Hydrogen sulfide inhibits Cav3.2 T-type Ca2+ channels

The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca2+ channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM-1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn2+ to this channel. Chelation of Zn2+ with N,N,N',N'-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn2+ binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca2+ channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.

Cell coupling in mouse pancreatic beta-cells measured in intact islets of Langerhans

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified beta-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent beta-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between beta-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell ( due to the. ring of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of beta-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to -55 mV) gave rise to a slow capacitive current indicative of coupling between beta-cells, but not in non-beta-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial beta-cell in an islet is in electrical contact with six to seven other beta-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell K(ATP) channel conductance (G(K,ATP)) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC(50) of approximately 4 mM. Theoretical considerations indicate that the coupling between beta-cells within the islet is sufficient to allow propagation of [Ca(2+)](i) waves to spread with a speed of approximately 80 mu m s(-1), similar to that observed experimentally in confocal [Ca(2+)](i) imaging.

Mode of cembranoid action on embryonic muscle acetylcholine receptor

The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H-1 cells was studied by using whole-cell and single-channel patch-clamp current measurements. With whole-cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 mu M EUAC with 50 mu M, 100 N, or 500 mu M carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 mu M CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01-40 N) obtained with 100 mu M and 500 mu M CCh displayed slope coefficients, n(H), significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single-channel studies was that EUAC did not affect single-channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open-channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed-channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations. (c) 2007 Wiley-Liss, Inc.

Efeitos da sinalização via creb sobre a sobrevivência e diferenciação meuronal

The cortical development requires a precise process of proliferation, migration, survival and differentiation of newly formed neurons to finally achieve the development of a functional network. Different kinases, such as PKA, CaMKII, MAPK and PI3K, phosphorylate the transcription factors CREB, and thus activate it, inducing CREB-dependent gene expression. In order to identify the involvement of such signaling pathways mediated by CREB over neuronal differentiation and survival, in vitro experiments of cell culture were conducted using pharmacological kinase inhibitors and genetic techniques to express different forms of CREB (A-CREB and CREB-FY) in cortical neurons. Inhibition of PKA and CaMKII decreased the length of neuronal processes (neurites); whereas inhibition of MAPK did not affect the length, but increased the number of neurites. Blockade of PI3K do not appear to alter neuronal morphology, nor the soma size changed with the kinase blockades. CREB activation (CREB-FY) along with MAPK and PI3K blockades presented a negative side effect over neuritic growth and the expression of A-CREB leaded to a significant decrease in neuronal survival after 60h in vitro and mimicked some of the effects on neuronal morphology observed with PKA and CaMKII blockade. In summary the signaling through CREB influences the morphology of cortical neurons, particularly when phosphorylated by PKA, and CREB signaling is also important for survival of immature neurons prior to the establishment of fully functional synaptic contacts. Our data contribute to understanding the role of CREB signaling, activated by different routes, on survival and neuronal differentiation and may be valuable in the development of regenerative strategies in different neurological diseases

Relationships between dendritic morphology, spatial distribution and firing patterns in rat layer 1 neurons

The cortical layer 1 contains mainly small interneurons, which have traditionally been classified according to their axonal morphology. The dendritic morphology of these cells, however, has received little attention and remains ill defined. Very little is known about how the dendritic morphology and spatial distribution of these cells may relate to functional neuronal properties. We used biocytin labeling and whole cell patch clamp recordings, associated with digital reconstruction and quantitative morphological analysis, to assess correlations between dendritic morphology, spatial distribution and membrane properties of rat layer 1 neurons. A total of 106 cells were recorded, labeled and subjected to morphological analysis. Based on the quantitative patterns of their dendritic arbor, cells were divided into four major morphotypes: horizontal, radial, ascendant, and descendant cells. Descendant cells exhibited a highly distinct spatial distribution in relation to other morphotypes, suggesting that they may have a distinct function in these cortical circuits. A significant difference was also found in the distribution of firing patterns between each morphotype and between the neuronal populations of each sublayer. Passive membrane properties were, however, statistically homogeneous among all subgroups. We speculate that the differences observed in active membrane properties might be related to differences in the synaptic input of specific types of afferent fibers and to differences in the computational roles of each morphotype in layer 1 circuits. Our findings provide new insights into dendritic morphology and neuronal spatial distribution in layer 1 circuits, indicating that variations in these properties may be correlated with distinct physiological functions.

