Pharmacological analysis of ionotropic glutamate and GABA recptor function in neuronal circuits of the zebrafish olfactory bulb

In the olfactory bulb and other brain areas, basic cellular and synaptic properties of individual neurons have been studied extensively in reduced preparations. Nevertheless, it is still poorly understood how intactions between multiple neurons shape spatio-temporal activity patterns and give rise to the computational properties of the the intact circuit. In this thesis, I used pharmacological manipulations of excitatory and inhibitory neurotransmitter receptors to examine the synaptic interactions underlying spontaneous and odor-evoked activity patterns in the intact olfactory bulb of zebrafish. Electrophysiological and one- and two-photon calcium imaging methods were used to record activity from the principal neurons of the OB (mitral cells, MCs), their sensory input, and local interneurons. The combined blockade of AMPA/kainate and NMDA receptors abolished odor-evoked excitation of MCs, indicating that sensory input to the OB is mediated by ionotropic glutamate receptors. Surprisingly, however, the blockade of AMPA/Kainiate receptors alone increased the mean response of MCs and decreased the mean response of interneurons (INs), and the blockade of NMDA receptors caused little or no change in the mean responses of MCs and INs. In addition, antagonists of both glutamate receptor types had diverse effects on the magnitude and time course of individual MC and IN responses and, thus, changed spatio-temporal activity patterns across neuronal populations. The blockade of GABA(A) receptors increased spontaneous and odor evoked firing rates of mitral cells and often induced rhythmic bursting. Moreover, the blockade of, GABA(A) or AMPA/kainate receptors abolished fast oscillatory activity in the local field potential. Blockade of GABA(B) receptors reduced calcium influx in afferent sensory axons and modulated response time courses of mitral cells. These results indicate that (1) IN activity during an odor response depends mainly on AMPA/Kainiate receptor input, (2) interactions between MCs and INs regulate the total OB output activity, (3) AMPA/Kainiate receptors and GABA(A) receptors underly the synchronization of odor-dependent neuronal ensembles and (4) odor-specific patterns of OB output activity are shaped by circuits containing iGlu receptors and GABA receptors. These results provide insights into the mechanisms underlying the processing of odor-encoding activity patterns in the OB.Im olfaktorischen Bulbus (OB) und anderen Hirnarealen wurden grundlegende zelluläre und synaptische Eigenschaften der Einzelneurone ausführlich in reduzierten Präparaten studiert. Trotzdem ist kaum bekannt, wie die Interaktionen mehrerer Nervenzellen untereinander räumlich-zeitlich strukturierte Aktivitätsmuster formen und dadurch die rechnerischen Eigenschaften der intakten Schaltkreise entstehen. In dieser Arbeit nutzte ich pharmakologische Manipulationen der erregenden und hemmenden Neurotransmitter-Rezeptoren, um die synaptischen Interaktionen zu untersuchen, die spontanen und geruchsinduzierten Aktivitätsmustern im intakten OB des Zebrafisch zugrunde liegen. Methoden der Elektrophysiology sowie der konventionellen und Zwei-Photonen-Mikroskopie wurden genutzt, um Aktivität von Ausgangsneuronen des OB (Mitralzellen, MCs), ihrem sensorischen Eingang, und Interneuronen (INs) zu messen. Die gleichzeitige Blockierung von AMPA/Kainate- und NMDA-Rezeptoren verhinderte die geruchsinduzierte Erregung von MCs, was darauf hinweist, dass der sensorische Eingang des OB durch ionotrope Glutamatrezeptoren vermittelt wird. Die Blockierung von AMPA/Kainate Rezeptoren allein jedoch erhöhte überraschender Weise im Mittel die Antwort von MCs und reduzierte im Mittel die Antwort von INs. Die Blockierung von NMDA Rezeptoren allein lösten im Mittel geringe oder keine Veränderung der Antworten von MCs and INs aus. Außerdem hatten die Antagonisten für beide Glutamatrezeptoren unterschiedliche Einflüsse auf Größe und Zeitverlauf individueller MC- und IN- Antworten und veränderten daher das räumlich-zeitliche Aktivitätsmuster innerhalb der Nervenzellpopulation. Die Blockierung von GABA(A)-Rezeptoren erhöhte spontane und geruchsinduzierte Feuerraten in MCs und induzierten oft rhythmische, stoßweise Aktivität. Die Blockierung von GABA(A)- und AMPA/Kainate-Rezeptoren hob überdies geruchsinduzierte Oszillationen im Feldpotenzial auf. Die Blockierung von GABA(B)-Rezeptoren verringerte den Kalziumeinstrom in die Endigungen afferenter sensorischer Axone und modulierte den Zeitverlauf von MC-Antworten. Die Ergebnisse zeigen, dass (1) die Aktivität der Interneurone während der Geruchsantwort hauptsächlich von AMPA/Kainate-Rezeptoren abhängt, (2) die Interaktionen zwischen Mitralzellen und Interneuronen die Gesamtaktivität des Ausgangssingnales des olfaktorischen Bulbus regulieren, (3) AMPA/Kainate-Rezeptoren und GABA(A)-Rezeptoren der Synchronisation geruchsabhängiger Gruppen von Nervenzellen zugrunde liegen und (4) geruchsspezifische Muster im Ausgangssignal des olfaktorischen Bulbus durch Schaltkreise geformt werden, die iGlu Rezeptoren und GABA Rezeptoren enthalten. Diese Ergebnisse ermöglichen Einblick in die Mechanismen die der Verarbeitung geruchskodierender Aktivitätsmuster im olfaktorischen Bulbus unterliegen

