Antagonistic properties of caged GABA compounds used for activation of GABAA receptors in neocortical pyramidal neurons

Caged photolysable compounds have served to be pivotal to neuroscientific investigations; allowing the cognizing of molecular kinetics and properties of neuronal micro-machinery such as neurotransmitter receptors. Precision in terms of temporal and spatial resolution of neurotransmitter release endowed by photolysis has multitudinal applicabilities in the realm of GABAA receptors (GABAARs), their neuronal niche and effects on neuronal and network activity. Caged compounds, in their caged form, may display certain unideal traits such as undesired interactions with the system and antagonistic activity on the target receptor. This study aims to reevaluate the GABAAR antagonistic actions of caged Rubi-GABA, which was found to antagonize these receptors at significantly lower concentrations than those reported in the literature. Furthermore, this study electrophysiologically characterizes the possible antagonistic properties of a novel quinoline-derived UV-photolysable caged GABA compound, 8 DMAQ GABA, whose activity, in its caged form appears to have a much more favorable antagonism profile compared to the widely used RuBi-GABA. To assess the antagonistic effects of these compounds on GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs) patch-clamp recordings were carried out in the whole-cell voltage clamp configuration on cortical layer 2/3 cortical pyramidal neurons in acute neocortical slices prepared from 16-18 day-old rat rats. The results of this study indicate a revised antagonism profile for caged Rubi-GABA, with marked GABAAR toxicity in the low micromolar range. The study also scrutinizes the photo-kinetic properties of both caged GABA compounds and reveals that the rate of GABA release from 8-DMAQ is slower than from RuBi-GABA

Super-resolution 2-photon microscopy reveals that the morphology of each dendritic spine correlates with diffusive but not synaptic properties

The structure of dendritic spines suggests a specialized function in compartmentalizing synaptic signals near active synapses. Indeed, theoretical and experimental analyses indicate that the diffusive resistance of the spine neck is sufficient to effectively compartmentalize some signaling molecules in a spine for the duration of their activated lifetime. Here we describe the application of 2-photon microscopy combined with stimulated emission depletion (STED-2P) to the biophysical study of the relationship between synaptic signals and spine morphology, demonstrating the utility of combining STED-2P with modern optical and electrophysiological techniques. Morphological determinants of fluorescence recovery time were identified and evaluated within the context of a simple compartmental model describing diffusive transfer between spine and dendrite. Correlations between the neck geometry and the amplitude of synaptic potentials and calcium transients evoked by 2-photon glutamate uncaging were also investigated

Aktiini-välitteisten liikkuvuuden rooli ääreis-astrosyyttejen kehityskulkussa synaptisessa toiminnassa

Among other glial cell types such as microglia, oligodendrocytes and radial glia, astrocytes are known to be involved in brain function; metabolically supporting neurons, regulating blood flow dynamics, participating in the development of pathological states, sensing and modulating synaptic activity. At the same time the complex astrocytic morphology, with a number of highly ramified peripheral processes located near the synaptic terminals, suggests them as a possible source for morpho-functional plasticity in the brain. This thesis summarizes the work on the in vitro development and further in vivo implementation, using a gene delivery system, of a tool for suppressing activity-dependent astrocytic motility. Calciuminduced astrocyte process outgrowth and its dependence on Profilin-1, novel in vivo gene delivery approaches, a demonstration of astrocytic motility in vivo and the independence of visual processing from astrocytic motility rates are the main findings of the project. The results described in this work increase our understanding of the interactions occurring between astrocytes and neurons as well as the consequences for brain function.Glia-soluihin kuuluvat astrosyytit ovat tiedettävästi osa aivojen toimintaa. Astrosyytit tukevat metabolisesti neuroneita, säätelevät verenkierron dynamiikkaa, ovat hyvin läsnä patoloogisten tilojen kehittymisessä, mukana aistimassa ja muuntamassa synaptista aktiivisuutta. Samanaikaisesti astrosyyttejen kompleksi morfologia, läheinen sijainti synaptisien terminalejen kanssa viittaavat mahdolliseen morfo-funtionaaliseen muovautumiseen aivoissa. Tämä väitöskirja keskittyy töihin jotka kehittävät in vitro ja in vivo tekniikoita jolla voidaan käyttää geeni jakelu menetelmää joka vaimentaa astrosyyttejen liikkuvuutta. Keskeiset havainnot tässä väitöskirjassa ovat astrosyyttejen kasliumin indusoima haarottuminen joka on riippuvainen Profiliini-1:stä ja havainnollistaminen astrosyyttejen liikkuvuudesta in vivo menetelmillä ja astrosyyttejen liikkuvuus aste joka on itsenäinen visuaalisesta prosessoinnista. Kuvaillut tulokset tässä väitöskirjassa kohentavat meidän ymmärrystä astrosyytetjen ja neuronejen välisestä kanssakäymisestä ja niiden seuraamus aivojen toiminnassa