Erythrina mulungu Alkaloids Are Potent Inhibitors of Neuronal Nicotinic Receptor Currents in Mammalian Cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caries progression and inhibition in human and bovine root dentine in situ

Since the use of bovine instead of human dentine to evaluate cariogenic and anticariogenic substances is not well established, this in situ study was conducted. Eleven volunteers wore palatal acrylic devices containing 4 dentine slabs (2 human and 2 bovine). Sucrose solution (20%) was dripped over all slabs 4 times a day, simulating a cariogenic challenge. Dentifrice slurries, fluoridated or not, were dripped over specified dentine slabs 3 times a day to evaluate caries reduction. After 14 days, the biofilm formed on the dentine slabs was collected for microbiological analysis. In dentine, mineral loss (DeltaZ) and lesion depth (LD) were determined by cross-sectional microhardness and by polarized light microscopy, respectively. The total streptococci and mutans streptococci counts in the biofilm formed either on human or on bovine slabs, whether treated or not with fluoride dentifrice, were not statistically different. The DeltaZ and the LID values of dentine treated with fluoride dentifrice were significantly lower than the values of dentine treated with non-fluoride dentifrice. The differences in the DeltaZ and LD values between the human and bovine dentine were not statistically significant. The results suggest that bovine dentine can be used instead of human to evaluate caries development and inhibition. Copyright (C) 2003 S. Karger AG, Basel.

Alveolar ridge augmentation with the perforated and nonperforated bone grafts

Purpose: Autogenous bones are frequently used because of their lack of antigenicity, but good osteoconductive and osteoinductive properties. This study evaluated the biological behavior of perforated and nonperforated cortical block bone grafts.Methods: Ten nonsmoking patients who required treatment due to severe resorption of the alveolar process and subsequent implant installation were included in the study. The inclusion criteria was loss of one or more teeth; the presence of atrophy of the alveolar process with the indication of reconstruction procedures to allow rehabilitation with dental implants; and the absence of systemic disease, local infection, or inflammation. The patients were randomly divided into two groups based on whether they received a perforated (inner surface) or nonperforated graft. After a 6-month healing period, a biopsy was performed and osseointegrated implants were installed in the same procedure.Results: Fibrous connective tissue was evident at the interface in patients who received nonperforated grafts. However, full union between the graft and host bed was visible in those who had received a perforated graft.Conclusions: We found that cortical inner side perforations at donor sites increased the surface area and opened the medullary cavity. Our results indicate an increased rate of graft incorporation in patients who received such perforated grafts.

Enhanced Expression of Heme Oxygenase-1 and Carbon Monoxide Excitatory Effects in Oxytocin and Vasopressin Neurones During Water Deprivation

A growing body of evidence indiates that carbon monoxide (CO) acts as a gas neurotransmitter within the central nervous system. Although CO has been shown to affect neurohypophyseal hormone release in response to osmotic stimuli, the precise sources, targets and mechanisms underlying the actions of CO within the magnocellular neurosecretory system remain largely unknown. In the present study, we combined immunohistochemistry and patch-clamp electrophysiology to study the cellular distribution of the CO-synthase enzyme heme oxygenase type 1 (HO-1), as well as the actions of CO on oxytocin (OT) and vasopressin (VP) magnocellular neurosecretory cells (MNCs), in euhydrated (EU) and 48-h water-deprived rats (48WD). Our results show the expression of HO-1 immunoreactivity both in OT and VP neurones, as well as in a small proportion of astrocytes, both in supraoptic (SON) and paraventricular (PVN) nuclei. HO-1 expression, and its colocalisation with OT and VP neurones within the SON and PVN, was significantly enhanced in 48WD rats. Inhibition of HO activity with chromium mesoporphyrin IX chloride (CrMP; 20 mu m) resulted in a slight membrane hyperpolarisation in SON neurones from EU rats, without significantly affecting their firing activity. In 48WD rats, on the other hand, CrMP resulted in a more robust membrane hyperpolarisation, significantly decreasing neuronal firing discharge. Taken together, our results indicate that magnocellular SON and PVN neurones express HO-1, and that CO acts as an excitatory gas neurotransmitter in this system. Moreover, we found that the expression and actions of CO were enhanced in water-deprived rats, suggesting that the state-dependent up-regulation of the HO-1/CO signalling pathway contributes to enhance MNCs firing activity during an osmotic challenge.

