No description available.

No description available.

Les variations de la concentration cytosolique de Ca[indice supérieur 2+] contrôlent divers processus biologiques tel la contraction, la division cellulaire et la transactivation de gènes. Chez les cellules non excitables, la stimulation de certains GPCRs (G protein coupled receptor) ou récepteurs tyrosine kinase mène à l'activation d'une phospholipase C qui produit de l'inositol 1,4,5-trisphosphate (IP[indice inférieur 3]). L'IP[indice inférieur 3] active un récepteur-canal au niveau du réticulum endoplasmique (RE) et permet une relâche de Ca[indice supérieur 2+] du RE. Il existe trois isoformes distinctes d'IP[indice inférieur 3]R (IP[indice inférieur 3]R-1 à -3) différemment exprimées dans les tissus. Dans ces travaux, nous avons étudié un mode de régulation de l'IP[indice inférieur 3]R-2 par la PKC, une kinase elle-même activée lors de la réponse calcique. Nous avons aussi vérifié l'implication de chaque isoforme d'IP[indice inférieur 3]R dans l'activité transcriptionnelle des facteurs de transcription sensibles au Ca[indice supérieur 2+] NFAT et CREB.Les cellules AR4-2J expriment principalement l'IP[indice inférieur 3]R-2 (86%), ce qui en fait un bon modèle d'étude pour les mécanismes de régulation de l'IP[indice inférieur 3]R-2. Dans la première étude, nous avons vérifié si la PKC influençait l'activité calcique de l'IP[indice inférieur 3]R-2. D'abord, nous avons montré que l'IP[indice inférieur 3]R-2 est majoritairement exprimé dans les cellules AR4-2J. Nous avons montré par phosphorylation in vitro et in cellulo que l'IP[indice inférieur 3]R-2 est phosphorylé par la PKC. Nous avons montré que le traitement par la PKC réduit la réponse calcique induite par l'IP[indice inférieur 3] sur des cellules perméabilisées. Finalement, nous avons démontré que la réponse calcique induite par le CCh ou l'EGF sur des cellules entières était réduite avec l'activation de la PKC. Ces résultats indiquent que l'IP[indice inférieur 3]R-2 est une autre cible de la PKC permettant de contrôler l'intensité de la réponse calcique. Dans la deuxième étude l'objectif était de déterminer l'implication des isoformes d'IP[indice inférieur 3]R sur l'activité des facteurs de transcription sensibles au Ca[indice supérieur 2+] NFAT et CREB. Par l'approche des gènes rapporteurs de l'activité de NFAT ou CREB, nous avons montré que NFAT était activé par la voie de la calcineurine et que CREB par les voies de CamKII et de la calcineurine dans les cellules HEK 293A. Nous avons démontré que l'invalidation de l'IP[indice inférieur 3]R-2 diminuait la réponse calcique induite par le CCh et que la costimulation avec le VIP pouvait la restaurer. Nous avons montré que l'activité transcriptionnelle de NFAT était affectée, contrairement à celle de CREB, par l'invalidation de l'IP[indice inférieur 3]R-2. Finalement, nous avons montré que l'IP[indice inférieur 3]R-2, et aussi l'IP[indice inférieur 3]R-1, semblent jouer un rôle secondaire, différent de celui de transporteur de Ca[indice supérieur 2+], dans le mécanisme d'activation de NFAT. Ces résultats laissent envisager que l'IP[indice inférieur 3]R serait impliqué dans la formation d'un complexe protéique facilitant l'activation de NFAT.

