The mitral cells of the accessory olfactory bulb (AOB) of anuran frogs project their axons directly to the medial amygdala (MeA) along the accessory olfactory tract. An en bloc preparation of the telencephalon of the American bullfrog Lithobates catesbeiana was utilized to study a form of low-frequency inducible long-term potentiation (LTP) expressed at the synapse formed between the terminals of the accessory olfactory tract and the neurons of the MeA. Delivery of repetitive 1Hz-stimulation or sets of 5Hz tetani to the accessory olfactory tract both induced potentiation that was stable for over an hour, as measured by extracellular field recordings. LTP induced by 5Hz tetanus was associated with a decrease in paired-pulse ratio, which would be consistent with an increased probability of release contributing to the increased synaptic strength. Blockade of neither NMDA nor kainate glutamate receptors, with AP5 and UBP310 respectively, prevented LTP induction by 5Hz tetanus; however expression of LTP was partially masked in the presence of UBP310. These results suggest that kainate receptors are involved in the expression of LTP at the AOB-MeA synapse, though the means by which LTP is induced remains unclear. / Graduate / 2016-09-28

Activity-dependent and synapse-specific translation of mRNAs is required for long-term changes in synaptic strength (or efficacy). However, many of the components mediating repression, transport and activation of mRNAs are unknown. Translational control in neurons is a highly conserved process and mediated by a ribonuclear particle (RNP). This study shows that RNPs in Drosophila neurons are similar not only to mammalian neuronal RNA granules but also to yeast P-bodies, cytoplasmic foci involved in translational repression and RNA decay. The evolutionarily conserved proteins Me31b and Trailer Hitch localize to RNA granules. Me31b and Trailer Hitch are required for normal dendritic growth. Mutations in Me31b and Trailer Hitch suppress phenotypes resulting from overexpression of Fragile X Mental Retardation protein, suggesting that both proteins may act as translational repressors. In addition, this study reports the identification of novel translational repressors in neurons. Using the overexpression phenotype of Fragile X Mental Retardation protein in a candidate-based genetic screen, I identified dominant suppressor mutations in five genes, including Doubletime/Discs Overgrown, Orb2/CPEB, PolyA Binding Protein, Rm62/Dmp68 and SmD3. Like Me31b and Trailer Hitch, all five proteins localize to neuronal RNPs. Overexpression of each proteins affects dendritic branching of sensory neurons in Drosophila. Identification and further characterization of these novel RNP granule components and dFMR1-interacting proteins may provide further insights into the mechanisms controlling translational in dendrites.

Non-competitive antagonists of the glutamate N-methyl-D-aspartate receptor (NMDAR) induce a broad range of schizophrenia-like symptoms in humans. Consequently hypothesis has emerged suggesting that glutamate or NMDAR hypofunction may occur in schizophrenia. The NMDAR is localised at dendritic spines of neurons and is embedded in a multi-protein complex called the post-synaptic density (PSD). The biochemical composition of the postsynaptic membrane and the structure of dendritic spines are continuously modulated by glutamatergic synaptic activity. The activity-dependent interaction between glutamate receptors and proteins of the PSD stimulate intracellular signalling pathways underlying learning and memory processes. These may be disturbed in schizophrenia. In the present study we hypothesised that molecules of the PSD may be disturbed in expression in the premotor cortex of patients with schizophrenia. Postmortem premotor cortex from patients with schizophrenia, major depressive disorder, bipolar disorder and healthy controls were processed for PSD extraction and purification. Protein expression of the PSD fraction was assessed using co-immunoprecipitation (co-IP) and Western blotting (WB) methods. The expression of NMDAR subunit NR2A, PSD-95, Ca2+/calmodulin-dependent protein kinase II subunit β (CaMKIIβ) and truncated isoform of the tropomyosin receptor kinase type B (TrkB-T1) were significantly reduced in schizophrenia. A significant decrease in the expression of NR2A was also observed in patients with major depressive disorder relative to controls. A decrease in the abundance of key PSD proteins in schizophrenia provides strong evidence that PSD function and possibly synaptic plasticity may be disturbed in the premotor cortex in the disease. There may also be more subtle disturbances in PSD function in major depressive disorder.