Calcium sensitivity of neurotransmitter release in a glutamatergic synapse of the central nervous system

Der Signalübertragung an chemischen Synapsen liegt ein Kopplungsmechanismus zwischen dem präsynaptischen Aktionspotential und der biochemischen Überträgerstoff-Ausschüttung zu Grunde. Dabei werden Ca2+-Kanäle geöffnet und ein Freisetzungssensor Ca2+-abhängig aktiviert, der schließlich die Überträgerstoff-Ausschüttung auslöst. In der vorliegenden Arbeit wurde die Abhängigkeit der Überträgerstoff-Ausschüttung von der intrazellulären Ca2+-Konzentration ([Ca2+]) in einer glutamatergen kelchförmigen Synapse in Stammhirnschnitten der Ratte unter Verwendung photolytischer Ca2+-Freisetzungen gemessen. Um [Ca2+]-Sprünge auf einer Zeitskala sowohl hervorrufen als auch messen zu können, die der schnellen Übertragungsgeschwindigkeit von glutamatergen Synapsen vergleichbar ist, wurde ein elektrophysiologischer Messstand mit einem schnellen Fluoreszenzdetektor und einem UV-Kurzpulslaser ausgestattet. Ein deutlich messbarer Anstieg der Überträgerstoff-Ausschüttung wurde bereits bei einer homogenen Erhöhung der präsynaptischen [Ca2+] von ca. 1 µM beobachtet. Der Spitzenwert der Freisetzungsrate wuchs mit mehr als der vierten Potenz der präsynaptischen [Ca2+]-Amplitude. Ein [Ca2+]-Sprung von mehr als 30 µM löste die Aktivierung aller zur Fusion unmittelbar bereitstehenden Vesikel innerhalb von 0,5 ms aus. Die in dieser Synapse beobachtete Beziehung zwischen der Freisetzungsrate und der präsynaptischen [Ca2+] wurde mit Hilfe eines kinetischen Modells quantitativ beschrieben. Ein Vergleich der Modellvorhersagen mit Freisetzungsraten, die in denselben Synapsen während eines Aktionspotentials gemessen worden waren, ergab, dass ein kurzer Anstieg der [Ca2+] auf weniger als 10 µM ausreicht, um den physiologischen Freisetzungsverlauf zu erklären. Die synaptische Überträgerstoff-Ausschüttung reagiert somit zumindest in manchen synaptischen Systemen empfindlicher auf Ca2+ als bisher angenommen

Neocortical Layer 4 to Layer 2/3 Sensory Information Processing Investigated with Digital-Light-Projection Neuronal Photostimulation

