The Role of Extracellular Adenosine in Chemical Neurotransmission in the Hippocampus and Basal Ganglia: Pharmacological and Clinical Aspects

Now there is general agreement that the purine nucleoside adenosine is an important neuromodulator in the central nervous system, playing a crucial role in neuronal excitability and synaptic/non-synaptic transmission in the hippocampus and basal ganglia. Adenosine is derived from the breakdown of extra- or intracellular ATP and is released upon a variety of physiological and pathological stimuli from neuronal and non-neuronal sources, i.e. from glial cells and exerts effects diffusing far away from release sites. The resultant elevation of adenosine levels in the extracellular space reaches micromolar level, and leads to the activation A1, A2A, A2B and A3 receptors, localized to pre- and postsynaptic as well as extrasynaptic sites. Activation of presynaptic A1 receptors inhibits the release of the majority of transmitters including glutamate, acetylcholine, noradrenaline, 5-HT and dopamine, whilst the stimulation of A2A receptors facilitates the release of glutamate and acetylcholine and inhibits the release of GABA. These actions underlie modulation of neuronal excitability, synaptic plasticity and coordination of neural networks and provide intriguing target sites for pharmacological intervention in ischemia and Parkinson’s disease. However, despite that adenosine is also released during ischemia, A1 adenosine receptors do not participate in the modulation of excitotoxic glutamate release, which is nonsynaptic and is due to the reverse operation of transporters. Instead, extrasynaptic A1 receptors might be responsible for the neuroprotection afforded by A1 receptor activation

Phenotype-dependent Ca2+ dynamics in single boutons of various anatomically identified GABAergic interneurons in the rat hippocampus

L

Kinetic properties and open probability of alpha7 nicotinic acetylcholine receptors.

The alpha7 nicotinic acetylcholine receptor (nAChR) has some peculiar kinetic properties. From the literature of alpha7 nAChR-mediated currents we concluded that experimentally measured kinetic properties reflected properties of the solution exchange system, rather than genuine kinetic properties of the receptors. We also concluded that all experimentally measured EC50 values for agonists must inherently be inaccurate. The aim of this study was to assess the undistorted kinetic properties of alpha7 nAChRs, and to construct an improved kinetic model, which can also serve as a basis of modeling the effect of the positive allosteric modulator PNU-120596, as it is described in the accompanying paper. Agonist-evoked currents were recorded from GH4C1 cells stably transfected with pCEP4/rat alpha7 nAChR using patch-clamp and fast solution exchange. We used two approaches to circumvent the problem of insufficient solution exchange rate: extrapolation and kinetic modeling. First, using different solution exchange rates we recorded evoked currents, and extrapolated their amplitude and kinetics to instantaneous solution exchange. Second, we constructed a kinetic model that reproduced concentration-dependence and solution exchange rate-dependence of receptors, and then we simulated receptor behavior at experimentally unattainably fast solution exchange. We also determined open probabilities during choline-evoked unmodulated and modulated currents using nonstationary fluctuation analysis. The peak open probability of 10 mM choline-evoked currents was 0.033 +/- 0.006, while in the presence of choline (10 mM) and PNU-120596 (10 muM), it was increased to 0.599 +/- 0.058. Our kinetic model could adequately reproduce low open probability, fast kinetics, fast recovery and solution exchange rate-dependent kinetics

Preszinaptikus moduláció megfigyelése 2-foton képalkotással = 2-photon imaging of presynaptic modulation