Statistical physics approach to dendritic computation: The excitable-wave mean-field approximation

We analytically study the input-output properties of a neuron whose active dendritic tree, modeled as a Cayley tree of excitable elements, is subjected to Poisson stimulus. Both single-site and two-site mean-field approximations incorrectly predict a nonequilibrium phase transition which is not allowed in the model. We propose an excitable-wave mean-field approximation which shows good agreement with previously published simulation results [Gollo et al., PLoS Comput. Biol. 5, e1000402 (2009)] and accounts for finite-size effects. We also discuss the relevance of our results to experiments in neuroscience, emphasizing the role of active dendrites in the enhancement of dynamic range and in gain control modulation.

Speech perception and cortical auditory evoked potentials in cochlear implant users with auditory neuropathy spectrum disorders

Objective: To characterize the PI component of long latency auditory evoked potentials (LLAEPs) in cochlear implant users with auditory neuropathy spectrum disorder (ANSD) and determine firstly whether they correlate with speech perception performance and secondly whether they correlate with other variables related to cochlear implant use. Methods: This study was conducted at the Center for Audiological Research at the University of Sao Paulo. The sample included 14 pediatric (4-11 years of age) cochlear implant users with ANSD, of both sexes, with profound prelingual hearing loss. Patients with hypoplasia or agenesis of the auditory nerve were excluded from the study. LLAEPs produced in response to speech stimuli were recorded using a Smart EP USB Jr. system. The subjects' speech perception was evaluated using tests 5 and 6 of the Glendonald Auditory Screening Procedure (GASP). Results: The P-1 component was detected in 12/14 (85.7%) children with ANSD. Latency of the P-1 component correlated with duration of sensorial hearing deprivation (*p = 0.007, r = 0.7278), but not with duration of cochlear implant use. An analysis of groups assigned according to GASP performance (k-means clustering) revealed that aspects of prior central auditory system development reflected in the P-1 component are related to behavioral auditory skills. Conclusions: In children with ANSD using cochlear implants, the P-1 component can serve as a marker of central auditory cortical development and a predictor of the implanted child's speech perception performance. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

Characterisation of the cell-transistor coupling

Die Verbindung von elektrisch aktiven, lebenden Zellen zu extrazellulären Sensorsystemen eröffnet vielfälige Möglichkeiten im Bereich der Biosensorik. Die vorliegende Arbeit leistet einen Beitrag zum tieferen Verständnis der elektrischen Kopplungsmechanismen zwischen den biologischen und elektronischen Teilen solcher Hybridsysteme. Es wurden dazu drei Hauptbereiche bearbeitet:Ein System zur extrazellulären Signalableitung an lebenden Zellen bestehend aus einem Sensorchip, einem Vorverstärkerkopf und einem Hauptverstärker wurde weiterentwickelt.Als Sensoren wurden entweder Metallmikroelektroden-Chips mit 64 Kanälen oder Feldeffekt Transistoren-Chips mit 16 Kanälen (FET) eingesetzt. Es wurden zusätzlich spezielle FET Sensoren mit Rückseitenkontakten hergestellt und eingesetzt.Die elektrische Kopplung von einzelnen Nervenzellen der neuronalen Zell-Linien SH-SY5Y und TR14 oder primär kultivierten Neuronen aus dem Hirnstamm oder dem Hippocampus von embryonalen Ratten mit den extrazellulären Sensoren wurde untersucht. In der 'whole-cell' Patch-Clamp Technik wurden die Beiträge der spannungsgesteuerten Na+- und K+-Ionenkanäle zur extrazellulären Signalform identifiziert. Die Simulation der Signale mit einem Ersatzschaltkreis (Punkt-Kontakt Modell), der in PSPICE implementiert wurde, deutet auf eine starke Abhängigkeit der Signalformen in bezug auf Konzentrationsänderungen von Na+- und K+-Ionen im Volumenbereich zwischen Zelle und den ionensensitiven Transistoren hin. Ein empirisch erweitertes Punkt-Kontakt Modell wurde daraufhin vorgestellt.Im dritten Teil der Arbeit wurden Zellschichten von Kardiomyocyten embryonaler Ratten auf den extrazellulären Sensoren kultiviert. Die Eignung eines solchen Hybridsensors als Modellherz fuer das pharmazeutische Screeing wurde durch Messungen mit Herzstimulanzien und -relaktanzien bestätigt.

