Os endocanabinóides (ECs) são neuromoduladores lipídicos que são produzidos por demanda tendo ação retrógrada. Existem duas moléculas que são atualmente reconhecidos como os principais ECs, a anandamida (AEA) e 2-araquidonoilglicerol (2-AG). O hipocampo sintetiza endocanabinóides e expressa seus receptores (CB1, CB2, TRPV1).Existe uma discrepância de efeitos dos 2 endocanabinóides na potenciação á longo prazo (LTP) hipocampal que pode ser um resultado da AEA agir tanto em receptores CB1 quanto em receptores TRPV1.Tendo em vista que a ativação dos receptores TRPV1 potenciam a LTP hipocampal, e não a inibem como é observado com a AEA, então qual seria o mecanismo de ação da AEA em inibir a LTP? Seria possível que a AEA estivesse preferencialmente inibindo a produção de 2-AG e assim inibindo a LTP? Para testar essa hipótese usamos inibidores farmacológicos da degradação hidrolítica do 2-AG e da AEA e também usamos antagonistas dos receptores TRPV1 e um animal knock-out para o receptor TRPV1. Camundongos machos BlackC57 e knockout(KO) para TRPV1 com idade entre 35 a 49 dias foram utilizados para obtenção de fatias do hipocampo (400µm), e a potenciação a longo prazo na via Schaffer-CA1 estudada. Não observamos efeito dos inibidores da degradação hidrolítica e oxidative dos endocanabinóides na LTP. A LTP do camundongo knock-out era inibida, porem o antagonismo farmacológico dos receptores TRPV1 não minetizou esse efeito. Já o agonista dos receptors canabinóides WIN55212-2 inibiu a indução da LTP. Concluimos que o aumento dos endocanabinóides pela inibição da sua degradação não foi eficiente em alterar a LTP hippocampal em nosso modelo experimental. Aprovação do CONCEA-FMRP- nº008/2017 / Endocannabinoids (ECs) are lipid neuromodulators that are produced on demand having retrograde action. There are two molecules that are currently recognized as the major ECs, anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The hippocampus synthesizes endocannabinoids and expresses their receptors (CB1, CB2, TRPV1). There is a discrepancy of endocannabinoid effects on hippocampal long term potentiation (LTP) which may be a result of AEA acting on both CB1 and TRPV1 receptors. Given that the activation of TRPV1 receptors potentiate hippocampal LTP, and do not inhibit it as observed with AEA, then what would be the mechanism of action of AEA in inhibiting LTP? Was it possible that AEA was preferentially inhibiting 2-AG production and thus inhibiting LTP? To test this hypothesis, we used pharmacological inhibitors of the hydrolytic degradation of 2-AG and AEA and also used TRPV1 receptor antagonists and a knock-out animal for the TRPV1 receptor. Male BlackC57 mice and TRPV1 knockout (KO) aged 35 to 49 days were used to obtain hippocampal slices (400 ?m), and the long-term potentiation in the Schaffer-CA1 pathway studied). The LTP of the knock-out mouse was inhibited, but the pharmacological antagonism of TRPV1 receptors did not mimic this effect, whereas the WIN55212-2 cannabinoid receptor agonist inhibited the induction of LTP. We conclude that increasing the levels of endocannabinoids by inhibiting their degradation was not efficient in altering hippocampal LTP in our experimental model Approval of CONCEA-FMRP- nº 008/2017

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

<p>Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.</p> / Dissertation

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.

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.

<p>At excitatory synapses, NMDAR-mediated synaptic plasticity occurs in response to activity inputs by modifying synaptic strength. While comprehensive studies have been focused on the induction and expression mechanisms underlying synaptic plasticity, it is less clear whether and how synaptic plasticity itself can be subjected to regulations. The presence of "plasticity of plasticity", or meta-plasticity, has been proposed as an essential mechanism to ensure a proper working range of plasticity, which may also offer an additional layer of information storage capacity. However, it remains elusive whether and how meta-plasticity occurs at single synapses and what molecular substrates are locally utilized. Here, I develop systems allowing for sustained alterations of individual synaptic inputs. By implementing a history of inactivity at single synapses, I demonstrate that individual synaptic inputs control synaptic molecular composition homosynaptically, while allowing heterosynaptic integration along dendrites. Furthermore, I report that subunit-specific regulation of NMDARs at single synapses mediates a novel form of input-specific metaplasticity. Prolonged suppression of synaptic releases at single synapses enhances synaptic NMDAR-mediated currents and increases the number of functional NMDARs containing NR2B. Interestingly, synaptic NMDAR composition is adjusted by spontaneous glutamate release rather than evoked activity. I also demonstrate that inactivated synapses with more NMDARs containing NR2B acquire a lower induction threshold for long-term synaptic potentiation. Together, these results suggest that at single synapses, spontaneous release primes the synapse by modifying its synaptic state with specific molecular compositions, which in turn determine the synaptic gain in an input-specific manner.</p> / Dissertation

The LIM-Kinase family of proteins (LIMK) plays an important role in actin dynamics through its regulation of ADF/cofilin. A subtype of LIMK, LIMK1, is mostly expressed in neuronal tissues with high levels in the mature synapse. Previous studies from the Zhen Ping Jia laboratory have shown that LIMK1-/- mice exhibit abnormal spine morphology as well as altered hippocampal synaptic plasticity. LIMK1 has been shown to interact with CREB during neuronal development (Yang et al., 2004). We propose that LIMK1 is able to phosphorylate CREB in response to a synaptic activity. We hypothesize that if LIMK1 activates CREB in mature neurons, then LIMK1 knockout mice will have decreased L-LTP and deficits in long-term memory. My results show that LIMK1 and CREB exist in a complex and are bound to each other in mature neurons. LIMK1-/- mice exhibit deficits in the late phase of long-term potentiation and specific deficits in long-term memory while short-term memory remains unaltered. Pharmacological activation of CREB attenuates the observed deficits in synaptic plasticity and long-term memory. These results show a potentially novel mechanism of CREB activation in response to synaptic activity. Moreover, using peptides to manipulate actin dynamics in LIMK1 lacking animals only has effects on early LTP and is not able to rescue the late phase LTP deficits found in LIMK1 -/- mice. These results indicate a specific role of LIMK1 long-term memory and synaptic plasticity through regulation of CREB and not through regulation of the actin cytoskeleton.

