The role of L-type voltage-gated calcium-channels in the discharge activity of primary hippocampal neurons

L-type spannungsabhängige Ca2+-Kanäle (LTCC) erfüllen verschiedene Aufgaben in erregbaren Zellen. Unter anderem wurde gezeigt, dass LTCCs zur Erregungs-Transkription Kopplung betragen [Pinato et al. 2009, West et al. 2002]. Darüberhinaus hat die Aktivierung von LTCCs sowohl einen direkten, als auch indirekten Einfluss auf die Membranspannung. Letzteres erfolgt z.B. über die Aktivierung von Ca2+-abhängigen K+- ]Lima and Marion 2007] und Kationen-Kanäle [Lee and Tepper 2007]. Im Zuge dieser Diplomarbeit wurde der Einfluss von LTCCs auf das Entladungsverhalten von hippocampalen Neuronen in vitro untersucht. Um das Membranpotential abzuleiten wurden „current clamp“-Experimente in der „perforated patch“-Konfiguration durchgeführt. ILTCC wurde durch die Applikation von den Dihydropyridine BayK 8644 (Agonist) und isradipine (Antagonist) verstärkt bzw. verringert. Um spannungsaktivierte Na+-Kanäle zu blockieren, deren Ströme die LTCC-induzierten Effekte überlagern würden, wurde bei allen „current clamp“-Experimenten TTX 500 nM zusätzlich appliziert. Die spanngungsinduzierte Aktivierung der LTCCs erfolgte mittels 5 aufeinander folgender Strominjektionen ansteigender Stromstärke, die entweder eine Rechteck- oder Rampenform aufwiesen. Das Ziel dieser Experimente war es (I) den Schwellenwert der LTCC-Aktivierung zu bestimmen und (II) die LTCC-induzierte Kopplung zu Ca2+-aktivierten Ionenkanälen zu untersuchen. Die Analyse der Aktivierungschwelle des L-type Ca2+-Kanals ergab eine Population von Neuronen, deren Schwellenwerte größtenteils deutlich negativer als -35 mV lagen. Da bis dato keine neuronalen Cav1.2-Kanalvarinaten beschrieben worden sind, die negativer als -35 mV aktivieren würden, verweisen meine Daten auf einen bedeutenden Beitrag von Cav1.3-Kanälen, deren Aktivierung allgemein um 10 - 15 mV negativer liegt als die der Cav1.2-Kanäle. Dieses Ergebnis steht im krassen Gegensatz zu Untersuchungen an transgenen Tieren, deren Daten auf eine untergeordnete Rolle von Cav1.3-Kanälen im ZNS verwiesen [Clark et al. 2003, Lacinova et al. 2008, Moosmang et al. 2005]. Nachpotentiale traten in meinen Ergebnissen in zwei unterschiedlichen Modalitäten auf: Nachhyperpolarisationen (AHPs) und Nachdepolarisationen (ADPs). Diesen wurden typischerweise schon unter Kontrollbedingungen (DMSO) ausgelöst und durch ILTCC-Erhöhung mit BayK noch weiter verstärkt. Im Falle von 8 s andauernden depolarisierenden Strompulsen, überwiegte die Häufigkeit der AHPs klar. Aus pharmakologischer Sicht, konnten die AHPs als Apamin- und UCL 1684-sensitive und somit K(Ca)2.x/SK-vermittelt charakterisiert werden. Mittels Blockade von SK-Kanäle konnten zwei unterschiedliche AHP-Typen ausgemacht werden. Dies betraf das gleichzeitige Auftreten von AHPs und ADPs, was nur in einer Teilpopulation von hippocampalen Neuronen (50 %) nachgewiesen wurde (N = 5, Nachpotentialsfläche normalisiert auf DMSO mit BayK: -3.25, 8.05 oder BayK + apamin: 18.99, 22.72, p 2 s) entstanden. Anschließend wurde die Validität des Kopplungs-Modells im Bezug auf die physiologische Feueraktivität von Hippocampus-Neuronen getestet. Da das Entladungsverhalten von hippocampalen Neuronen in der Regel aus eher kurzen Depolarisationen (< 1 s) besteht, würde man anhand des Kopplungsmodells für diese Neurone eine stärkere LTCC-CAN- als LTCC-SK-Kopplung erwarten. Diese Vorhersage stimmte in der Tat mit den experimentellen Beobachtungen in unserer Kultur überein, indem 80 % der Neurone mit BayK ihre Feueraktivität verstärkten. Daraus schlussfolgere ich, dass die funktionale Kopplung von L-type Ca2+-Kanälen an ADP-bildende CAN-Kanäle sowie AHP-bildende SK-Kanäle einen wichtigen Mechanismus zur Regulation hippocamplar Entladungsaktivität darstellt. Etwaige physiologische Implikationen sind im Falle der ADPs die Verstärkung von unterschwelligen Potentialen und im Falle der AHPs ein negativer Rückkopplungsmechanismus zur Unterbindung excitoxischer Plateaupotentiale [Ping and Sheppard 1999]. Zusammenfassend weisen meine Ergebnisse darauf hin, dass in primären hippocampalen Neuronen sowohl Cav1.2, als auch Cav1.3 aktiv sind und zeigen, dass (I) SK- und CAN-Kanäle funktional an L-type Ca2+-Kanäle koppeln, dass (II) in den meisten getesteten Neuronen eine Überlagerung beider Nachpotential-Modalitäten stattfindet und dass (III) sich diese beiden Nachpotentiale in der Effizienz der LTCC-Kopplung unterscheiden.L-type voltage-gated calcium channels (LTCCs) were shown to have several distinct roles in