Sensibilisierung hitzeevozierter Ionenströme an nozizeptiven Spinalganglienneuronen der Ratte

Die freien Endigungen von Spinalganglienneuronen sind für die Detektion schmerzhafter Reize verantwortlich. Dabei rufen thermische, chemische oder mechanische Reize Ionenströme über die Membran und dadurch Membranpotentialänderungen hervor. Diese noxisch induzierten Ströme sind in großem Ausmaß durch chemische Substanzen und andere Reize modulierbar. Der Ionenkanal TRPV1 ist für die Detektion zahlreicher chemischer Reize und zumindest eines Teils der noxischen Hitzereize verantwortlich. Im Rahmen dieser Arbeit wurden einige der Mechanismen geklärt, die zur schnellen Sensibilisierung hitzeevozierter Ionenströme führen. Hierfür wurden akut dissoziierte Spinalganglienneurone der Ratte als Modell ihrer peripheren Endigung verwendet und mittels Ganzzellableitung in der patch-clamp-Technik untersucht. Die Verwendung von Trypsin während der Präparation von Spinalganglienneuronen hat keinen funktionellen Einfluss auf hitze- oder capsaicininduzierte Ströme, verbessert aber die Untersuchungsbedingungen für das patch-clamp-Verfahren. Bei 144 akut dissoziierten Spinalganglienneuronen wurden die Stromantworten auf drei im Abstand von 40 s durch Überspülen mit 45,3 bis 46,3°C heißer Extrazellularlösung applizierte einsekündige Hitzereize gemessen. Dabei ließen sich repetitiv reproduzierbare hitzeinduzierte Einwärtsströme von etwa 160 pA erzielen; es konnte keine Tachyphylaxie und nahezu keine Inaktivierung beobachtet werden. Direkt vor dem zweiten Hitzereiz wurden die Neurone für zwei Sekunden mit Extrazellularlösung überspült, die Kontrolllösung, 0,5 μM Capsaicin, 10 μM Natriumnitroprussid oder 10 μM YC-1 enthielt. Es fand sich kein Hinweis, dass Stickstoffmonoxid oder die Guanylatzyklase einen signifikanten Beitrag zur Sensibilisierung von hitzeinduzierten Strömen in Spinalganglienneuronen leisten, wobei ein durch den Versuchsaufbau bedingtes Auswaschen zytosolischer Faktoren, die für den Signalweg notwendig sind, nicht ausgeschlossen werden kann. Bei einer Konzentration von 0,5 μM löst Capsaicin für zwei Sekunden einen sehr kleinen Einwärtsstrom von etwa 33 pA aus und führt innerhalb von zwei Sekunden zu einer schnell reversiblen Sensibilisierung von hitzeinduzierten Einwärtsströmen in Spinalganglienneuronen (p<0,01). Das Ausmaß der Sensibilisierung ist proportional zur Größe des capsaicininduzierten Stromes (r=−0,7, p<0,001). Konstant halten der intrazellulären Calciumkonzentration mittels des Calciumchelators BAPTA verhindert die capsaicininduzierte Sensibilisierung hitzeinduzierter Ströme an Spinalganglienneuronen. Demzufolge beruht die capsaicininduzierte Sensibilisierung trotz der schnellen Kinetik nicht auf einer synergistischen Wirkung der beiden Agonisten Capsaicin und Hitze auf ihren gemeinsamen Rezeptor; vielmehr ist sie von einer Erhöhung der intrazellulären freien Calciumkonzentration abhängig. Funktionelle Änderungen der zellulären Funktion werden häufig durch Proteinkinasen vermittelt. Die zur Gruppe der MAP-Kinasen gehörende ERK (extracellular signal related kinase) wird bei Membrandepolarisation und Calciumeinstrom in die Zelle durch MEK (MAPK/extracellular signal related kinase kinase) aktiviert. Blockade der MEK/ERK-Kaskade durch den spezifischen MEK-Hemmstoff U0126 führt ebenfalls zu einer Aufhebung der Sensibilisierung der Hitzeantworten durch Capsaicin. Applikation von Capsaicin führt innerhalb von zwei Sekunden zu einer schnell reversiblen Sensibilisierung hitzeevozierter Ionenströme an nozizeptiven Spinalganglienneuronen. Diese Sensibilisierung wird durch einen Calciumeinstrom in die Zelle und die dadurch eintretende Aktivierung von Proteinkinasen hervorgerufen. Die MEK/ERK-Kaskade ist ein sehr schnell (deutlich unter 2 s) aktivierbares intrazelluläres Signalsystem, welches bei der Regulation der Empfindlichkeit nozizeptiver Spinalganglienneurone eine entscheidende Rolle spielt; die schnelle Kinetik ist dabei nur durch eine membranständige oder zumindest membrannahe Lokalisation dieser Proteinkinasen erklärbar. Durch Applikation zehnsekündiger Hitzereize lässt sich ebenfalls eine Sensibilisierung hitzeevozierter Ionenströme auslösen, die ebenso ausgeprägt ist, wie die Sensibilisierung durch 0,5 μM Capsaicin (p<0,005). Durch das immer größere Verständnis der Funktionsweise des nozizeptiven Systems ergeben sich ständig neue Ansätze für die Entwicklung neuer Analgetika. So könnte durch Modulation spezifischer intrazellulärer Proteinkinasen der Phosphorylierungszustand und damit die Aktivierbarkeit von Ionenkanälen, die der Transduktion noxischer Reize dienen, positiv beeinflusst werden. Neuere, noch spezifischere Inhibitoren der MEK können der Forschung und später auch der Therapie neue Möglichkeiten eröffnen.