The release of chemical transmitter from nerve terminals is critically dependent on a transient increase in intracellular Ca2+.6,25 The increase in Ca2+ may be due to influx of Ca2+ from the extracellular fluid15 or release of Ca2+ from intracellular stores such as mitochondria.1,8,18 Whether Ca2+ utilized in transmitter release is liberated from organelles other than mitochondria is uncertain. Smooth endoplasmic reticulum is known to release Ca2+, e.g., on activation by inositol trisphosphate or cyclic adenosine diphosphate-ribose,2 so the possibility exists that Ca2+ from this source may be involved in the events leading to exocytosis. We examined this hypothesis by testing whether inositol trisphosphate and cyclic adenosine diphosphate-ribose modified transmitter release. We used liposomes to deliver these agents into the cytoplasmic compartment and binomial analysis to determine their effects on the quantal components of transmitter release. Administration of inositol trisphosphate (10-4M) caused a rapid, 25% increase in the number of quanta released. This was due to an increase in the number of functional release sites, as the other quantal parameters were unaffected. The effect was reversed with 40min of wash. Virtually identical results were obtained with cyclic adenosine diphosphate-ribose (10-4M). Inositol trisphosphate caused a 10% increase in quantal size, whereas cyclic adenosine diphosphate-ribose had no effect. The results suggest that quantal transmitter release can be increased by Ca2+ released from smooth endoplasmic reticulum upon stimulation by inositol trisphosphate or cyclic adenosine diphosphate-ribose. This may involve priming of synaptic vesicles at the release sites or mobilization of vesicles to the active zone. Inositol trisphosphate may have an additional action to increase the content of transmitter within the vesicles. These findings raise the possibility of a role of endogenous inositol phosphate and smooth endoplasmic reticulum in the regulation of cytoplasmic Ca2+ and transmitter release.

No description available.

Understanding how plants respond to stress is of importance, considering the increasing need to feed a growing population and supply its energy. Plants have complex systems for detecting, and responding to stresses. One stress-responsive system involves myo-inositol (Ins). Ins is a precursor for cell wall components, inositol trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol phosphate signaling molecules, and an alternate ascorbic acid (AsA) synthesis pathway. The enzyme, myo-inositol oxygenase (MIOX) is encoded by four genes in Arabidopsis and catalyzes the first step of Ins catabolism producing D-glucuronic acid (DGlcA). This research focuses on MIOX metabolism of Ins during plant growth and stress responses. I have examined miox mutants for alterations in metabolism and signaling. MIOX2 and MIOX4 expression patterns correlate with miox mutant root growth in varying nutrient conditions, and changes in flowering time. In miox2 mutants, I found an increase in Ins in most tissues, which was accompanied by cold- and abscisic (ABA)- sensitivity; however, miox4 mutants are ABA- insensitive, and have a small increase of Ins in flowers. MIOX2:GFP fusion protein accumulates in the cytoplasm and MIOX4:GFP accumulates in the cytoplasm and nucleus. Overexpresser MIOX4+ plants provide a model system to examine how directing carbon from Ins into DGlcA impacts Ins levels and Ins signaling. I have examined MIOX4+ plants for alterations in MIOX4 RNA and protein, and measured Ins by gas chromatography (GC). My results indicate that MIOX4+ tissues are impacted differently by the MIOX4 transgene, with decreases in Ins after seed imbibition, and increased Ins levels later in development. Ins depletion in seedlings was correlated with a decrease in Ins(1,4,5)P3. To determine the impact of reducing Ins and Ins(1,4,5)P3 in MIOX4+ seedlings, I examined processes known to involve Ins(1,4,5)P3 signaling. MIOX4+ seed have increased seed dormancy, NaCl-sensitivity, and ABA-insensitivity. These results suggest MIOX affects Ins signaling in response to ABA. Together, these data indicate that transcriptional control of MIOX2 and MIOX4 results in distinct roles in plant growth, and that MIOX2 and MIOX4 function in metabolic and signaling processes critical for growth, nutrient sensing, and stress responses. / Ph. D.

No description available.

