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ABSTRACT: Introduction. Studies of synaptic plasticity using the marine mollusk Aplysia californica as model system have been successfully used to identify proteins involved in learning and memory. The importance of molecular elements regulated by the learning- related neurotransmitter serotonin in Aplysia can then be explored in rodent models and finally tested for their relevance for human physiology and pathology. Materials and methods. Herein, 2-DE gel-based electrophoresis has been used to investigate protein level changes after treatment with serotonin in Aplysia abdominal ganglia. Results. Twenty-one proteins have been found to be regulated by serotonin, and protein level changes of actin depolymerizing factor (ADF), deleted in azoospermia associated protein (DAZAP-1), and Flotillin-1 have been verified by Western blotting. Discussion. Flotillin-1, a member of the flotillin/reggie family of scaffolding proteins, has been previously found to be involved in neuritic branching and synapse formation in hippocampal neurons in vitro. However, its importance for hippocampal- dependent learning and memory in the mouse has not been examined. Here, elevated levels of Flotillin-1 in hippocampal tissue of mice trained in the Morris water maze confirmed the relevance of Flotillin-1 for memory-related processes in a mammalian system. Thus, a translational approach-from invertebrates to rodents-led to the identification of Flotillin-1 as evolutionary-conserved memory-related protein.
Annals of medicine 04/2013; · 3.52 Impact Factor
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ABSTRACT: The lymphocyte-specific protein tyrosine kinase (Lck), which belongs to the Src kinase-family, is expressed in neurons of the hippocampus, a structure critical for learning and memory. Recent evidence demonstrated a significant downregulation of Lck in Alzheimer's disease. Lck has additionally been proposed to be a risk factor for Alzheimer's disease, thus suggesting the involvement of Lck in memory function. The neuronal role of Lck, however, and its involvement in learning and memory remain largely unexplored. Here, in vitro electrophysiology, confocal microscopy, and molecular, pharmacological, genetic and biochemical techniques were combined with in vivo behavioral approaches to examine the role of Lck in the mouse hippocampus. Specific pharmacological inhibition and genetic silencing indicated the involvement of Lck in the regulation of neuritic outgrowth. In the functional pre-established synaptic networks that were examined electrophysiologically, specific Lck-inhibition also selectively impaired the long-term hippocampal synaptic plasticity without affecting spontaneous excitatory synaptic transmission or short-term synaptic potentiation. The selective inhibition of Lck also significantly altered hippocampus-dependent spatial learning and memory in vivo. These data provide the basis for the functional characterization of brain Lck, describing the importance of Lck as a critical regulator of both neuronal morphology and in vivo long-term memory.
Cellular and Molecular Life Sciences CMLS 09/2012; · 6.57 Impact Factor
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ABSTRACT: Aplysia californica (AC) is a widely used model for testing learning and memory. Although ESTs have been generated, proteomics studies on AC proteins are limited. Studies at the protein level, however, are mandatory, not only due to the fact that studies at the nucleic acid level are not allowing conclusions about PTMs. A gel-based proteomics method was therefore applied to carry out protein profiling in abdominal ganglia from AC. Abdominal ganglia were extirpated, proteins extracted and run on 2DE with subsequent in-gel digestion with trypsin, chymotrypsin, and partially by subtilisin. Peptides were identified using a nano-LC-ESI-LTQ-FT-mass spectrometer. MS/MS data were analyzed by searching the NCBI nonredundant public AC EST database and the NCBI nonredundant public AC protein database. A total of 477 different proteins represented by 363 protein spots were detected and were assigned to different protein pathways as for instance signaling (receptors, protein kinases, and phosphatases), metabolism, protein synthesis, handling and degradation, cytoskeleton and structural, oxido-redox, heat shock and chaperone, hypothetical, predicted and unnamed proteins. The generation of a protein map of soluble proteins shows the existence of so far hypothetical and predicted proteins and is allowing and challenging further work at the protein level, in particular in the field of neuroscience.
Proteomics 06/2012; 12(15-16):2482-6. · 4.43 Impact Factor
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ABSTRACT: The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large-scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2-D gel electrophoresis approach. Protein spots were quantified and protein-level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin-1 and deleted in azoospermia-associated protein (DAZAP-1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP-1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK-1 and actin-depolymerizing factor (ADF) in pleural ganglia and DAZAP-1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel-based mass spectrometrical method (FT-MS). Taken together, protein-level changes induced by the learning-related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.