A pályázat első évében a megvalósítás érdekében elsősorban olyan munkák elvégzése történt meg, amelyek a legoptimálisabb kísérleti felállás módozatait keresték, de természetesen elkezdtük az axonális Ca2+ dinamika preszinapikus modulációjának vizsgálatát is. Elsőnek a már korábban a témavezető által behatóan vizsgált jelenség, a dendrit tüskék motilitásának farmakológiai modulálását vizsgáltuk. Ezzel a farmakonok adásának, a fluorescens gerjesztés beállítása történt meg a dendritekben. Majd a dendritikus Ca2+ dinamika farmakológiai befolyásolását vizsgáltuk meg részletesebben piramissejtekben és interneuronokban. A technikai nehézségek miatt (vékony nyúlványok töltődése igen lassú, viszont a kísérleti időablakunk viszonylag rövid) kisebb elemszámot tudtunk csak összegyűjteni az axonok esetében. Ennek a megoldásán jelenleg is dolgozunk. Az axon varikozitásokból történő mérések nem tartoznak a legegyszerűbb 2-foton mikroszkópos kísérletek közé, az ebből születő eredmények nemzetközi szinten is újdonságnak számítanak. A biztosan mérhető posztszinaptikus hatás vizsgálata azért is fontos, mert a technikai újdonságot jelentő 2-foton mikroszkópiának folyamatosan új ismereteket kell szolgáltatnia, hogy jelentős befektetés kutatási szempontból sikeresnek bizonyuljon. | In the first year of the OTKA support we made the setup and fine-tuning of experimental design to optimally image structure and function. First, we studied the pharmacological modulation of dendritic spine motility. Next we investigated the calcium dynamics of dendrites and spines of pyramidal neurons and interneurons. Because of the technical difficulties, we could reach only smaller sample size for the imaging of axonal varicosities (dye loading in fine process). The safe and reliable measurement of postsynaptic effects in spines and dendrites is extremely important as the 2-photon studies, representing a technical breakthrough for the neuroscience, must provide new information to be a beneficial and reasonable investment

Mode of action of the positive modulator PNU-120596 on alpha7 nicotinic acetylcholine receptors.

We investigated the mode of action of PNU-120596, a type II positive allosteric modulator of the rat alpha7 nicotinic acetylcholine receptor expressed by GH4C1 cells, using patch-clamp and fast solution exchange. We made two important observations: first, while PNU-120596 rapidly associated to desensitized receptors, it had at least hundredfold lower affinity to resting conformation, therefore at 10 muM concentration it dissociated from resting receptors; and second, binding of PNU-120596 slowed down dissociation of choline molecules from the receptor radically. We propose that when agonist concentration is transiently elevated in the continuous presence of the modulator (as upon the neuronal release of acetylcholine in a modulator-treated animal) these two elements together cause occurrence of a cycle of events: Binding of the modulator is limited in the absence of the agonist. When the agonist is released, it binds to the receptor, and induces desensitization, thereby enabling modulator binding. Modulator binding in turn traps the agonist within its binding site for a prolonged period of time. Once the agonist finally dissociated, the modulator can also dissociate without re-binding, and the receptor assumes its original resting conformation. In kinetic simulations this "trapped agonist cycle" mechanism did not require that the orthosteric and allosteric ligands symmetrically modify each other's affinity, only the modulator must decrease agonist accessibility, and the agonist must induce a conformation that is accessible to the modulator. This mechanism effectively prolongs and amplifies the effect of the agonist

Purinerg receptorok által közvetített hatások komplex vizsgálata: új neuroprotektív terápiás lehetőségek elméleti alapjai = Complex studies on purinergic receptor-mediated actions: a theoretical basis for neuroprotection