The LIM-Kinase family of proteins (LIMK) plays an important role in actin dynamics through its regulation of ADF/cofilin. A subtype of LIMK, LIMK1, is mostly expressed in neuronal tissues with high levels in the mature synapse. Previous studies from the Zhen Ping Jia laboratory have shown that LIMK1-/- mice exhibit abnormal spine morphology as well as altered hippocampal synaptic plasticity. LIMK1 has been shown to interact with CREB during neuronal development (Yang et al., 2004). We propose that LIMK1 is able to phosphorylate CREB in response to a synaptic activity. We hypothesize that if LIMK1 activates CREB in mature neurons, then LIMK1 knockout mice will have decreased L-LTP and deficits in long-term memory. My results show that LIMK1 and CREB exist in a complex and are bound to each other in mature neurons. LIMK1-/- mice exhibit deficits in the late phase of long-term potentiation and specific deficits in long-term memory while short-term memory remains unaltered. Pharmacological activation of CREB attenuates the observed deficits in synaptic plasticity and long-term memory. These results show a potentially novel mechanism of CREB activation in response to synaptic activity. Moreover, using peptides to manipulate actin dynamics in LIMK1 lacking animals only has effects on early LTP and is not able to rescue the late phase LTP deficits found in LIMK1 -/- mice. These results indicate a specific role of LIMK1 long-term memory and synaptic plasticity through regulation of CREB and not through regulation of the actin cytoskeleton.

Learning and memory mechanisms that are normally related to natural rewards, such as long-term potentiation (LTP) and depression (LTD), may be usurped by the voluntary intake of drugs of abuse. The maladaptive behaviour that characterizes addiction is thought to arise from persistent changes in excitatory synaptic function in brain reward circuits. The oval region of the dorsal bed nucleus of the stria terminalis (ovBST) is one such region susceptible to drug-induced synaptic remodeling and is implicated in drug-driven behaviors, reinforcement and stress-induced relapse to drug-seeking. Using whole-cell voltage clamp recordings of ovBST neurons in brain slices prepared from adult Long-Evans rats, we demonstrated an unrestrained increase in AMPAR-mediated excitatory transmission with maintenance of cocaine self-administration. This is unlike self-administration of a natural reward, in which we observed an enhancement and then decline of AMPAR-mediated transmission with continued intake. Additionally, we demonstrate impairment in NMDAR-mediated LTD in ovBST neurons with cocaine self-administration. This impairment may be due to resilient GluN2B-containing NMDARs, as application of a GluN2B-antagonist rescued impaired LTD. Based on models of NMDAR-mediated bidirectional plasticity we suggest that a drug-induced de-regulation between GluN2A and GluN2B subunits impairs LTD, which may underlie the enhancement AMPAR-mediated transmission. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2012-05-31 09:46:39.312

<p>Long-term plasticity, the long-lasting, activity-dependent change in synaptic efficacy, is a fundamental property of the nervous system. Presynaptic forms of long-term plasticity are widely expressed throughout the brain, having been described in regions such as the cortex, cerebellum, hippocampus, thalamus, amygdala and striatum. Presynaptic long-term potentiation (LTP) is associated with an increase in presynaptic release probability, but further evidence of the cellular basis for the change in release probability is not known. At the molecular level, presynaptic LTP is known to require protein kinase A, the synaptic vesicle protein, Rab3A, and the active zone protein, RIM1alpha. RIM1alpha, a presynaptic scaffold protein, binds to many molecules with known functions at different stages of the neurotransmitter release process and the synaptic vesicle cycle. Understanding which interactions of RIM1alpha mediate presynaptic LTP would shed light on the molecular and cellular mechanisms for presynaptic long-term plasticity.</p><p>Here I developed a novel platform to achieve robust acute genetic</p><p>manipulation of presynaptic proteins at hippocampal mossy fiber synapses, where presynaptic LTP is expressed. With this platform, I perform structure-function analysis of RIM1alpha in presynaptic LTP. I find that RIM1alpha phosphorylation by PKA at serine 413 is not required for mossy fiber LTP, nor does RIM1alpha-Rab3A interation. These findings suggest that RIM1alpha, Rab3A and PKA signaling, instead of functioning synergistically, may represent separate requirements for presynaptic long-term plasticity. I then tested whether Munc13-1, a priming protein, is an effector for RIM1alpha in presynaptic LTP and provide the first evidence for the involvement of Munc13-1 in presynaptic long-term synaptic plasticity. I further demonstrate that the interaction between RIM1alpha and Munc13-1 is required for this plasticity. These results further our understanding of the molecular mechanisms of presynaptic plasticity and suggest that modulation of vesicle priming may provide the cellular substrate for expression of LTP at mossy fiber synapses.</p> / Dissertation