To survive, an organism must constantly adjust its internal state to changes in the environment from which it receives signals. The signals set off a chain of events referred to signal transduction. Signal transduction systems are especially important in multicellular organisms, such as plants and animals, because of the need to coordinate the activities of hundreds to trillions of cells. Plants, in particular, have a special need for perceiving signals from their environment because of their static nature. As in the animal cell, the first steps in perception of a signal include signal interaction with a receptor, signal amplification through second messenger production, and signal termination through second messenger hydrolysis. Myo-inositol polyphosphate 5-phosphatases (5PTases) (EC 3.1.3.56) have unique signal terminating abilities toward the second messenger inositol trisphosphate (Ins (1,4,5)P3, InsP3). In Arabidopsis thaliana there are 15 members of the 5PTase family, the majority of which contain a single 5PTase catalytic domain. Four members of the Arabidopsis 5PTase family, however, have a unique protein domain structure, with additional N-terminal WD40 repeats that are implicated in protein-protein interactions. The research presented here focused on the identification of 5PTase interacting proteins and the characterization of their functional role in Arabidopsis. To accomplish this goal, I examined a 5PTase13-interacting protein, the sucrose (Suc) nonfermenting-1-related kinase, SnRK1.1, an important energy sensor that is highly conserved among eukaryotes. My identification of a 5PTase13:SnRK1.1 complex points to the novel interaction of this metabolic modulator and inositol signaling/metabolism. 5PTase13 , however, plays a regulatory role in other plant specific processes as well, since I also identified the Arabidopsis homolog (Atp80) of the human WDR48 (HsWDR48, Hsp80) as a novel protein interactor of 5PTase13. My results indicate that Atp80 is important for leaf emergence, development and senescence likely via a regulatory interaction with 5PTase13 and PINOID â binding protein (PBP1). / Ph. D.

Intricate interactions among constituent components are defining hallmarks of biological systems and sculpt physiology across different scales spanning gene networks to behavioural repertoires. Whereas interactions among channels and receptors define neuronal physiology, interactions among different cells specify the characteristic features of network physiology. From a single-neuron perspective, it is now evident that the somato-dendritic plasma membrane of hippocampus pyramidal neurons is endowed with several voltage-gated ion channels (VGICs) with varying biophysical properties and sub cellular expression profiles. Structural and physiological interactions among these channels define generation and propagation of electrical signals, thereby transforming neuronal dendrites to actively excitable membrane endowed with complex computational capabilities. In parallel to this complex network of plasma membrane channels is an elegantly placed continuous intraneuronal membrane of the endoplasmic reticulum (ER) that runs throughout the neuronal morphology. Akin to the plasma membrane, the ER is also endowed with a variety of channels and receptors, prominent among them being the inositol trisphosphate (InsP3) receptors (InsP3Rs) and ryanodine receptors (RyR), both of which are calcium release channels. Physiological interactions among these receptors transform the ER into a calcium excitable membrane, capable of active propagation of calcium waves and of spatiotemporal integration of neuronal signals. Thus, a neuron is endowed with two continuously parallel excitable membranes that actively participate in the bidirectional flow of intraneuronal information, through interactions among different channels and receptors on either membrane. Although the interactions among sets of channels and receptors present individually on either membrane are very well characterized, our understanding of cross-membrane interactions among channels and receptors across these two membranes has been very limited. Recent literature has emphasized the critical nature of such cross-membrane interactions and the several physiological roles played by such interactions. Such cross-channel interactions include ER depletion-induced signaling involving store-operated calcium channels, generation and propagation of calcium waves through interactions between plasma membrane and ER membrane receptors, and the plasticity of plasma membrane VGICs and receptors induced by ER Ca2+. Such tight interactions between these two membranes have highlighted several roles of the ER in the integration of intraneuronal information, in regulating signalling microdomains and in regulating the downstream signaling pathways that are regulated by these Ca2+ signals. Yet, our understanding about the functional interactions between the ion channels and receptors present on either of these membranes is very limited from the perspective of the combinatorial possibilities that