Numerous experiments have demonstrated that the activity of neurons can alter the strength of excitatory synapses. This synaptic plasticity is bidirectional and synapses can be strengthened (potentiation) or weakened (depression). Synaptic plasticity offers a mechanism that links the ongoing activity of the brain with persistent physical changes to its structure. For this reason it is widely believed that synaptic plasticity mediates learning and memory. The hypothesis that synapses store memories by modifying their strengths raises an important issue. There should be a balance between the necessity that synapses change frequently, allowing new memories to be stored with high fidelity, and the necessity that synapses retain previously stored information. This is the plasticity stability dilemma. In this thesis the plasticity stability dilemma is studied in the context of the two dominant paradigms of activity dependent synaptic plasticity: Spike timing dependent plasticity (STDP) and long term potentiation and depression (LTP/D). Models of biological synapses are analysed and processes that might ameliorate the plasticity stability dilemma are identified. Two popular existing models of STDP are compared. Through this comparison it is demonstrated that the synaptic weight dynamics of STDP has a large impact upon the retention time of correlation between the weights of a single neuron and a memory. In networks it is shown that lateral inhibition stabilises the synaptic weights and receptive fields. To analyse LTP a novel model of LTP/D is proposed. The model centres on the distinction between early LTP/D, when synaptic modifications are persistent on a short timescale, and late LTP/D when synaptic modifications are persistent on a long timescale. In the context of the hippocampus it is proposed that early LTP/D allows the rapid and continuous storage of short lasting memory traces over a long lasting trace established with late LTP/D. It is shown that this might confer a longer memory retention time than in a system with only one phase of LTP/D. Experimental predictions about the dynamics of amnesia based upon this model are proposed. Synaptic tagging is a phenomenon whereby early LTP can be converted into late LTP, by subsequent induction of late LTP in a separate but nearby input. Synaptic tagging is incorporated into the LTP/D framework. Using this model it is demonstrated that synaptic tagging could lead to the conversion of a short lasting memory trace into a longer lasting trace. It is proposed that this allows the rescue of memory traces that were initially destined for complete decay. When combined with early and late LTP/D iii synaptic tagging might allow the management of hippocampal memory traces, such that not all memories must be stored on the longest, most stable late phase timescale. This lessens the plasticity stability dilemma in the hippocampus, where it has been hypothesised that memory traces must be frequently and vividly formed, but that not all traces demand eventual consolidation at the systems level.