Charakterisierung von inhibitorischen, glyzinergen Synapsen bei Amakrinzellen der Säugernetzhaut

Amakrinzellen sind hemmende Interneurone der Netzhaut. Sie exprimieren erregende, ionotrope Glutamat-Rezeptoren und hemmende Glyzin- bzw. GABA-Rezeptoren. In der vorliegenden Arbeit wurden die Glyzinrezeptoren von Amakrinzellen mit Hilfe der „Patch Clamp“ Technik in Wildtyp- und Glyzin-Rezeptor Knock-out-Mäusen (Glra1spd-ot, Glra2-/-, Glra3-/-) untersucht. In Schnitten und Ganzpräparaten von akut isolierten Netzhäuten wurden Glyzin-induzierte und spontane inhibitorische postsynaptische Ströme (sIPSCs) gemessen. Die Untersuchungen beschränkten sich auf eine Gruppe von Amakrinzellen, die sich durch ein relativ kleines Dendritenfeld auszeichnen, das alle Schichten der IPL durchzieht. Dabei wurden die Ströme von zwei Typen von Amakrinzellen, den AII-Zellen und den NF-Zellen, miteinander verglichen. Alle untersuchten Amakrinzellen reagierten mit einem Stromfluss über die Membran, wenn Glyzin appliziert wurde. Bei AII-Zellen war die Amplitude des Stromes bei der Glra3-/--Maus um etwa 50 % reduziert, während bei den anderen Mauslinien kein Unterschied zum Wildtyp festgestellt wurde. Bei NF-Zellen wurde nur ein geringer Unterschied der Stromamplituden zwischen Wildtyp und Mutanten gefunden. Er war am deutlichsten bei der Glra2-/--Maus. Picrotoxinin ist ein effektiver Antagonist von homomeren Glyzinrezeptoren, während heteromere Glyzinrezeptoren relativ unempfindlich sind. Die Wirkung von Picrotoxinin war bei allen untersuchten Zellen ähnlich und reduzierte die Glyzinantwort um etwa 25 - 30 %. Dieser Effekt war unabhängig von der Mauslinie. Amakrinzellen exprimieren also zum Großteil heteromere Rezeptoren Zur Untersuchung der synaptischen Glyzinrezeptoren der Amakrinzellen wurden die spontanen inhibitorischen postsynaptischen Ströme dieser Zellen gemessen und deren Amplituden und Kinetiken bestimmt. Dabei unterschieden sich die Zeitkonstanten der Deaktivierungs/Desensitivierungskinetik (τw) von AII- und NF-Zellen, wohingegen die Aktivierungszeit nicht voneinander abwich. Spontane IPSCs, die von AII-Amakrinzellen abgeleitet wurden, hatten eine mittlere Zeitkonstante von τ = 11 ms und streuten zwischen 5 und 30 ms. Die Zeitkonstanten der sIPSCs von NF-Amakrinzellen lagen zwischen 10 und 50 ms und wiesen eine mittlere Zeitkonstante von τw = 27 ms auf. Die unterschiedlichen Zeitkonstanten spiegeln die Zusammensetzung der α-Untereinheiten des Glyzinrezeptors wider. AII-Zellen in der Glra1-/-- und in der Glra2-/--Maus hatten vergleichbare Zeitkonstanten wie die AII-Zellen im Wildtyp. Bei der Glra3-/--Maus konnten bei 50 untersuchten AII-Amakrinzellen keine sIPSCs gemessen werden. Dies und die Ergebnisse der Glyzin-induzierten Ströme von AII-Zellen lassen darauf schließen, dass die glyzinergen Synapsen dieser Zellen bevorzugt die α3-Untereinheit enthalten. Bei NF-Amakrinzellen konnte kein Unterschied zwischen Wildtyp-, Glra1spd-ot- und Glra3-/--Mäusen festgestellt werden. Dagegen zeigten die sIPSCs der NF-Amakrinzellen der Glra2-/--Maus signifikant längere Zeitkonstanten. Der Mittelwert verlängerte sich von 27 ms auf 69 ms und es war eine breitere Streuung mit Zeitkonstanten zwischen 15 und 200 ms zu sehen. Die glyzinergen Synapsen der NF-Zellen enthalten vor allem die α2-Untereinheit des Glyzinrezeptors. Die Zeitkonstanten der sIPSCs sind unabhängig von der Verteilung ihrer jeweiligen Amplituden, und zwischen Wildtyp- und KO-Mäusen wurden keine Unterschiede in den Amplituden der sIPSCs beobachtet. Während der Untersuchungen wurden sporadisch noch weitere Amakrinzellen, vor allem „widefield“- (WF) Zellen abgeleitet. Die Verteilungen der Zeitkonstanten der sIPSCs dieser Zellen streuten zwischen 8 und über 100 ms. Dabei wurden Zeitkonstanten gemessen, die noch langsamer waren als die von NF-Amakrinzellen und bei einigen WF-Zellen wurden mittlere Zeitkonstanten von mehr als 50 ms beobachtet. Diese Ergebnisse zeigen, dass unterschiedliche Klassen von Amakrinzellen verschiedene α-Untereinheiten des Glyzinrezeptors in den Synapsen exprimieren. Dies hat Auswirkung auf die Kinetik der glyzinergen Hemmung bei diesen Zellen und lässt darauf schließen, dass sie bei der zeitlichen Modulation der Lichtsignale unterschiedliche Aufgaben haben.