excitable cells. For example, Ca2+-influx via LTCCs was shown to be an important component of the excitation-transcription coupling [West et al. 2002; Pinato et al. 2009]. Additionally, LTCC-mediated Ca2+-influx can also affect membrane voltage and discharge properties, either directly or via activation of Ca2+-dependent conductances, such as K+- [Lima and Marrion 2007], or cation-channels [Lee and Tepper 2007]. Here I evaluated the contribution of LTCCs to the electrical activity of central neurons in vitro. Current-clamp experiments were performed on hippocampal neurons in culture using the perforated patch-clamp method to record the membrane voltage. The neurons were continuously superfused and LTCC-activity was modulated by application of the dihydropyridines BayK 8644 3 µM (enhanced LTCC-activity) and isradipine 3 µM (LTCC-inhibition), all in the presence of TTX. Activation of LTCCs was provoked by incremental current injections to depolarize the neurons experimentally. Doing so, LTCC-activation thresholds and LTCC-induced afterpotentials, occurring after the current pulse, were investigated. Analysis of LTCC-activation revealed a distinct population of neurons with thresholds negative to -35 mV under control conditions. Since neuronal Cav1.2-channel variations have never been found to activate at such negative potentials, these data argue for an important role of Cav1.3-channels in hippocampal neurons. This findings stand in clear contrast to what has previously been concluded from results obtained with transgenic mice [Clark et al. 2003; Moosmang et al. 2005; Lacinova et al. 2008]. The afterpotential occurring as after-hyperpolarizations (AHPs) or after-depolarizations (ADPs) were typically seen already under control conditions, and increased with BayK. Using 8 s-long current pulses, in the majority (80 %) of neurons, the afterpotential was an AHP, which could be blocked by apamin 100 nM or UCL 1684 30 nM indicating the involvement of K(Ca)2.x/SK-channels. K(Ca)2.x-blockade also revealed that the hyperpolarizing afterpotential can either represent a pure AHP (N = 10, afterpotential-area normalized to control under BayK: -1.37, 1.30 or BayK + apamin: 0.11, 1.00, p < 0.001) or an overlap of an AHP and ADP (N = 5, afterpotential-area normalized to control under BayK: -3.25, 8.05 or BayK + apamin: 18.99, 22.72, p < 0.01). In neurons, which showed a depolarizing afterpotential, the co-application of BayK and apamin as compared to BayK alone revealed a further enhancement of the ADP and therefore a concomitantly occurring AHP. Reduction of external [Na+] to 1.5 mM led to a significant diminishment of the ADP-area, suggesting that a Ca2+-activated non selective cation (CAN) channel mediates the primary excitatory LTCC-coupling in the hippocampus (N = 7, afterpotential-area normalized to DMSO under BayK + apamin = 2.22, 3.99 or low Na+/BayK + apamin = 0.34, 0.68, p < 0.01, all values are given as median and interquartile range). Moreover, variations of the current pulse duration (0.1 to 10 s) as well as the pulse strength indicated that LTCC-coupling to CAN- and SK-channels differs in its coupling efficiency: CAN-channels activate at weaker stimuli (shorter and/or smaller depolarization) than SK-channels and long-lasting (> 2 s) favored the SK-mediated AHP-formation. As the control discharge pattern of hippocampal neurons mainly consists of rather short-lasting (500 ms to 1 s) depolarizations, the predominant abundance of cells increasing their excitation, when ILTCC is enhanced, is in accordance with the coupling-model described above. From that, I suggest a certain balance between de- and hyperpolarizing LTCC-couplings as an important mechanism regulating the discharge activity in the hippocampus. This could include a CAN-mediated enhancement of short subthreshold potentials, such as in LTCC-dependent and NMDA-independent hippocampal LTP [Moosmang et al. 2005] as well as a SK-mediated protection against long-lasting excitotoxic LTCC-mediated plateau potentials occurring after SK-channels blockade [Ping et al. 1999]. In accordance with the latter assumption, the up-regulation of ICAN is currently discussed in relation to Parkinson’s desease [Lee and Tepper 2007]. To sum up, my data indicate that Cav1.2 as well as Cav1.3 contribute to hippocampal firing activities and suggest that in primary hippocampal neurons (I) LTCCs couple to both SK- and CAN-channels, (II) that in most hippocampal neurons both coupling modes operate in parallel and (III) that coupling efficiency differs among the two Ca2+-dependent conductances