encompass the span of channels and receptors across these two membranes. In this context, the first part of this thesis deals with two specific instances of such cross-membrane functional interactions, presented as two subparts with each probing different direction of impact. Specifically, whereas the first of these subparts deals with the impact of plasma membrane VGICs on the physiology of ER receptors, the second subpart presents an instance of the effect of ER receptor activation on plasma membrane VGIC. In the first subpart of the thesis, we establish a novel role for the A-type potassium current in regulating the release of calcium through inositol triphosphate receptors (InsP3R) that reside on the endoplasmic reticulum (ER) of hippocampus pyramidal neurons. Specifically, the A-type potassium current has been implicated in the regulation of several physiological processes including the regulation of calcium influx through voltage-gated calcium channels (VGCCs). Given the dependence of InsP3R open probability on cytosolic calcium concentration ([Ca2+]c) we asked if this regulation of calcium influx by A-type potassium current could translate into the regulation of release of calcium through InsP3Rs by the A-type potassium current. To answer this, we constructed morphologically realistic, conductance-based neuronal models equipped with kinetic schemes that govern several calcium signalling modules and pathways, and constrained the distributions and properties of constitutive components by experimental measurements from these neurons. Employing these models, we establish a bell-shaped dependence of calcium release through InsP3Rs on the density of A-type potassium current, during the propagation of an intraneuronal calcium wave initiated through established protocols. Exploring the sensitivities of calcium wave initiation and propagation to several underlying parameters, we found that ER calcium release critically depends on dendrite diameter and wave initiation occurred at branch points as a consequence of high surface area to volume ratio of oblique dendrites. Further, analogous to the role of A-type potassium channels in regulating spike latency, we found that an increase in the density of A-type potassium channels led to increases in the latency and the temporal spread of a propagating calcium wave. Next, we incorporated kinetic models for the metabotropic glutamate receptor (miler) signalling components and a calcium-controlled plasticity rule into our model and demonstrate that the presence of mGluRs induced a leftward shift in a BCM-like synaptic plasticity profile. Finally, we show that the A-type potassium current could regulate the relative contribution of ER calcium to synaptic plasticity induced either through 900 pulses of various stimulus frequencies or through theta burst stimulation. These results establish a novel form of interaction between active dendrites and the ER membrane and suggest that A-type K+ channels are ideally placed for inhibiting the suppression of InsP3Rs in thin-caliber dendrites. Furthermore, they uncover a powerful mechanism that could regulate biophysical/biochemical signal integration and steer the spatiotemporal spread of signalling micro domains through changes in dendritic excitability. In the second subpart, we turned our focus to the role of calcium released through InsP3Rs in regulating the properties of VGICs present on the plasma membrane, thereby altering neuronal intrinsic properties that are dependent on these VGICs. Specifically, the synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signalling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. Here, we complemented the recently established necessity of inositol trisphosphate (InsP3) receptors (InsP3R) in a form of intrinsic plasticity by asking if ER InsP3R activation was sufficient to induce plasticity in intrinsic properties of hippocampus neurons. To do this, we employed whole-cell patch-clamp recordings to infuse D-myo-InsP3, the endogenous ligand for InsP3Rs, into hippocampus pyramidal neurons and assessed the impact of InsP3R activation on neuronal intrinsic properties. We found that such activation reduced input resistance, maximal impedance amplitude and temporal summation, but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead of hippocampus pyramidal neurons. Strikingly, the magnitude of plasticity in all these measurements was dependent upon [InsP3], emphasizing the graded dependence of such plasticity on InsP3R activation. Mechanistically, we found that this InsP3-induced plasticity depended on hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP3Rs, the influx of calcium through N-methyl-D -aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. These results delineate a causal role for InsP3Rs in graded adaptation of neuronal response dynamics through changes in plasma membrane ion channels, thereby revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis. Whereas the first part of the thesis dealt with bidirectional interactions between ER membrane and plasma membrane