SGZ) of the hippocampus incorporate into the dentate gyrus and mature. Numerous studies have focused on hippocampal neurogenesis because of its importance in learning and memory. However, it is largely unknown whether hippocampal neurogenesis is involved in memory extinction per se. Here, we sought to examine the possibility that hippocampal neurogenesis may play a critical role in the formation and extinction of hippocampus-dependent contextual fear memory. By methylazoxymethanol acetate (MAM) or gamma-ray irradiation, hippocampal neurogenesis was impaired in adult mice. Under our experimental conditions, only a severe impairment of hippocampal neurogenesis inhibited the formation of contextual fear memory. However, the extinction of contextual fear memory was not affected. These results suggest that although adult newborn neurons contribute to contextual fear memory, they may not be involved in the extinction or erasure of hippocampus-dependent contextual fear memory.

Graham Leon Collingridge FRS, MRC Centre for Synaptic Plasticity, U of Bristol HP

2010

* Disruption of the interaction between myosin VI and SAP97 is associated with a reduction in the number of AMPARs at hippocampal synapses 2010.
[Article (PDF)]

Myosin VI is an actin-based motor protein that is enriched at the postsynaptic density and appears to interact with alphaamino- 3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptors (AMPARs) via synapse associated protein 97 (SAP97). Here, we find that a Flag epitope-tagged dominant negative construct that inhibits the interaction between SAP97 and myosin VI (Flag-myoVI-DN) causes a dramatic reduction in the number of synapses and the surface expression of AMPARs in cultured hippocampal neurons. Furthermore, we find that Flag-myoVI-DN also prevents the rapid delivery of AMPARs to synapses that can be induced by the transient activation of N-methyl-D-aspartate receptors. The Flag-myoVIDN induced decrease in surface AMPARs is not because of reduced AMPAR subunit protein synthesis. Using whole-cell recording, we show that Flag-myoVI-DN also prevents the activity-induced increase in miniature excitatory postsynaptic current frequency that is normally associated with recruitment of AMPARs to the cell surface at synaptic sites that lack these receptors (i.e. ‘silent’ synapses). Together, these results indicate that myosin VI/SAP97 plays an important role in trafficking and activity-dependent recruitment of AMPARs to synapses.

2009

* A systematic investigation of the protein kinases involved in NMDA receptor-dependent LTD: evidence for a role of GSK-3 but not other serine/threonine kinases
[Abstract] [Full Text] [PDF] [PubMed] [Related articles] [Cited on BioMed Central]

The signalling mechanisms involved in the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term depression (LTD) in the hippocampus are poorly understood. Numerous studies have presented evidence both for and against a variety of second messengers systems being involved in LTD induction. Here we provide the first systematic investigation of the involvement of serine/threonine (ser/thr) protein kinases in NMDAR-LTD, using whole-cell recordings from CA1 pyramidal neurons. RESULTS: Using a panel of 23 inhibitors individually loaded into the recorded neurons, we can discount the involvement of at least 57 kinases, including PKA, PKC, CaMKII, p38 MAPK and DYRK1A. However, we have been able to confirm a role for the ser/thr protein kinase, glycogen synthase kinase 3 (GSK-3). CONCLUSION: The present study is the first to investigate the role of 58 ser/thr protein kinases in LTD in the same study. Of these 58 protein kinases, we have found evidence for the involvement of only one, GSK-3, in LTD.

* A novel mechanism of hippocampal LTD involving muscarinic receptor-triggered interactions between AMPARs, GRIP and liprin-α
[Abstract] [Full Text] [PDF] [PubMed] [Related articles] [Cited on BioMed Central]

Long-term depression (LTD) in the hippocampus can be induced by activation of different types of G-protein coupled receptors, in particular metabotropic glutamate receptors (mGluRs) and muscarinic acethycholine receptors (mAChRs). Since mGluRs and mAChRs activate the same G-proteins and isoforms of phospholipase C (PLC), it would be expected that these two forms of LTD utilise the same molecular mechanisms. However, we find a distinct mechanism of LTD involving GRIP and liprin-alpha. RESULTS: Whilst both forms of LTD require activation of tyrosine phosphatases and involve internalisation of AMPARs, they use different molecular interactions. Specifically, mAChR-LTD, but not mGluR-LTD, is blocked by peptides that inhibit the binding of GRIP to the AMPA receptor subunit GluA2 and the binding of GRIP to liprin-alpha. Thus, different receptors that utilise the same G-proteins can regulate AMPAR trafficking and synaptic efficacy via distinct molecular mechanisms. CONCLUSION: Our results suggest that mAChR-LTD selectively involves interactions between GRIP and liprin-alpha. These data indicate a novel mechanism of synaptic plasticity in which activation of M1 receptors results in AMPAR endocytosis, via a mechanism involving interactions between GluA2, GRIP and liprin-alpha.