Immunogenicity and Tolerability after Two Doses of Non-Adjuvanted, Whole-Virion Pandemic Influenza A (H1N1) Vaccine in HIV-Infected Individuals

BACKGROUND: During the influenza pandemic of 2009/10, the whole-virion, Vero-cell-derived, inactivated, pandemic influenza A (H1N1) vaccine Celvapan® (Baxter) was used in Austria. Celvapan® is adjuvant-free and was the only such vaccine at that time in Europe. The objective of this observational, non-interventional, prospective single-center study was to evaluate the immunogenicity and tolerability of two intramuscular doses of this novel vaccine in HIV-positive individuals. METHODS AND FINDINGS: A standard hemagglutination inhibition (HAI) assay was used for evaluation of the seroconversion rate and seroprotection against the pandemic H1N1 strain. In addition, H1N1-specific IgG antibodies were measured using a recently developed ELISA and compared with the HAI results. Tolerability of vaccination was evaluated up to one month after the second dose. A total of 79 HIV-infected adults with an indication for H1N1 vaccination were evaluated. At baseline, 55 of the 79 participants had an HAI titer ≥1:40 and two patients showed a positive IgG ELISA. The seroconversion rate was 31% after the first vaccination, increasing to 41% after the second; the corresponding seroprotection rates were 92% and 83% respectively. ELISA IgG levels were positive in 25% after the first vaccination and in 37% after the second. Among the participants with baseline HAI titers <1:40, 63% seroconverted. Young age was clearly associated with lower HAI titers at baseline and with higher seroconversion rates, whereas none of the seven patients >60 years of age had a baseline HAI titer <1:40 or seroconverted after vaccination. The vaccine was well tolerated. CONCLUSION: The non-adjuvanted pandemic influenza A (H1N1) vaccine was well tolerated and induced a measurable immune response in a sample of HIV-infected individuals

Pahenu1 is a mouse model for tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency and promotes analysis of the pharmacological chaperone mechanism in vivo

The recent approval of sapropterin dihydrochloride, the synthetic form of 6[R]-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4), for the treatment of phenylketonuria (PKU) as the first pharmacological chaperone drug initiated a paradigm change in the treatment of monogenetic diseases. Symptomatic treatment is now replaced by a causal pharmacological therapy correcting misfolding of the defective phenylalanine hydroxylase (PAH) in numerous patients. Here, we disclose BH4 responsiveness in Pahenu1, a mouse model for PAH deficiency. Loss of function resulted from loss of PAH, a consequence of misfolding, aggregation, and accelerated degradation of the enzyme. BH4 attenuated this triad by conformational stabilization augmenting the effective PAH concentration. This led to the rescue of the biochemical phenotype and enzyme function in vivo. Combined in vitro and in vivo analyses revealed a selective pharmaceutical action of BH4 confined to the pathological metabolic state. Our data provide new molecular-level insights into the mechanisms underlying protein misfolding with loss of function and support a general model of pharmacological chaperone-induced stabilization of protein conformation to correct this intracellular phenotype. Pahenu1 will be essential for pharmaceutical drug optimization and to design individually tailored therapie