Les micro-ARNs (miRNAs) sont de petits ARNs (20-25 nt) qui ont un rôle important dans les mécanismes d'interférence ARN. Les miRNAs sont des inhibiteurs de l'expression génique qui interviennent au niveau post-traductionnel en s'hybridant à des sites spécifiques de leurs ARNm cibles. Ce mécanisme induit la dégradation de l'ARNm ou l'inhibition de sa traduction. Puisque l'hybridation partielle du miRNA est suffisante pour induire une inhibition, chaque miRNA peut avoir des centaines de cibles. Les miRNAs sont impliqués dans de nombreuses fonctions biologiques et en particulier dans processus neuronaux. Plus de la moitié des miRNAs connus sont exprimés dans le cerveau de mammifère avec une distribution spécifique du miRNA considéré. A l'échelle sub-cellulaire il y a également une distribution hétérogène des miRNAs. De plus, il a été montré récemment une implication des miRNAs dans la régulation de la traduction locale dans les neurones. En effet, des miRNAs et des protyeines impliquées dans la biogenèse et la fonction des miRNAs ont été retrouvés dans le soma, les dendrites et les axones. Il a été montré que la dérégulation des miRNAs été impliquée dans de nombreux mécanismes pathologiques. Cette thèse a pour objectif de révéler le rôle des miRNAs dans la plasticité synaptique. Nous avons étudié l'implication des miRNAs dans les mécanismes de la plasticité synaptique homéostatique et dans la plasticité dysfonctionnelle rencontrée en condition de douleur cancéreuse.Notre hypothèse était que la régulation de la traduction locale des récepteurs AMPA dans les dendrites en condition d'homéostasie synaptique implique les miRNAs. Par bio-informatique, qRT-PCR et test luciférase, nous avons identifié le miRNA miR-92a comme régulateur de la traduction de l'ARNm de GluA1. Des immunomarquages des récepteurs AMPA et des enregistrements des courants miniatures AMPA montrent que miR-92a régule spécifiquement l'incorporation synaptique de nouveau récepteurs AMPA contenant GluA1 en réponse à un blocage de l'activité synaptique. La douleur est un symptôme très fréquemment associé au cancer et constitue un challenge pour les médecins puisque aucun traitement spécifique et efficace n'existe. C'est sans doute le résultat d'un manque de connaissances des mécanismes moléculaires responsables de la douleur cancéreuse. En combinant les screening des miRNA et des ARNm, nous avons mis en évidence une voie de régulation impliquant miR-124, un miRNA enrichi dans le système nerveux. Ainsi, dans un modèle de douleur cancéreuse chez la souris, la diminution de miR-124 est associée à une augmentation de ces cibles : calpain 1, synaptopodine et tropomyosine 4. Toutes ces protéines ont précédemment été identifiées comme des molécules clef de la fonction et de la plasticité synaptique. Des experiences in vitro ont confirmé que miR-124 exercait une inhibition multiple de calpain 1, synaptopodine et tropomyosine 4. La pertinence clinique de cette découverte a été vérifiée par le screening du liquide cérébro-spinal de patients souffrant de douleur cancéreuse qui montre également une diminution de miR-124. Ce résultat suggère un fort potentiel thérapeutique du ciblage de miR-124 dans les douleurs cancéreuses. Enfin, l'injection intrathécale de miR-124 dans des souris cancéreuses a permis de normaliser l'expression de la synaptopodine et de stopper la douleur cancéreuse lors de la phase initiale de la maladie. / MicroRNAs (miRNAs) are a type of small RNA molecules (21-25nt), with a central role in RNA silencing and interference. MiRNAs function as negative regulators of gene expression at the post-transcriptional level, by binding to specific sites on their targeted mRNAs. A process results in mRNA degradation or repression of productive translation. Because partial binding to target mRNA is enough to induce silencing, each miRNA has up to hundreds of targets. miRNAs have been shown to be involved in most, if not all, fundamental biological processes. Some of the most interesting examples of miRNA activity regulation are coming from neurons. Almost 50% of all identified miRNAs are expressed in the mammalian brain. Furthermore, miRNAs appear to be differentially distributed in distinct brain regions and neuron types. Importantly, miRNAs are reported to be differentially distributed at the sub-cellular level. Recently, miRNAs have been suggested to be involved in the local translation of neuronal compartments. This has been derived from the observations reporting the presence of miRNAs and the protein complexes involved in miRNA biogenesis and function in neuronal soma, dendrites, and axons. Deregulation of miRNAs has been shown to be implicated in pathological conditions. The present thesis aimed at deciphering the role of miRNA regulation in neuronal plasticity. Here we investigated the involvement of miRNA in synaptic plasticity, specifically in homeostatic synaptic plasticity mode. In addition, we investigated the involvement of miRNAs in the maladaptive nervous system state, specifically, in bone cancer pain condition.We hypothesized that local regulation of AMPA receptor translation in dendrites upon homeostatic synaptic scaling may involve miRNAs. Using bioinformatics, qRT-PCR and luciferase reporter assays, we identified several brain-specific miRNAs including miR-92a, targeting the 3’UTR of GluA1 mRNA. Immunostaining of AMPA receptors and recordings of miniature AMPA currents in primary neurons showed that miR-92a selectively regulates the synaptic incorporation of new GluA1-containing AMPA receptors during activity blockade.Pain is a very common symptom associated with cancer and is still a challenge for clinicians due to the lack of specific and effective treatments. This reflects the crucial lack of knowledge regarding the molecular mechanisms responsible for cancer-related pain. Combining miRNA and mRNA screenings we were able to identify a regulatory pathway involving the nervous system-enriched miRNA, miR-124. Thus, miR-124 downregulation was associated with an upregulation of its predicted targets, Calpain 1, Synaptopodin and Tropomyosin 4 in a cancer-pain model in mice. All these targets have been previously identified as key proteins for the synapse function and plasticity. Clinical pertinence of this finding was assessed by the screening of cerebrospinal fluid from cancer patient suffering from pain who presented also a downregulation of miR-124, strongly suggesting miR-124 as a therapeutic target. In vitro experiments confirmed that miR-124 exerts a multi-target inhibition on Calpain 1, Synaptopodin and Tropomyosin 4. In addition, intrathecal injection of miR-124 was able to normalize the Synaptopodin expression and to alleviate the initial phase of cancer pain in mice.