The mammalian brain forms neuronal networks and microcircuits with cell-type- and anatomical-specific synaptic connections. Despite great advances in elucidating the cellular physiology of the nervous system, little is known about the computational processes occurring at the level of neuronal microcircuits. Much success has been reported in describing the synaptic input patterns of many brain regions and cell types using photostimulation systems; however, these systems are severely limited in their ability to study the integration of synaptic input from multiple synchronous or temporally correlated presynaptic locations. Here we describe a system that allows the generation of arbitrary 2-D stimulus patterns with thousands of independently controlled sites to manipulate the activity of populations of neurons with high spatial and temporal precision. The PC-controlled Digital-Light-Processing (DLP) based system updates the 780,000 parallel photostimulation beams, or pixels, at a maximum rate of 13 kHz. With the currently used projection objective, the pixel sizes at the plane of focus are 7.3 µm2 . The high-power UV laser source used in this system provides a light flux density sufficient for bins of 8x8 pixels (21.6 µm x 21.6 µm) with dwell times as low 3 ms to reliably induce action potentials in 2.5 mM MNI-caged glutamate. At these settings the effective diameter of a glutamate uncaging site is \u3c 86 µm, which is equivalent to most other UV photostimulation rigs. With DLP photostimulation, sub-threshold responses and action potentials can be synchronously induced at thousands of sites over a 2.76 mm x 2.07 mm area, a capability unmatched by any other current system. This DLP-based system has the unique capability to investigate normal and diseased circuit properties by investigating neuronal responses to spatiotemporally complex activity patterns. This technique was used to investigate the temporal integration of synaptic input in the whisker barrel cortex of mice. The neocortex is organized into layers, with neuronal networks and circuits formed by layer-specific connections. While the anatomical organization of these circuits has been well characterized, the information processing and coding performed by these ensembles is poorly understood. A key component of this investigation concerns the transmission and transformation of the neuronal representation from one neuronal pool to the next. In the rodent somatosensory barrel cortex, histologically-distinguishable “barrels” in layer 4 (L4) receive principal input from a single whisker. L4 projects to layer II/III (L2/3), where the circuit diverges to multiple postsynaptic targets. Using the DLP-photostimulation system, we modulated the synchronicity of action potentials in L4 cells while recording from L2/3 in an acute slice preparation. This data shows that synchronous activity in L4 neurons is highly effective at eliciting strong spiking responses in L2/3 pyramidal cells, while asynchronous L4 activity fails to drive L2/3 to action-potential threshold. Pharmacological manipulation of the slice-bathing solution has suggested that this phenomenon is AMPA-receptor dependent and modulated by NMDA receptor activity. Intracellular pharmacological manipulations suggest that postsynaptic conductances also play a role in the nonlinear L2/3 synaptic integration of L4 activity

Dendritic integration in olfactory bulb granule cells: Thresholds for lateral inhibition and role of active conductances upon 3D multi-site photostimulation of spines using a holographic projector module

The inhibitory axonless olfactory bulb granule cells (OB GCs) form reciprocal dendrodendritic synapses with mitral and tufted cells (MCs and TCs) via large spines, mediating recurrent and lateral inhibition. Rat GC dendrites are excitable by local Na⁺ spine spikes and global Ca²⁺- and Na⁺-spikes. Since reaching global threshold potentials also represents the onset of lateral inhibition, the goal of my work was to investigate the exact transition from local to global signalling: How many spines, in which position and distribution on the dendritic tree have to be activated to trigger global spikes and what are the molecular key players, i.e. which ion channels are involved. In the first part of this study we have integrated a holographic projector into the existing commercial two-photon (2P) Galvanometer-based 2D laser scanning microscope with an uncaging unit (Uncaging: Activation of photolabile biologically inactive derivatives of neurotransmitters by photolysis), which allows the simultaneous photostimulation of several spines in three dimensions (3D) in acute brain slices. Patterned 2P photolysis via holographic illumination is a powerful method to investigate neuronal function because of its capability to emulate multiple synaptic inputs in three dimensions (3D) simultaneously. However, like any optical system, holographic projectors have a finite space-bandwidth product that restricts the spatial range of patterned illumination or field-of-view (FOV) for a desired resolution. Such trade-off between holographic FOV and resolution restricts the coverage within a limited domain of the neuron’s dendritic tree to perform highly resolved patterned 2P photolysis on individual spines. Here, we integrate a holographic projector into a commercial 2P galvanometer-based 2D scanning microscope with an uncaging unit and extend the accessible holographic FOV by using the galvanometer scanning mirrors to reposition the holographic FOV arbitrarily across the imaging FOV. The projector system utilizes the microscope’s built-in imaging functions. Stimulation positions can be selected from within an acquired 3D image stack (the volume of interest, VOI) and the holographic projector then generates 3D illumination patterns with multiple uncaging foci. The imaging FOV of our system is 800×800 μm² within which a holographic VOI of 70×70×70 μm³ can be chosen at arbitrary positions and also moved during experiments without moving the sample. We describe the design and alignment protocol as well as the custom software plugin that controls the 3D positioning of stimulation sites. We demonstrate the neurobiological application of the system by simultaneously uncaging glutamate at multiple spines within dendritic domains and consequently observing summation of postsynaptic potentials at the soma, eventually resulting in APs. At the same time, it is possible to perform 2P Ca²⁺ imaging in 2D in the dendrite and thus to monitor synaptic Ca²⁺ entry in selected spines and also local regenerative events such as dendritic APs. In the second part of this study we applied the system to study dendritic integration in GCs. Less than 10 coactive reciprocal spines were sufficient to generate diverse regional and global signals that also included local dendritic Ca²⁺- and Na⁺-spikes (D-spikes). Individual spines could sense the respective signal transitions as increments in Ca²⁺ entry. Dendritic integration was mostly linear until a few spines below global Na⁺-spike threshold, where often D-spikes set in. NMDARs strongly contributed to active integration, whereas morphological parameters barely mattered. In summary, thresholds for GC-mediated bulbar lateral inhibition are low