Rapid Diagnostic Algorithms as a Screening Tool for Tuberculosis: An Assessor Blinded Cross-Sectional Study

Background: A major obstacle to effectively treat and control tuberculosis is the absence of an accurate, rapid, and low-cost diagnostic tool. A new approach for the screening of patients for tuberculosis is the use of rapid diagnostic classification algorithms. Methods: We tested a previously published diagnostic algorithm based on four biomarkers as a screening tool for tuberculosis in a Central European patient population using an assessor-blinded cross-sectional study design. In addition, we developed an improved diagnostic classification algorithm based on a study population at a tertiary hospital in Vienna, Austria, by supervised computational statistics. Results: The diagnostic accuracy of the previously published diagnostic algorithm for our patient population consisting of 206 patients was 54% (CI: 47%–61%). An improved model was constructed using inflammation parameters and clinical information. A diagnostic accuracy of 86% (CI: 80%–90%) was demonstrated by 10-fold cross validation. An alternative model relying solely on clinical parameters exhibited a diagnostic accuracy of 85% (CI: 79%–89%). Conclusion: Here we show that a rapid diagnostic algorithm based on clinical parameters is only slightly improved by inclusion of inflammation markers in our cohort. Our results also emphasize the need for validation of new diagnostic algorithms in different settings and patient populations

JRC – Ispra Atmosphere – Biosphere – Climate Integrated monitoring Station 2013 report

The Institute for Environment and Sustainability provides long-term observations of the atmosphere within international programs and research projects. These observations are performed from the research infrastructure named ABC-IS: Atmosphere – Biosphere – Climate Integrated monitoring Station. Most measurements are performed at the JRC-Ispra site, but observations are also carried out from two other platforms: the forest station in San Rossore, and a ship cruising in the Western Mediterranean sea. This document reports about the measurement programs, the equipment which is deployed, the data quality assessment, and the results obtained for each site. Our observations are presented, compared to each other, as well as to historical data obtained over more than 25 years at the Ispra site.JRC.H.2-Air and Climat

JRC – Ispra Atmosphere – Biosphere – Climate Integrated monitoring Station: 2014 Report

The Institute for Environment and Sustainability provides long-term observations of the atmosphere within international programs and research projects. These observations are performed from the research infrastructure named ABC-IS: Atmosphere – Biosphere – Climate Integrated monitoring Station. Most measurements are performed at the JRC-Ispra site, but observations are also carried out from two other platforms: the forest station in San Rossore, and a ship cruising in the Western Mediterranean sea. This document reports about the measurement programs, the equipment which is deployed, the data quality assessment, and the results obtained for each site. Our observations are presented, compared to each other, as well as to historical data obtained over close to 30 years at the Ispra site.JRC.H.2-Air and Climat

Spike timing of identified interneurons in the medial prefrontal cortex of anaesthetised and freely-moving rats

