Astrocytes are the most abundant non-neuronal cells in vertebrate brains. Although Drosophila melanogaster has fewer astrocytic cells relative to neuronal and other glial cell populations, they, like vertebrate astrocytes, are located in synaptic regions, organized into exclusive, minimally-overlapping domains, and play developmental roles in synaptogenesis. But, do Drosophila astrocytes have parallel roles in the regulation of synaptic signaling? Preliminary electron microscopic (EM) data indicates that astrocytic processes are located at a greater distance, on average, from Drosophila synapses than they are from vertebrate synapses, thus raising questions about their capacity to alter synaptic signals. Do astrocytic cells and processes occupy stereotyped synaptic regions across repeating segmental structures and across individuals? In the studies presented here, we have addressed these questions directly in the ventral nerve cord (VNC) of the third-instar larva. We collected the first whole-cell patch-clamp recordings from Drosophila astrocytes. These indicate that intrinsic membrane properties, such as low membrane resistance, high capacitance, a hyperpolarized resting potential relative to neurons, a passive current-voltage relationship, coupling to other astrocytic cells, and an absence of voltage-gated currents, are shared between astrocytes of highly divergent species. Next, we optogenetically activated of a group of glutamatergic pre-motor neurons and showed that astrocytes respond with a glutamate transporter current that is mediated by Eaat1, and that acute, pharmacological and chronic, genetic blockades of this transporter have subsequent effects on the decay of post-synaptic motor neuron currents. Then, we used three-dimensional EM to locate the pre-motor glutamatergic neurons that were activated in the physiological study and measured the distance from each presynaptic site to the nearest astrocytic process. We found that these distances vary 100-fold even along a single neurite and that these structures are rarely in direct contact, but that no synapse is positioned greater than one micron from an astrocytic process. Thus, it is in this anatomical configuration that the regulation of post-synaptic currents by Eaat1 occurs. Finally, we generated a library of single, fluorescently-labeled astrocytes that were co-labeled with fiduciary landmarks, and used this library to compare the placement of astrocyte cell bodies and arbors across VNC segments and individuals. We found substantial variation in the gross shape, size, and territory covered by astrocytes, and conclude that their neuropil domains are not reliably stereotyped. Given the consistent placement of neuronal connectome elements, this indicates that signals of a specific synapse are not regulated by a designated astrocyte. Together, these findings reveal new functional parallels between Drosophila and vertebrate astrocytes. These findings argue for the relevance and applicability of mechanistic discovery in Drosophila astrocytes, and set the stage for further inquiry into the genetic determinants of astrocyte morphology and physiology.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Une brève stimulation du Noyau Sous-thalamique (STN) dans la tranche des ganglions de la base induit une réponse polysynaptique de longue durée dans ses cibles : Substantia Nigra Pars reticulata (SNr), GP (Globus Pallidus). Cette réponse consiste en un courant NMDA suivi d'un barrage de courant excitateurs de type AMPA qui génère des bouffées de potentiels d'action. La même stimulation dans des branches provenant de souris déplétée en dopamine génère aussi des réponses complexes de plus longue durée. Leur seuil de déclenchement étant 2 à 3 fois plus faibles et ne peuvent dans certains cas se réverbérer. Afin de comprendre les mécanismes sous-sous-jacents dans le STN, nous avons étudié la réponse de ces neurones. en contrôle de courtes réponses sont enregistrées tandis qu'en conditions "parkinsoniennes" cette réponse est considérablement augmentée. Nous proposons qu'un réseau glutamatergique est présent dans le STN et est sous le contrôle négatif des récepteurs dopaminergiques / Single pulse stimulation of the Subthalamic Nucleus (STN) induces a polysynaptic response in the Basal Ganglia Slice in its targets : Substantia Nigra Pars Reticulata (SNr), Globus Pallidus (GP). This response consists of a slow NMDA current with superimposed AMPA transients which generate burst of action potential. The same stimulation in slices depleted in dopamine generates complex response of longer duration. The threshold is also lowered (2-3 times) and in some cases this response reverberates. Theses bursts of synaptic activities could be generated spontaneously. In order to understand the mechanisms, we recorded the evoked response in the STN. In control, only short responses are recorded whereas in "Parkinsonian" conditions, the polysynaptic response is increased. We propose that a polysynaptic network of dopaminegic receptors