Vizsgálataink fő célja a neurotranszmitter felszabadulást serkentő P2 nukleotid receptorok szerepének tisztázása volt fiziológiás és patológiás állapotokban. Feltérképeztük a P2X7 receptort kódoló mRNS eloszlását a központi idegrendszer számos területén. Elsőként azonosítottunk a GABA és glutamát felszabadulás szabályozásában résztvevő, serkentő P2X7 receptorokat a hippokampuszban. Feltártuk a P2X7 receptorok celluláris és szubcelluláris eloszlását ezen agyterületen, igazoltuk a P2X7 receptor részvételét az ATP GABA és glutamát felszabadító hatásában farmakológiai analízis, valamint transzgenikus technológia igénybevételével. Neurokémiai és elektrofizológiai módszerekkel igazoltuk, hogy a P2X7 receptorok funkcionális válaszkészsége fokozódik energiadepriváció hatására. Kimutattuk, hogy a noradrenalin felszabadulást a hippokampuszban serkentő P2X1 és/vagy P2X3 receptorok szabályozzák. Megállapítottuk, hogy az ATP és egyéb purinok képesek önerősítő módon saját felszabadulásukat fokozni a homo- illetve heteroexchange által. Tisztáztuk a mitokondriális inhibitorok és az oxidatív stressz szupraadditív kölcsönhatását a noradrenalin/dopamin felszabadulás kiváltásában a hippokampuszban, illetve a rotenon indukált Parkinson modellben. Feltártuk az IL-1béta purin felszabadulást előidéző hatását. Eredményeink alátámasztották a pályázatban felállított hipotézist, mely szerint a P2X7 vagy egyéb P2X receptorok befolyásolása ígéretes terápiás célpont lehet neurodegeneratív betegségekben. | The main objective of the studies was to identify the role of the facilitatory P2 nucleotide receptors under physiological and pathological conditions. We explored the mRNA expression of P2X7 receptors in several areas of the CNS. We demonstrated for the first time that the activation of P2X7 receptors facilitate the release of GABA and glutamate in the hippocampus, and the cell-type specific distribution of this receptor was also explored. The involvement of P2X7 receptor in the GABA and glutamate releasing effect of ATP was proved by pharmacological analysis and by the utilization of transgenic technology. We also demonstrated by electrophysiological and neurochemical techniques that the functional responsiveness of P2X7 receptors is increased during energy deprivation. On the other hand, the release of noradrenaline is subject to facilitation by P2X1 and /or P2X3 receptors. We identified the homo-and heteroexchange, as a new mechanism, whereby purines could promote the release of each other and themselves. We revealed the supraadditive impact of mitochondrial inhibitors and oxidative stress on noradrenaline release in the hippocampus and on dopamine release in the rotenon induced Parkinson model. In addition the effect of IL-1beta on the release of purines from the hippocampus was also described. In conclusion our findings support our initial hypothesis that P2X7 or other P2X receptors could be attractive therapeutic targets in neurodegenerative diseases

Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy

The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl--dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects

Friends of Hungary Conference: EXPO

For 16th September 2022, the first day of the Eighth Friends of Hungary Conference we prepared an illustrated compilation of the history and activities of the Friends of Hungary Foundation, while on 17th September we will present the Hungarian partner organizations of the Friends of Hungary Foundation from all over the world

The tricyclic antidepressant desipramine inhibited the neurotoxic, kainate-induced [Ca] increases in CA1 pyramidal cells in acute hippocampal slices.

Kainate (KA), used for modelling neurodegenerative diseases, evokes excitotoxicity. However, the precise mechanism of KA-evoked [Ca2+]i increase is unexplored, especially in acute brain slice preparations. We used [Ca2+]i imaging and patch clamp electrophysiology to decipher the mechanism of KA-evoked [Ca2+]i rise and its inhibition by the tricyclic antidepressant desipramine (DMI) in CA1 pyramidal cells in rat hippocampal slices and in cultured hippocampal cells. The effect of KA was dose-dependent and relied totally on extracellular Ca2+. The lack of effect of dl-2-amino-5-phosphonopentanoic acid (AP-5) and abolishment of the response by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) suggested the involvement of non-N-methyl-d-aspartate receptors (non-NMDARs). The predominant role of the Ca2+-impermeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the initiation of the Ca2+ response was supported by the inhibitory effect of the selective AMPAR antagonist GYKI 53655 and the ineffectiveness of 1-naphthyl acetylspermine (NASPM), an inhibitor of the Ca2+-permeable AMPARs. The voltage-gated Ca2+ channels (VGCC), blocked by omega-Conotoxin MVIIC+nifedipine+NiCl2, contributed to the [Ca2+]i rise. VGCCs were also involved, similarly to AMPAR current, in the KA-evoked depolarisation. Inhibition of voltage-gated Na+ channels (VGSCs; tetrodotoxin, TTX) did not affect the depolarisation of pyramidal cells but blocked the depolarisation-evoked action potential bursts and reduced the Ca2+ response. The tricyclic antidepressant DMI inhibited the KA-evoked [Ca2+]i rise in a dose-dependent manner. It directly attenuated the AMPA-/KAR current, but its more potent inhibition on the Ca2+ response supports additional effect on VGCCs, VGSCs and Na+/Ca2+ exchangers. The multitarget action on decisive players of excitotoxicity holds out more promise in clinical therapy of neurodegenerative diseases