Die räumliche und zeitliche Koordination neuronaler Aktivität ist ein grundlegendes Prinzip kognitiver Prozesse wie Kurzzeitgedächtnis oder Entscheidungsfindung. GABAerge Interneurone mit ihrer zeitlich präzise kontrollierten synaptischen Transmitterfreisetzung auf spezifische subzelluläre Bereiche der Membran von Pyramidalneuronen sind der Schlüssel zum Verständnis synchronisierter Gehirnaktivität. Mittels in vivo extrazellulärer Aufnahmen gefolgt von juxtazellulärer Zellfärbung im medialen präfrontalen Kortex narkotisierter Ratten habe ich gezeigt, dass Parvalbumin expremierende Korbzellen und Axo-axonic Zellen unterschiedliche Feueraktivitäten während kortikaler 1Hz-Oszillationen besitzen. Außerdem weisen meine Daten darauf hin, dass die Aktivität von Korbzellen, nicht aber von Axo-axonic Zellen zeitlich mit dem Auftreten von Schlafspindeln, ein EEG-Signal charakteristisch für bestimmte Schlafphasen, korreliert. Experimente mit anästhesierten Tieren haben sich als Modell für eine Reihe verschiedener Netzwerkoszillationen des wachen Gehirns bewährt, doch ist das Interpretationspotential der daraus resultierenden Daten immer durch etwaige Effekte der Anästhesie selbst beschränkt geblieben. Daher habe ich begonnen juxtazelluläre Aufnahmen in frei beweglichen Ratten durchzuführen. In der CA1 Region des dorsalen Hippocampus habe ich dazu beigetragen nachzuweisen, dass sich die Aktivität von Parvalbumin expremierende Korbzellen abhängig vom Verhalten der Ratte dynamisch verändert. Wie wir gezeigt haben, steht das im Gegensatz zur Aktivität von so genannten Ivy-Zellen, die ihre Aktionspotentialsfrequenz während unterschiedlicher Verhaltenszustände konstant halten und stattdessen vermutlich eine bedeutende Rolle in der Aufrechterhaltung der Homöostase des neuronalen Netzwerkes inne haben. Nicht nur durch die im vorhergegangenen Projekt beobachtete Spezifizität der Aktivität bestimmter Interneurone, sondern auch durch die steigende Anzahl aktueller Studien, die auf eine Zelltypen-spezifische Beteiligung an kognitiven Prozessen hinweisen, habe ich mich dazu entschlossen juxtazelluläre Aufnahmen an Ratten durchzuführen, die gelernt haben eine Gedächtnisaufgabe zu meistern. Durch diese Experimente wird es mir möglich sein, die Aktivität von identifizierten Interneuron-Typen mit bestimmten kognitiven Prozessen zu korrelieren. Bisher zeigen meine Resultate, dass die Aktivität von Parvalbumin expremierenden Interneuronen durch bestimmte Aspekte der Gedächtnisaufgabe beeinflusst wird. Wichtig ist dabei darauf hinzuweisen, dass ich unter den bisher analysierten Aktivitätsmustern Heterogenität festgestellt habe, die zum Teil mit bestimmten Eigenschaften der Neurone, wie axo-dendritische Verästelungen in bestimmten kortikalen Schichten, übereinstimmt. Insgesamt weisen die Ergebnisse meiner Arbeit darauf hin, dass verschiedenen Typen von GABAergen Interneuronen im medialen präfrontalen Kortex an Netzwerkoszillationen und kognitiven Prozessen in spezifischer Weise beteiligt sind.Spatiotemporal coordination of neuronal activity is a fundamental principle underlying cognitive functions such as working memory and decision making. GABAergic interneurons providing tightly controlled synaptic inputs at millisecond precision onto distinct subcellular domains of pyramidal neurons are key to synchronise brain activity. By performing in vivo extracellular recording followed by juxtacellular labelling in the medial prefrontal cortex of anaesthetised rats, I have demonstrated that PV expressing basket and axo-axonic cells display distinct spike timing during cortical slow oscillations. Moreover, I have shown that basket, but not axo-axonic cells are temporally correlated with the occurrence of thalamocortical sleep spindles suggesting a pivotal role of basket cells in the generation of prefrontal spindle oscillations. Anaesthetised animals have been proven useful as a model for a number of different network states of the drug-free brain and yet always have limited potential interpretations. Therefore, I started employing juxtacellular recordings in freely-moving rats. In the dorsal CA1 region of the rodent hippocampus I contributed in showing that PV expressing basket, but not Ivy cells dynamically change their firing activity during different behavioural states. Encouraged not only by the spike timing specificity of identified prefrontal interneurons observed in my previous project, but also by a number of recent studies pointing at their cell type specific cognitive implications, I started to apply freely-moving juxtacellular recordings to rats performing a behavioural paradigm engaging working memory and decision making. I have shown that the firing activity of PV expressing interneurons in the prelimbic cortex is modulated by the different aspects of a working memory task. I have found heterogeneity among PV expressing interneurons that correlated with the layer-specific axo-dendritic arborisation of some of the cells. Altogether, the findings presented in this thesis indicate that distinct types of GABAergic interneuron in the medial prefrontal cortex contribute to network oscillations, behavioural and cognitive states in a distinct manner.submitted by Michael LaglerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2014OeBB(VLID)171359