Min Zhuo, Dept of Physiology, U of Toronto HP

2010

* CaMKIV forebrain overexpression boosts cortical 4-7Hz oscillations during learning and 1-4Hz delta oscillations during sleep
[Abstract] [PubMed] [Related articles]

Mounting evidence suggests that neural oscillations are related to the learning and consolidation of newly formed memory in the mammalian brain. Four to seven Hertz (4-7Hz) oscillations in the prefrontal cortex are also postulated to be involved in learning and attention processes. Additionally, slow delta oscillations (1-4Hz) have been proposed for memory consolidation or down scaling during sleep. The molecular mechanisms which link learning-related oscillations during wakefulness to sleep-related oscillations remain unknown. We show that increasing the expression of calcium/calmodulin dependent protein kinase IV (CaMKIV), a key nucleic protein kinase, selectively enhances 4-7.5Hz oscillation power during trace fear learning and slow delta oscillations during subsequent sleep. These oscillations were found to be boosted in response to the trace fear paradigm and are likely to be localized to regions of the prefrontal cortex. Correlation analyses demonstrate that a proportion of the variance in 4-7.5Hz oscillations, during fear conditioning, could account for some degree of learning and subsequent memory formation, while changes in slow delta power did not share this predictive strength. Our data emphasize the role of CaMKIV in controlling learning and sleep related oscillations and suggest that oscillatory activity during wakefulness may be a relevant predictor of subsequent memory consolidation.

* DREAM contributes to synaptic depression and contextual fear memory
[Abstract] [Full Text] [PubMed] [Related articles]

The downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, binds specifically to DNA and several nucleoproteins regulating gene expression and with proteins outside the nucleus to regulate membrane excitability or calcium homeostasis. DREAM is highly expressed in the central nervous system including the hippocampus and cortex; however, the roles of DREAM in hippocampal synaptic transmission and plasticity have not been investigated. Taking advantage of transgenic mice overexpressing a Ca2+-insensitive DREAM mutant (TgDREAM), we used integrative methods including electrophysiology, biochemistry, immunostaining, and behavior tests to study the function of DREAM in synaptic transmission, long-term plasticity and fear memory in hippocampal CA1 region. We found that NMDA receptor but not AMPA receptor-mediated current was decreased in TgDREAM mice. Moreover, synaptic plasticity, such as long-term depression (LTD) but not long-term potentiation (LTP), was impaired in TgDREAM mice. Biochemical experiments found that DREAM interacts with PSD-95 and may inhibit NMDA receptor function through this interaction. Contextual fear memory was significantly impaired in TgDREAM mice. By contrast, sensory responses to noxious stimuli were not affected. Our results demonstrate that DREAM plays a novel role in postsynaptic modulation of the NMDA receptor, and contributes to synaptic plasticity and behavioral memory.

2009

* Enhanced synaptic long-term potentiation in the anterior cingulate cortex of adult wild mice as compared with that in laboratory mice
[Abstract] [Full Text] [PubMed] [Related articles]

Activation of N-methyl D-aspartate (NMDA) receptor is important for learning, memory and persistent pain. Genetic enhancement of NMDA receptor function by overexpressing NR2B subunit significantly enhances hippocampal long-term potentiation (LTP), behavioral learning as well as persistent pain. Recent studies found that NMDA NR2B subunits can undergo long-term upregulation in the brain under certain conditions including peripheral injury and environmental enrichment. Considering the fact that laboratory grown animals live in an artificial comfort environment, we wondered if NMDA receptor functions and its related LTP would differ in animals living in a natural wild environment. In this report we performed whole-cell patch-clamp recordings from both laboratory wild-type mice and wild mice from a natural environment. We found that LTP was significantly enhanced in the anterior cingulate cortex (ACC) of the wild mice as compared with that of laboratory mice. In parallel, NMDA receptor NR2B/total NMDA receptor mediated EPSC ratio was significantly increased in slices of wild mice. Our findings provide the first evidence that NMDA NR2B receptors play an important role in experience-dependent synaptic potentiation within the ACC in wild mice as previously reported in laboratory mice.

* Sexual attraction enhances glutamate transmission in mammalian anterior cingulate cortex
[Abstract] [Full Text] [PubMed] [Related articles] [Cited on BioMed Central]

Functional human brain imaging studies have indicated the essential role of cortical regions, such as the anterior cingulate cortex (ACC), in romantic love and sex. However, the neurobiological basis of how the ACC neurons are activated and engaged in sexual attraction remains unknown. Using transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the promoter of the activity-dependent gene c-fos, we found that ACC pyramidal neurons are activated by sexual attraction. The presynaptic glutamate release to the activated neurons is increased and pharmacological inhibition of neuronal activities in the ACC reduced the interest of male mice to female mice. Our results present direct evidence of the critical role of the ACC in sexual attraction, and long-term increases in glutamate mediated excitatory transmission may contribute to sexual attraction between male and female mice.

* Plasticity of NMDA receptor NR2B subunit in memory and chronic pain
[Abstract] [Full Text] [PubMed] [Related articles] [Cited on BioMed Central]

Glutamatergic synapses play critical roles in brain functions and diseases. Long-term potentiation (LTP) is a most effective cellular model for investigating the synaptic changes that underlie learning as well as brain disease - although different molecular mechanisms are likely involved in LTP in physiological and pathological conditions. In the case of learning, N-methyl-D-aspartate (NMDA) receptor is known to be important for triggering learning-related plasticity; alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptors are thought to be important for the expression of synaptic changes. In this review, I will examine recent evidence on the novel roles of NMDA receptors, in particular NR2B subunit-containing NMDA receptors in learning and chronic pain. A positive feedback control of NR2B receptor subunit is proposed to explain cortical sensitization involved in chronic pain, but not learning and memory.