Interstitial cystitis (IC)/painful bladder syndrome (PBS) is a functional visceral disorder characterized by increased bladder activity and chronic pelvic pain in the absence of a pathobiological condition. Enhanced sensory transduction of peripheral bladder afferents is hypothesized to contribute to the pain and mechanical hypersensitivity of IC/PBS patients. The aim of this thesis is to test the hypothesis that purinergic receptors, including ionotropic P2X and metabotropic P2Y, are important for sensory transmission in bladder afferent neurons and may be involved in bladder hypersensitivity after bladder tissue insults. Electrophysiological, single cell RT-PCR and immunohistochemistry techniques were performed in bladder afferent neurons from naïve and bladder inflamed mice to test the hypothesis. In Chapter 2, I characterized the distribution and function of P2X receptors in thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia (DRG) neurons innervating the urinary bladder, and found that LS and TL bladder neurons have differential purinergic signaling and distinct membrane electrical properties. In Chapter 3, I examined the sensitization of bladder afferent neurons and the plasticity of P2X receptor function in a mouse model of chemical induced bladder inflammation. P2X-mediated signals in LS and TL bladder neurons after bladder inflammation were enhanced compared with those in saline-treated controls, suggesting the importance of P2X in bladder hypersensitivity associated with cystitis. In Chapter 4, the modulation of P2Y on P2X function and the co-localization of P2Y and P2X were examined in bladder sensory neurons. It has been found that P2Y2 receptor enhances bladder sensory neuron excitability and facilitates the response of homomeric P2X2 receptor to the purinergic agonist (ATP). The present study provides evidence that LS and TL mouse bladder sensory neurons exhibit distinct P2X signaling, and the function of P2X receptors could be facilitated during bladder inflammation and modulated by activation of P2Y2 receptor, indicating an involvement of P2X and P2Y2 receptors as mechano- and chemosensors in bladder sensory transmission under normal conditions and in bladder hypersensitivity associated with inflammation.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

beta-Bungarotoxin (beta-BuTx), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide subunits. A phospholipase A2 subunit named A chain, and a non-phospholipase A2 subunits named B chain. The A chain and B chain are covalently linked by one disulfide bridge. Although it has been widely accepted that the toxic effect of beta-BuTx is attributed to the disturbance of presynaptic transmitter release, however the inhibition of transmitter release by beta-BuTx is still obscure. Here we investigate the mechanism that mediates facilitation of transmitter release at the neuromuscular junction induced by beta-BuTx, using Xenopus nerve-muscle coculture. Application of beta-BuTx and isotoxins BM12, BM13 led to a marked increase in the frequency of spontaneous synaptic currents (SSCs) after a short period (12~18 min) of latency. The synaptic potentiation induced by these toxins was abolished when Ca2+ in the medium is substituted by Ba2+ (a potent phospholipase A2 inhibitor). Application of PLP-BM12 and PLP-BM13, which have been chemical-modification to lose their PLA2 activity from BM12 and BM13, failed to potentiate the transmitter release.

Reproduction de : Thèse de doctorat : Biologie cellulaire et moléculaire : Lyon 1 : 2004. / Titre provenant de l'écran titre. 256 réf. bibliogr.

Zugl.: Giessen, Univ., Diss., 2007

Adiponectin (ADP) is an adipokine, which acts as an insulin sensitizing hormone. Recent studies have shown that adiponectin receptors (AdipoR1, AdipoR2) are present in the CNS; however, there is some debate as to whether or not ADP crosses the blood brain barrier (BBB). Circumventricular organs (CVO) are CNS sites outside the BBB, and thus represent sites at which circulating adiponectin may act to influence the CNS without having to cross the BBB. The subfornical organ (SFO) is a CVO that is responsive to a number of different circulating satiety signals including amylin, CCK, and ghrelin. We report here that the SFO also shows a high density of mRNA for both adiponectin receptors. These observations support the concept that the SFO may be a key player in sensing circulating ADP. To test the hypothesis that ADP influences the excitability of SFO neurons, we used current-clamp electrophysiology on dissociated SFO neurons to observe changes in membrane potential. ADP (10 nM) application effected the excitability of SFO neurons, where the cells either depolarized (8.9±0.9 mV, 21 of 97 cells) or hyperpolarized (-8.0±0.5 mV, 34 of 97 cells). Using single-cell RT-PCR we found that the majority of the responsive neurons expressed AdipoR1 or R2 and the non-responsive neurons expressed neither. In view of the recognized role of ADP in the regulation of energy balance, we next examined the effects of food deprivation for 48 hours on ADP signaling in the SFO. Our previous microarray analysis of SFO showed increases in AdipoR2 mRNA, with no significant change in AdipoR1 mRNA. We have also assessed the effects of such changes in receptor expression on ADP signaling in SFO neurons using calcium imaging and patch clamp techniques. In SFO neurons obtained from control animals, ADP induced increases in intracellular Ca2+ were observed in 25% of cells, while following food deprivation 0% of cells showed this response. Furthermore, 77% of neurons from starved animals showed clear depolarization, while no hyperpolarizing responses were observed. The results presented in this study suggest that adiponectin modulates the excitability of SFO neurons and that the response to ADP changes during starvation. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2008-09-17 18:07:35.099