LPS-induced modifications in spontaneous network activity causes neuronal apoptosis in neonatal cerebral cortex

During the perinatal period the developing brain is most vulnerable to inflammation. Prenatal infection or exposure to inflammatory factors can have a profound impact on fetal neurodevelopment with long-term neurological deficits, such as cognitive impairment, learning deficits, perinatal brain damage and cerebral palsy. Inflammation in the brain is characterized by activation of resident immune cells, especially microglia and astrocytes whose activation is associated with a variety of neurodegenerative disorders like Alzheimer´s disease and Multiple sclerosis. These cell types express, release and respond to pro-inflammatory mediators such as cytokines, which are critically involved in the immune response to infection. It has been demonstrated recently that cytokines also directly influence neuronal function. Glial cells are capable of releaseing the pro-inflammatory cytokines MIP-2, which is involved in cell death, and tumor necrosis factor alpha (TNFalpha), which enhances excitatory synaptic function by increasing the surface expression of AMPA receptors. Thus constitutively released TNFalpha homeostatically regulates the balance between neuronal excitation and inhibition in an activity-dependent manner. Since TNFalpha is also involved in neuronal cell death, the interplay between neuronal activity MIP-2 and TNFalpha may control the process of cell death and cell survival in developing neuronal networks. An increasing body of evidence suggests that neuronal activity is important in the regulation of neuronal survival during early development, e.g. programmed cell death (apoptosis) is augmented when neuronal activity is blocked. In our study we were interested on the impact of inflammation on neuronal activity and cell survival during early cortical development. To address this question, we investigated the impact of inflammation on neuronal activity and cell survival during early cortical development in vivo and in vitro. Inflammation was experimentally induced by application of the endotoxin lipopolysaccharide (LPS), which initiates a rapid and well-characterized immune response. I studied the consequences of inflammation on spontaneous neuronal network activity and cell death by combining electrophysiological recordings with multi-electrode arrays and quantitative analyses of apoptosis. In addition, I used a cytokine array and antibodies directed against specific cytokines allowing the identification of the pro-inflammatory factors, which are critically involved in these processes. In this study I demonstrated a direct link between inflammation-induced modifications in neuronal network activity and the control of cell survival in a developing neuronal network for the first time. Our in vivo and in vitro recordings showed a fast LPS-induced reduction in occurrence of spontaneous oscillatory activity. It is indicated that LPS-induced inflammation causes fast release of proinflammatory factors which modify neuronal network activity. My experiments with specific antibodies demonstrate that TNFalpha and to a lesser extent MIP-2 seem to be the key mediators causing activity-dependent neuronal cell death in developing brain. These data may be of important clinical relevance, since spontaneous synchronized activity is also a hallmark of the developing human brain and inflammation-induced alterations in this early network activity may have a critical impact on the survival of immature neurons.