Inward rectifier potassium current in dopaminergic periglomerular cells of mouse olfactory bulb.

openInward rectifier potassium (Kir) channels are important for neuronal signalling and membrane excitability. In this work, patch-clamp techniques were used to characterize Kir channels in mouse dopaminergic (DA) periglomerular (PG) cells. These interneurons are critically placed at the entry of the bulbar circuitry, in contact with terminals of olfactory sensory neurons and with dendrites of projection neurons. Perforated-patch configuration was adopted to record Kir current in DA-PG cells in thin slice. IKir could be distinguished from the hyperpolarization-activated current by showing full activation in < 10 ms, no inactivation, suppression by Ba2+ in a typical voltage-dependent manner and reversal potential nearly coincident with EK. DA-PG cells are autorhythmic and are target of numerous afferents releasing a variety of neurotransmitters, although their properties and role remain elusive. Depolarization induced by Ba2+ blocks spontaneous activity, although the Kir current is not an essential component of the pacemaker machinery. The current is negatively modulated by intracellular cAMP, as shown by a decrease of its amplitude induced by forskolin. Several neuromodulatory effects were tested on the Kir current of DA-PG cell. Activation of metabotropic receptors - known to be present on these cells - shows that the current can be modulated by a multiplicity of pathways. The Kir current can be increased, as observed with agonists of muscarinic, α1 noradrenergic and GABAA receptors, or IKir modulation can caused the opposite effect, i.e. agonists of D2, 5-HT and histamine receptors. These characteristics of the Kir currents provide the basis for additional flexibility of DA-PG cells signaling and function.openFisiologia Sperimentale e ClinicaBorin, MirtaBorin, Mirt

Inward rectifier potassium current in dopaminergic periglomerular cells of mouse olfactory bulb.

Inward rectifier potassium (Kir) channels are important for neuronal signalling and membrane excitability. In this work, patch-clamp techniques were used to characterize Kir channels in mouse dopaminergic (DA) periglomerular (PG) cells. These interneurons are critically placed at the entry of the bulbar circuitry, in contact with terminals of olfactory sensory neurons and with dendrites of projection neurons. Perforated-patch configuration was adopted to record Kir current in DA-PG cells in thin slice. IKir could be distinguished from the hyperpolarization-activated current by showing full activation in < 10 ms, no inactivation, suppression by Ba2+ in a typical voltage-dependent manner and reversal potential nearly coincident with EK. DA-PG cells are autorhythmic and are target of numerous afferents releasing a variety of neurotransmitters, although their properties and role remain elusive. Depolarization induced by Ba2+ blocks spontaneous activity, although the Kir current is not an essential component of the pacemaker machinery. The current is negatively modulated by intracellular cAMP, as shown by a decrease of its amplitude induced by forskolin. Several neuromodulatory effects were tested on the Kir current of DA-PG cell. Activation of metabotropic receptors - known to be present on these cells - shows that the current can be modulated by a multiplicity of pathways. The Kir current can be increased, as observed with agonists of muscarinic, α1 noradrenergic and GABAA receptors, or IKir modulation can caused the opposite effect, i.e. agonists of D2, 5-HT and histamine receptors. These characteristics of the Kir currents provide the basis for additional flexibility of DA-PG cells signaling and function

Cracking the Odor Code: Molecular and Cellular Deconstruction of the Olfactory Circuit of Drosophila Larvae