channels/receptors within a neuron, second part focuses on cross-cellular interactions, specifically between ER membrane on astrocytes and dendritic plasma membrane of neurons. Specifically, the universality of ER-dependent calcium signalling ensures that its critical influence extends to regulating the physiology of astrocytes, an abundant form of glial cells in the hippocampus. Due to the presence of calcium release channels on ER membrane, astrocytes are calcium excitable, whereby they respond to neuronal activity by increase in their cytosolic calcium levels. Specifically, astrocytes respond to the release of neurotransmitters from neuronal presynaptic terminals through activation of metabotropic receptors expressed on their plasma membrane. Such activation results in the mobilization of cytosolic InsP3 and subsequent release of calcium through InsP3 on the astrocytes ER membrane. These ER-dependent [Ca2+]c elevations in astrocytes then result in the release of gliotransmitters from astrocytes, which bind to corresponding receptors located on neuronal plasma membrane resulting in voltage-deflections and/or activation of signaling pathways in the neuron. Although it is well established that gliotransmission constitutes an important communication channel between astrocytes and neurons, the impact of gliotransmission on neurons have largely been centered at the cell body of the neurons. Consequently, the impact of the activation of astrocytic InsP3R on neuronal dendrites, and the role of dendritic active conductances in regulating this impact have been lacking. This lacuna in mapping the spatial spread of gliotransmission in neurons is especially striking because most afferent synapses impinge on neuronal dendrites, and a significant proportion of information processing in neurons is performed in their dendritic arborization. Additionally, given that active dendritic conductances play a pivotal role in regulating the impact of fast synaptic neurotransmission on neurons, we hypothesized that such active-dendritic regulation should extend to the impact of slower extrasynaptic gliotransmission on neurons. The second part of the thesis is devoted to testing this hypothesis using dendritic and paired astrocyte-neuron electrophysiological recordings, where we also investigate the specific roles of active dendritic conductances in regulating the impact of gliotransmission initiated through activation of astrocytic InsP3Rs. In testing this hypothesis, in the second part of the thesis, we first demonstrate a significantly large increase in the amplitude of astrocytically originating spontaneous slow excitatory potentials (SEP) in distal dendrites compared to their perisomatic counterparts. Employing specific neuronal infusion of pharmacological agents, we show that blocking HCN channels increased the frequency, rise-time and width of dendritically-recorded spontaneous SEPs, whereas blockade of A-type potassium channels enhanced their amplitude. Next, through paired neuron-astrocytes recordings, we show that our conclusions on the differential roles of HCN and A-type potassium channels in modulating spontaneous SEPs also extended to SEPs induced through infusion of InsP3 in a nearby astrocyte. Additionally, employing subtype-specific receptor blockers during paired neuron-astrocyte recordings, we provide evidence that GluN2B-and GluN2D-containing NMDARs predominantly mediate perisomatic and dendritic SEPs, respectively. Finally, using morphologically realistic conductance-based computational models, we quantitatively demonstrate that dendritic conductances play an active role in mediating compartmentalization of the neuronal impact of gliotransmission. These results unveil an important role for active dendrites in regulating the impact of gliotransmission on neurons, and suggest astrocytes as a source of dendritic plateau potentials that have been implicated in localized plasticity and place cell formation. This thesis is organized into six chapters as follows: Chapter 1 lays the motivations for the questions addressed in the thesis apart from providing the highlights of the results presented here. Chapter 2 provides the background literature for the thesis, introducing facts and concepts that forms the foundation on which the rest of the chapters are built upon. In chapter 3, we present quantitative analyses of the physiological interactions between A-type potassium conductances and InsP3Rs in CA1 pyramidal neurons. In chapter 4, using electrophysiological recordings, we investigate the role of calcium released through InsP3Rs in induction of plasticity of intrinsic response dynamics, and demonstrate that this form of plasticity is consequent to changes in neuronal HCN channels. In chapter 5, we systematically map the spatial dynamics of the impact of gliotransmission on neurons across the somato-apical trunk, also unveiling the role of neuronal HCN and A-type potassium channels in compartmentalizing such impact. Finally, chapter 6 concludes the thesis highlighting its major contributions and discussing directions of future research.