Long-term depression (LTD) induced by low frequency stimulation (LFS; 900x1Hz) at medial perforant path (MPP) synapses in the rat dentate gyrus (DG) has been described as both developmentally regulated and N-methyl D-aspartate receptor (NMDAr) independent, yet sufficient evidence suggest that the processes is not entirely independent of NMDAr activity. In the present study, in vitro DG-LTD LFS was induced in hippocampal slices prepared from rats at postnatal day (PND) 14, 21 and 28 to investigate how the sensitivity of DG-LTD~LFS to the NMDAr antagonist amino-5-phosphonovaleric acid (AP5; 50µM) changes throughout the juvenile developmental period (jDP; PNDs 12-29) that occurs immediately after the period of peak neurogenesis. We further examined the acute effects of the partial NMDAr antagonist ethanol (EtOH) on DG-LTD LFS and NMDAr excitatory post synaptic currents (NMDAr-EPSCs) in dentate granule cells (DGCs) using 50 and 100mM concentrations (50mM ~0.2%BAC) of EtOH. The magnitude of LTD induced at all three time points was not statistically different between age groups, but the probability of successfully inducing LTD did decrease with age. We found that AP5 was insufficient to inhibit DG-LTD LFS at PND14, but significantly inhibited DG-LTD LFS at PND21 and PND28. We also found that 50mM EtOH, but not 100mM EtOH, significantly attenuated the mag-nitude of DG-LTD LFS induced at each time point. Acute effects of 50mM EtOH had relatively little effect on NMDAr-EPSCs at PND14, and showed a slight potentiation of the response at PND21. 50mM EtOH at PND28, and 100mM EtOH at all three developmental time points showed inhibition of the NMDAr-EPSC. These findings provide insight on how developmental changes to the DG network and dentate gran-ule cells (DGCs) influences mechanisms and processes involved in the induction and expression of synaptic plasticity in the DG. / Graduate

No description available.