Proteomics 12/2011; 12(3):490-9. · 4.43 Impact Factor
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ABSTRACT: The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase abundantly expressed in the mammalian brain and highly enriched in neuronal growth cones. Inhibitory and facilitatory activities of FAK on neuronal growth have been reported and its role in neuritic outgrowth remains controversial. Unlike other tyrosine kinases, such as the neurotrophin receptors regulating neuronal growth and plasticity, the relevance of FAK for learning and memory in vivo has not been clearly defined yet. A comprehensive study aimed at determining the role of FAK in neuronal growth, neurotransmitter release and synaptic plasticity in hippocampal neurons and in hippocampus-dependent learning and memory was therefore undertaken using the mouse model. Gain- and loss-of-function experiments indicated that FAK is a critical regulator of hippocampal cell morphology. FAK mediated neurotrophin-induced neuritic outgrowth and FAK inhibition affected both miniature excitatory postsynaptic potentials and activity-dependent hippocampal long-term potentiation prompting us to explore the possible role of FAK in spatial learning and memory in vivo. Our data indicate that FAK has a growth-promoting effect, is importantly involved in the regulation of the synaptic function and mediates in vivo hippocampus-dependent spatial learning and memory.
Neurosignals 09/2011; 20(1):1-14. · 2.11 Impact Factor
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ABSTRACT: Substantial experimental evidence indicates a major role for the circadian system in mood disorders. Additionally, proinflammatory cytokines have been proposed to be involved in the pathogenesis of depression. However, the molecular elements determining the functional interplay between these two systems in depression have not been described as yet. Here we investigate whether long-term light deprivation in the constant darkness (DD) paradigm affects depression-like behavior in mice and concomitantly modulates the levels of proinflammatory cytokines. We find that after 4 weeks of DD, mice display depression-like behavior, which is paralleled by reduced hippocampal cell proliferation. This chronobiologically induced depressive state is associated with elevated levels of plasma IL-6 (interleukin-6) and IL-6 and Il1-R1 (interleukin 1 receptor, type I) protein levels in the hippocampus and also alters hippocampal protein levels of the clock genes per2 and npas2. Using pharmacological blockers of the NF-κB pathway, we provide evidence that the effects of DD on depression-like behavior, on hippocampal cell proliferation, on altered expressional levels of brain and plasma IL-6, and on the modulation of clock gene expression are mediated through NF-κB signaling. Moreover, NF-κB activity is enhanced in hippocampal tissue of DD mice. Mice with a deletion of IL-6, one of the target genes of NF-κB, are resistant to DD-induced depression-like behavior, which suggests a pivotal role for this cytokine in the constant darkness mouse model of depression. We here first describe some of the molecular elements bridging chronobiological and inflammatory processes in the constant darkness mouse model of depression.
Journal of Neuroscience 06/2011; 31(25):9075-83. · 7.11 Impact Factor
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ABSTRACT: Abstract Background. The gastrin-releasing peptide receptor (GRPR) is highly expressed in the limbic system, where it importantly regulates emotional functions and in the suprachiasmatic nucleus, where it is central for the photic resetting of the circadian clock. Mice lacking GRPR presented with deficient light-induced phase shift in activity as well altered emotional learning and amygdala function. The effect of GRPR deletion on depression-like behavior and its molecular signature in the amygdala, however, has not yet been evaluated. Methods. GRPR knock-out mice (GRPR-KO) were tested in the forced-swim test and the sucrose preference test for depression-like behavior. Gene expression in the basolateral nucleus of the amygdala was evaluated by micorarray analysis subsequent to laser-capture microdissection-assisted extraction of mRNA. The expression of selected genes was confirmed by RT-PCR. Results. GRPR-KO mice were found to present with increased depression-like behavior. Microarray analysis revealed down-regulation of several glucocorticoid-responsive genes in the basolateral amygdala. Acute administration of dexamethasone reversed the behavioral phenotype and alterations in gene expression. Discussion. We propose that deletion of GRPR leads to the induction of depression-like behavior which is paralleled by dysregulation of amygdala gene expression, potentially resulting from deficient light-induced corticosterone release in GRPR-KO.
Annals of medicine 01/2011; 43(5):389-402. · 3.52 Impact Factor
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ABSTRACT: Aplysia proteins have not been studied systematically and it was therefore the aim of the study to carry out protein profiling in ganglia from Aplysia californica (AC). AC ganglia were extirpated, proteins extracted and run on 2DE with subsequent in-gel digestion, followed by identification of proteins by nano-LC-ESI-MS/MS on an ion trap. Proteins were identified based upon a public Aplysia EST database. Out of 408 picked spots, 276 spots were identified corresponding to 172 ESTs and 118 individual proteins. The range of sequence coverage was between 14 and 80% and the average amount of peptides used for the identification of proteins was 9 (from 3 to 24). Mean score for protein identification was 516. Comparison of protein levels between cerebral, pleural, pedal and abdominal ganglia revealed a series of significant differences including: signaling, metabolism, cytoskeleton and structural, redox, chaperone, replication/transcription and electron/proton transport proteins. The generation of a protein map complements transcriptional studies carried out in AC ganglia. The findings provide the basis for investigation into post-translational modifications, splice variants and assist in the generation of antibodies against AC proteins. Moreover, differences in protein expression between ganglia may be valuable for the design of future studies in neurobiology of AC.