The role of resting Ca2+ in astrocyte Ca2+ signalling

Astrocytes form gap-junction coupled networks and their fine processes cover many synapses enabling astrocytes to powerfully modulate synapse function. Such modulation is thought to involve Ca2+ -dependent release of signalling molecules from astrocytes. However, astrocyte Ca2+ signalling and its role in synaptic physiology remains a matter of debate. An incomplete and mostly qualitative understanding of the fundamental mechanisms of intracellular Ca2+ signalling in astrocytes could be a knowledge-limiting factor. Previous studies predict that astrocyte resting [Ca2+] profoundly affects astrocyte Ca2+ signalling, especially IP3 and store-dependent Ca2+ transients. I therefore quantitatively investigated the role of resting [Ca2+] in shaping spontaneous and evoked Ca2+ transients in astrocytes. I used two-photon excitation fluorescence microscopy and whole-cell patch clamp to document Ca2+ signalling of individual passive astrocytes in the CA1 stratum radiatum of acute hippocampal slices in young adult rat. I used fluorescence lifetime imaging to obtain a quantitative readout of astrocyte [Ca2+] and reveal the relationship between resting [Ca2+] and Ca2+ transients. I combined these techniques with UV-uncaging of Ca2+ or Ca2+ buffer to manipulate the astrocyte resting [Ca2+] to further investigate its effect on Ca2+ signalling. Using these methods, we have found that low resting [Ca2+] were associated with smaller amplitudes of spontaneous Ca2+ transients. This was also true for metabotropic glutamate receptor agonist (DHPG) evoked Ca2+ transients when different cells or regions of interest of the same cell were compared. The well-established increase of most IP3 receptors’ open probability at higher cytosolic [Ca2+] could explain this observation. In contrast, changes of resting [Ca2+] within a single astrocyte region were associated with inverse changes in amplitude of evoked Ca2+ transients. The DHPG-induced equilibration of [Ca2+] across cytosol and store compartments could be a potential explanation for this effect. Thus, resting [Ca2+] could shape the amplitude of astrocyte Ca2+ transients by at least two distinct mechanisms

Enhanced Astrocytic Ca\u3csup\u3e2+\u3c/sup\u3e Signals Contribute to Neuronal Excitotoxicity after Status Epilepticus

Status epilepticus (SE), an unremitting seizure, is known to cause a variety of traumatic responses including delayed neuronal death and later cognitive decline. Although excitotoxicity has been implicated in this delayed process, the cellular mechanisms are unclear. Because our previous brain slice studies have shown that chemically induced epileptiform activity can lead to elevated astrocytic Ca2+ signaling and because these signals are able to induce the release of the excitotoxic transmitter glutamate from these glia, we asked whether astrocytes are activated during status epilepticus and whether they contribute to delayed neuronal death in vivo. Using two-photon microscopy in vivo, we show that status epilepticus enhances astrocytic Ca2+ signals for 3 d and that the period of elevated glial Ca2+ signaling is correlated with the period of delayed neuronal death. To ask whether astrocytes contribute to delayed neuronal death, we first administered antagonists which inhibit gliotransmission: MPEP [2-methyl-6-(phenylethynyl)pyridine], a metabotropic glutamate receptor 5 antagonist that blocks astrocytic Ca2+ signals in vivo, and ifenprodil, an NMDA receptor antagonist that reduces the actions of glial-derived glutamate. Administration of these antagonists after SE provided significant neuronal protection raising the potential for a glial contribution to neuronal death. To test this glial hypothesis directly, we loaded Ca2+ chelators selectively into astrocytes after status epilepticus.We demonstrate that the selective attenuation of glial Ca2+ signals leads to neuronal protection. These observations support neurotoxic roles for astrocytic gliotransmission in pathological conditions and identify this process as a novel therapeutic target