The Drosophila larva offers a powerful model system to investigate the general principles by which the olfactory system processes behaviorally relevant sensory stimuli. The numerically reduced larval olfactory system relieves the formidable molecular and cellular complexity found in other organisms. This thesis presents a study in four parts that investigates molecular and neuronal mechanisms of larval odor coding. First, the larval odorant receptor (OR) repertoire was characterized. ORs define the olfactory receptive range of an animal. Each of the 21 larval olfactory sensory neurons (OSNs) expresses one or rarely two ORs, along with the highly conserved olfactory co-receptor Or83b. Second, odor response profiles of 11 larval OSNs were characterized by calcium imaging. A subset of larval neurons showed overlapping responses to the set of odorants tested, while other neurons showed either very narrow or very broad tuning. Third, the olfactory circuit for ethyl butyrate was investigated in detail. Three OSNs, expressing Or35a, Or42a and Or42b, responded with different sensitivity to ethyl butyrate. Second order projection neurons synapsing with each of these OSNs showed similar concentration tuning, but inhibitory interneurons showed high response thresholds and were only activated at high odor concentrations. We correlated these concentration-dependent response properties with larval chemotaxis behavior. Fourth, the relevance of olfaction to animals was investigated in competitive rearing experiments. Or83b mutants experienced a selective disadvantage when they had to forage for limiting food sources, particularly when competing against larvae with normal olfactory function. Thus, odor coding is achieved both by peripheral tuning and central circuit modulation

Molecular characterization of CNS interneurons: subtype diversity and fate determinants

In this project two approaches have been used to gain insights into the identity and molecular regulation of interneurons in the brain. First, a general molecular characterization of GABAergic neuron subtypes has been performed and second, novel fate determinants specifically for olfactory bulb interneurons have been identified and investigated in detail. GABAergic neurons, the largest population of inhibitory neurons in the brain, play crucial roles in information processing. While most of these neurons are interneurons, some, for example in the striatum, represent projection neurons. So far, biochemical, morphological, and electrophysiological properties served as exclusive criteria for the classification of GABAergic neurons. Although these parameters allow for a partial description of subpopulations, a systematic dichotomy is not available. Therefore, the general molecular characteristics of GABAergic neurons were analyzed and differences among distinct brain regions were defined. Transgenic GAD67-GFP mice in concert with flow cytometric cell sorting were used to isolate GABAergic neurons from defined regions of the postnatal mouse brain, namely olfactory bulb, cortex, striatum and cerebellum. Subsequently, gene expression profiling as well as cell surface proteome analysis were carried out and identified genes were validated by in situ hybridization and qPCR. Potential new marker genes for GABAergic neurons and candidate factors necessary for their differentiation and general functionality were determined. Clustering of gene expression data revealed major differences between hind- and forebrain GABAergic neurons indicating a correlation between their development and localization. For example, while GABAergic neurons of the forebrain are characterized mainly by three groups of transcription factors, namely the Distal-less-family, the POU-family and the ETS/FOX-family; specific members of the ZIC- and LHX-family define hindbrain inhibitory neurons. Olfactory bulb interneurons are generated throughout live by adult neuronal stem cells localized in the subventricular zone. While considerable information is available concerning the generation and migration of these cells, the molecular mechanisms regulating their terminal differentiation are barely understood. Therefore, mature interneurons from the periglomerular layer and their specific precursors were isolated by microdissection and magnetic cell sorting. Gene expression analysis was performed by microarray analysis. Several candidate factors to be involved in the differentiation of olfactory bulb interneurons were identified. The bHLH transcription factors NeuroD1 and NeuroD2 were analyzed for their functional relevance in vivo. Using in vivo electroporation, overexpression of these transcription factors was induced in postnatal forebrain stem cell populations as well as their progeny, namely neuronal precursors and mature neurons of the olfactory system. It was shown that high expression of NeuroD2 delayed the differentiation of Type A neuronal precursor cells into granule- and periglomerular neurons. In contrast, overexpression of NeuroD1 induced the premature and ectopic differentiation of precursor cells. Furthermore, NeuroD1 induced specifically a dopaminergic phenotype, indicating that it represents a novel key fate determinant for the specification of periglomerular interneurons, possibly with a GABA/dopamine bifunctional neurotransmitter phenotype. In conclusion, this study represents a comprehensive molecular basis for the understanding of spatially as well as temporally defined GABAergic neuron subpopulations and led to the identification of novel fate determinants for the differentiation of inhibitory interneuron subpopulations

Inward rectifier potassium current in dopaminergic periglomerular cells of mouse olfactory bulb.