Amino Acids 11/2010; 41(4):955-68. · 3.25 Impact Factor
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ABSTRACT: Depression, among other mood disorders, represents one of the most common health problems worldwide, with steadily increasing incidence and major socio-economic consequences. However, since the knowledge about the underlying pathophysiological principles is still very scanty, depression and other mood disorders are currently diagnosed solely on clinical grounds. Currently used treatment modalities would therefore benefit enormously from the development of alternative therapeutic interventions. The implementation of proper animal models is a prerequisite for increasing the understanding of the neurobiological basis of mood disorders and is paving the way for the discovery of novel therapeutic targets. In the past thirty years, since the seminal description of the Forced Swim Test as a system to probe antidepressant activity in rodents, the use of animals to model depression and antidepressant activity has come a long way. In this review we describe some of the most commonly used strategies, ranging from screening procedures, such as the Forced Swim Test and the Tail Suspension Test and animal models, such as those based upon chronic stress procedures, to genetic approaches. Finally we also discuss some of the inherent limitations and caveats that need to be considered when using animals as models for mental disorders in basic research.
Annals of medicine 04/2010; 42(4):252-64. · 3.52 Impact Factor
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ABSTRACT: Fear conditioning is one of the most widely used animal models for studying the neurobiological basis of fear and anxiety states. Conditioned inhibition of fear (or learned safety), however, is a relatively unexplored behavioral paradigm addressing the aspect of regulation of fear, which is central to survival and mental health. Although fear conditioning is achieved by pairing a previously neutral, conditioned stimulus (CS) with an aversive, unconditioned stimulus (US), learned safety training consists of a series of explicitly unpaired CS-US presentations. Animals are trained for 3 d, one session per day, and learn to associate the CS with protection from the impending danger of the aversive events. The entire procedure can be completed within 7 d. The protocol has been successfully used to study the molecular underpinnings of a behavioral intervention for depression. This paradigm complements currently used animal tests in neuropsychiatric research addressing the dysregulation of emotional behaviors in genetic, pharmacological or environmental mouse models of human affective disorders.
Nature Protocol 01/2010; 5(5):954-62. · 8.36 Impact Factor
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ABSTRACT: To explore how gene products, required for the initiation of synaptic growth, move from the cell body of the sensory neuron to its presynaptic terminals, and from the cell body of the motor neuron to its postsynaptic dendritic spines, we have investigated the anterograde transport machinery in both the sensory and motor neurons of the gill-withdrawal reflex of Aplysia. We found that the induction of long-term facilitation (LTF) by repeated applications of serotonin, a modulatory transmitter released during learning in Aplysia, requires upregulation of kinesin heavy chain (KHC) in both pre- and postsynaptic neurons. Indeed, upregulation of KHC in the presynaptic neurons alone is sufficient for the induction of LTF. However, KHC is not required for the persistence of LTF. Thus, in addition to transcriptional activation in the nucleus and local protein synthesis at the synapse, our studies have identified a third component critical for long-term learning-related plasticity: the coordinated upregulation of kinesin-mediated transport.
Cell 12/2008; 135(5):960-73. · 32.40 Impact Factor
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ABSTRACT: Although conditioned inhibition of fear (or learned safety) is a learning process critical for preventing chronic stress, a predisposing factor for depression and other psychopathologies, little is known about its functional purposes or molecular mechanisms. To obtain better insight into learned safety, we investigated its behavioral and molecular characteristics and found that it acts as a behavioral antidepressant in two animal models. Learned safety promotes the survival of newborn cells in the dentate gyrus of the hippocampus, while its antidepressant effect is abolished in mice with ablated hippocampal neurogenesis. Learned safety also increases the expression of BDNF in the hippocampus and leads to downregulation of genes involved in the dopaminergic and neuropeptidergic but not the serotonergic system in the basolateral amygdala. These data suggest that learned safety is an animal model of a behavioral antidepressant that shares some neuronal hallmarks of pharmacological antidepressants but is mediated by different molecular pathways.
Neuron 11/2008; 60(1):149-61. · 14.74 Impact Factor