Our memories are fundamental components of who we are as individuals. They influence almost every aspect of our lives such as our decisions, our personalities, our emotions, and our purpose in life. Diseases that affect memory have devastating impacts on the individuals who bear them. Imagine not being able to recall pleasant memories or even the faces of close family members. It's important to understand the biology of memory formation not only because it's an intriguing scientific question, but because of its consequences when these processes are lost. N-methyl-D-aspartate-type glutamate receptors (NMDARs) and calcium/calmodulin-dependent kinase II (CaMKII) are essential molecules involved in learning and its physiological correlate, synaptic plasticity. Calcium influx through NMDARs activates CaMKII, which translocates to the postsynaptic signaling sites through its interactions with the NMDAR subunits NR1 and NR2B. The significance of CaMKII's translocation is not fully known, however we hypothesize that it is an early molecular event that is necessary for the expression of synaptic plasticity and learning. Our laboratory has developed two strains of mice with targeted mutations to NR1 and NR2B (NR1KI and NR2BKI) that are deficient in their ability to bind to CaMKII to test the role of CaMKII binding to NMDARs in synaptic plasticity and learning. We found that CaMKII binding to NR2B is necessary for long-term potentiation (LTP), the most commonly studied form of synaptic plasticity. NR2BKI mice are able to learn spatial and cued tasks normally, however they are unable to consolidate spatial tasks for long-term memory storage. On the other hand, we found that CaMKII binding to NR1 is not necessary for LTP. Furthermore NR1KI mice do not show impairments in contextual or cued learning. We found that NR1 mutations resulted in an age-dependent truncation of the intracellular domains of NR1 that reduced its activity leading to severe impairments in synaptic transmission, LTP, and learning. Our results suggest that CaMKII binding to NR2B is the more important for synaptic plasticity and memory formation than NR1. However, we found that the intracellular domains of NR1 are critical for NMDAR and synapse function.

The computational ability of principal neurons and interneurons in the brain and their ability to work together in concert are thought to underlie higher order cognitive processes such as learning, memory, and attention. Dendrites play a very important role in neuronal information processing because they receive and integrate incoming input and can undergo experience-dependent changes that will alter the future output of the neuron. Here, I have used whole-cell patch clamp recordings and fluorescent Ca2+-imaging to examine the modulation of dendritic excitability in principal neurons of the rat and human hippocampus and neocortex. First, I determined that dendrites of dentate granule cells of the hippocampus are tuned to high frequencies of both afferent input and backpropagating action potentials. Under these conditions they are also capable of generating regenerative dendritic activity that can propagate to the soma, which is prone to modulation. In particular, Neuropeptide Y (NPY) Y1 receptors can decrease frequency-dependent dendritic Ca2+ influx. Dopamine D1 receptors (D1Rs) have an opposite effect; they potentiate frequency-dependent dendritic excitability. These two neuromodulators also have an opposing effect on plasticity, with dopamine acting to induce, and NPY acting to inhibit long-term potentiation (LTP). Parallel observations of D1-induced LTP and an NPY-mediated decrease in dendritic excitability in rodents were complemented by findings in human dentate granule cells. Second, I examined the role of NPY receptors on dendrites of layer 5 pyramidal neurons. In these neurons I found that NPY acts post-synaptically on distal dendrites via the Y1 receptor to inhibit frequency-dependent Ca2+-currents, similar to the findings in dentate granule cells. NPY also decreased regenerative Ca2+ currents caused by the appropriate pairing of pre- and post-synaptic input. Together, these observations demonstrate that the role of NPY in the hippocampus and neocortex is not solely as an anti-epileptic agent. NPY release, likely to occur during high frequency oscillatory activity, can act locally to limit dendritic excitability, which can have a profound effect on plasticity. In the dentate gyrus, NPY can inhibit a D1R induced increased dendritic excitability and resultant changes in synaptic strength. These findings will further the understanding of dendritic information processing in the hippocampus and neocortex.

Fragile X syndrome (FXS) is caused by the lack of fragile X mental retardation protein (FMRP). The animal model of FXS, Fmr1 knockout (KO) mice, shows impairment in hippocampus-dependent learning and memory. However, results for long-term potentiation (LTP), remain inconclusive in the hippocampus of Fmr1 KO mice. Here, we demonstrate that FMRP is required for glycine-induced LTP (Gly-LTP) in the CA1 of hippocampus. The Gly-LTP requires activation of postsynaptic NMDA receptors and metabotropic glutamateric receptors, as well as the subsequent activation of extracellular signal-regulated kinase (ERK) 1/2. However, paired-pulse facilitation was not affected by glycine treatment. Our study provide evidences that FMRP participates in Gly-LTP by regulating the phosphorylation of ERK1/2, and that improper regulation of these signaling pathways may contribute to the learning and memory deficits observed in FXS.