Inward rectifier potassium (Kir) channels are important for neuronal signalling and membrane excitability. In this work, patch-clamp techniques were used to characterize Kir channels in mouse dopaminergic (DA) periglomerular (PG) cells. These interneurons are critically placed at the entry of the bulbar circuitry, in contact with terminals of olfactory sensory neurons and with dendrites of projection neurons. Perforated-patch configuration was adopted to record Kir current in DA-PG cells in thin slice. IKir could be distinguished from the hyperpolarization-activated current by showing full activation in < 10 ms, no inactivation, suppression by Ba2+ in a typical voltage-dependent manner and reversal potential nearly coincident with EK. DA-PG cells are autorhythmic and are target of numerous afferents releasing a variety of neurotransmitters, although their properties and role remain elusive. Depolarization induced by Ba2+ blocks spontaneous activity, although the Kir current is not an essential component of the pacemaker machinery. The current is negatively modulated by intracellular cAMP, as shown by a decrease of its amplitude induced by forskolin. Several neuromodulatory effects were tested on the Kir current of DA-PG cell. Activation of metabotropic receptors - known to be present on these cells - shows that the current can be modulated by a multiplicity of pathways. The Kir current can be increased, as observed with agonists of muscarinic, α1 noradrenergic and GABAA receptors, or IKir modulation can caused the opposite effect, i.e. agonists of D2, 5-HT and histamine receptors. These characteristics of the Kir currents provide the basis for additional flexibility of DA-PG cells signaling and function

Physiology of rodent olfactory bulb interneurons

The sense of olfaction is a central gateway of perceiving and evaluating an animal’s environment filled with volatile chemicals. It affects individual and social behavior in an evaluative way, i.e. by helping to find food sources, warning from dangers like toxins or predators or influencing mating choice. Already the first central station for vertebrate olfactory processing, the olfactory bulb (OB), is astonishingly complex. Its structure features several horizontal layers of signal transformation that includes a large variety of local interneurons (INs). Most of these cells are subject to adult neurogenesis, which rejuvenates and remodels the circuitry throughout life. One of those interneuron subtypes, the granule cell (GC), poses the most numerous cell type of the olfactory bulb. As the major synaptic connection of the bulb, linking different glomerular units, it participates in numerous reported tasks like odor discrimination or memory formation. Many of those capacities are attributable to the function of peculiar spines with long necks and enormous bulbar heads called gemmules. They accommodate pre- and postsynaptic specializations of the reciprocal synapse with mitral cells (MCs) that are topographically and functionally linked and feature many modes of signal integration and transmission. As of yet, the mechanistic underpinnings of activation and neurotransmitter release are not yet resolved in great detail. This gave rise to the first project of this thesis, which focusses on the detailed granule cell gemmule physiology during local glutamatergic activation. With the help of two-photon glutamate uncaging and concomitant calcium imaging, the spine could be selectively stimulated and its physiological dynamics tested. By the use of different pharmacological agents, we could verify the importance of voltage gated sodium channels (Nav) for local signal amplification and the involvement of NMDA and high voltage activated calcium channels (HVACCs) in the calcium elevation during local stimulation, which is important for γ-aminobutyric acid (GABA) release from the spine. The superthreshold depolarizing signal and strong calcium elevation during local input are exclusively restricted to the spine, which affirms the chemical and electrical isolation of gemmules from the rest of the cell. In this study we thereby confirmed the theoretical prediction of active computation within single spines in our system, emphasizing the functional importance of morphological compartmentalization for the cell’s physiology. The second largest population of interneurons in the olfactory bulb is located in the glomerular layer (GL) of the olfactory bulb and subsumes a plethora of different cell types, categorized in terms of molecular characteristics (mostly neurotransmitter), morphology and function. Among those, dopaminergic (DAergic) juxtaglomerular cells (JGCs) form a subpopulation, which the second part of this thesis is focused on. Innervated by the first or second synapse in the olfactory pathway, these cells exert strong influence in very early stages of olfactory signaling. The gating and transformation of inputs locally and very importantly also laterally over large distances originate from several factors. This cell grouping usually expresses two neurotransmitters at the same time, GABA and dopamine (DA), and encompass many different morphologies and synaptic arrangements with other cell types. Utilizing dopamine transporter (DAT) based staining methods in three animal populations differing in age and species, this study revealed a larger diversity of dopaminergic cell types in the glomerular layer. New ‘uniglomerular’ and a ‘clasping’ cell types were discriminated, showing distinct dendritic formations and glomerulus innervations, which was assessed with a new morphometric tool kit. The clasping cell type features dendritic specializations, densely clasping around single cell bodies. These morphological traits occur in higher abundance and complexity specifically among adult animals and could be structures of neurotransmitter output since they show strong calcium influx upon soma depolarization. Comparisons of the three animal populations showed age- and/or species-dependent changes in the subtype composition of dopaminergic JGCs. Concordant with recent research, the inclusion of age-dependent comparisons in bulbar studies turned out to be of great significance