Role of the AMPA receptor auxiliary subunit CKAMP44 in the olfactory bulb

The olfactory bulb is the first processing station of olfactory information. Mitral and tufted cells, which are the output neurons of the olfactory bulb, receive input from olfactory sensory neurons. The activity of the output neurons is controlled by inhibitory periglomerular and granule cells. Periglomerular cells also receive excitatory input from olfactory sensory neurons and provide feedforward inhibition. Little is known about how AMPA receptor number and function in synapses of periglomerular cells is controlled. It is also not well understood how changes in AMPA receptor number of periglomerular cells affect the olfactory bulb network. Here I show with fluorescence in situ hybridization that the AMPA receptor auxiliary protein CKAMP44 is expressed at high levels in periglomerular cells, but not or only at low levels in other neurons of the olfactory bulb. With ex vivo slice electrophysiology I found that deletion of CKAMP44 decreases AMPA receptor-mediated currents in periglomerular cells. This in turn reduces the inhibition from periglomerular cells onto mitral cells and increases mitral cell activity upon olfactory sensory neuron activation. Data from a computational model of olfactory bulb neuron activity corroborate these findings and indicate that feedforward inhibition from periglomerular cells reduces mitral cell activity in particular during weak or intermediate excitation from olfactory sensory neurons. Behavior experiments showed that the function of the olfactory bulb, at least concerning the differentiation of odors, is not affected by decreased periglomerular cell excitability. Whether other odor functions (e.g. detection threshold) and in vivo network activity is affected remains to be investigated

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

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

Electrophysiological analysis of voltage-dependent currents in two types of periglomerular cells in the mouse olfactory bulb

Periglomerular cells represent an important class of interneurons of the olfactory bulb, contributing to the processing of olfactory information coming from the olfactory epithelium. Despite their involvement in the glomerular circuitry, a larger number of data is required to clarify their function. To further complicate matters, several types of periglomerular cells are described in literature. The main goal of my thesis is to provide new information about two types of periglomerular cells: dopaminergic and calretinin-positive neurons. They belong to different classes of periglomerular cells, suggesting distinct roles in modulating olfactory stimuli. Using patch clamp technique, in both whole cell and perforated patch configuration, properties of these periglomerular neurons have been illustrated. Electrophysiological experiments have been performed in two transgenic mouse strains, each one specific for a distinct periglomerular class. The first part of my thesis is focused on the investigation of the properties of h-current in the dopaminergic periglomerular neurons. Most of the experiments have been carried out in perforated patch, in order to minimize the disturbance of the intracellular fluid. Biophysical properties of h-current have been described, calculating the values of the main kinetic parameters. In addition, the influence of both thermic conditions and intracellular levels of cAMP on this conductance has been analyzed. The blockage of h-current suppresses the spontaneous firing of these neurons through its effect on the resting membrane potential. Finally, only noradrenaline have been observed to act on Ih, decreasing its amplitude. The second part of the thesis regards the calretinin-positive periglomerular cells. In whole cell configuration and under voltage clamp conditions, activation protocols have been applied to the cell membrane, in order to elicit a complete set of depolarization-evoked currents. The three main conductances (a fast transient sodium current, an A-type potassium current, and an L-type calcium current) have been further isolated and analyzed, determining their voltage dependence and kinetic properties. As a whole, all the data collected contribute to a better understanding of the functional role of the periglomerular cells in the glomerular network. The characterization of h-current in the dopaminergic cells enlarges the previous body of knowledge regarding this class of interneurons. On the other hand, the description of the properties of calretinin-positive cells represents their first electrophysiological analysis in literature

The role of different subtypes of olfactory bulb interneurons in olfactory behavior

Le bulbe olfactif (BO) représente dans le cerveau le premier relai dans le traitement des informations olfactives. Au niveau de cette structure, plusieurs types de neurones sont impliqués dans la modulation de l’information odorante, avant même que celle-ci ne soit envoyée vers des structures corticales supérieures. Parmi eux se trouvent les cellules granulaires (CGs), une population d’interneurones régulant de manière importante l’activité des cellules principales du BO. De manière intéressante, le BO est capable à l’âge adulte de produire et régénérer une partie de sa population interneuronale via le processus de neurogénèse adulte. Il est ainsi possible de faire la distinction entre les CGs générées au cours de la période postnatale (CGs postnatales) des CGs générées à l’âge adulte (CGs nouvellement générées). Le rôle que jouent ces CGs dans le traitement olfactif mais aussi dans les différents comportements olfactifs a pendant très longtemps donné lieu à des interprétations contradictoires. Le manque de cohérence au niveau des données peut s’expliquer par le fait que pendant longtemps, les CGs ont été considérées comme étant une population homogène de cellules. Néanmoins, des études ont montré que les CGs peuvent exprimer différents marqueurs neurochimiques. Notamment, nous nous sommes intéressés dans le cadre de notre étude à deux de ces marqueurs : la protéine kinase calcium calmoduline dépendante IIα (CaMKIIα) et la Calrétinine (CR). Une telle hétérogénéité au sein des cellules interneuronales du BO pourrait également refléter une hétérogénéité fonctionnelle, chaque sous-population de CGs pouvant contribuer de façon propre et unique au traitement des informations olfactives et donc au comportement olfactif. Dans la première partie de ces travaux, nous avons étudié le rôle fonctionnel des cellules exprimant la CaMKIIα et l’avons comparé à la population générale de CGs. De manière intéressante, nous montrons que, bien que ces deux populations de cellules soient en tous points semblables au niveau morphologique, les cellules CaMKIIα reçoivent un niveau d’inhibition moindre par rapport à leurs homologies négatives, les rendant plus susceptibles d’être activées suite à des tâches comportementales spécifiques. De plus, l’inhibition spécifique des cellules CaMKIIα- positive entraine une perturbation des performances de discrimination fine. Dans la seconde partie de ces travaux, nous nous sommes intéressés cette foisci à la sous-population de CGs exprimant la CR, en tenant compte également de la période développementale de ces cellules (i.e CGs post-natales ou nouvellement générées). Nous montrons que les cellules nouvellement générées exprimant ou non la CR, ainsi que les cellules CR-positives postnatales diffèrent quant à leurs propriétés électrophysiologiques. De plus, tout comme les cellules exprimant la CaMKIIα, les cellules exprimant la CR présentent un niveau d’activation plus important à la suite de certaines tâches comportementales et sont également nécessaires à la bonne réalisation de tâches de discrimination olfactive.The olfactory bulb (OB) is considered as the first relay in the brain during olfactory processing. Several types of neurons are involved at the level of this structure in the refinement of the olfactory information before it is sent to higher cortical structures. Among the cell types involved is the population of granule cells (GC), a population of interneurons largely regulating the activity of OB principal cells. Interestingly, the OB retain during adulthood the ability to produce and renew part of its interneuronal pool through a process called adult neurogenesis. Therefore, it is possible to distinguish in the adult OB between GCs born during the early postnatal period (early-born GCs) to the one that were generated during adulthood (adult-born GCs). Several studies aimed at determining the precise role played by GC in olfactory processing and olfactory behavior, giving rise quite often to conflicting results. This absence of coherence in the data could come from the fact that for long, the population of GCs was considered as a homogeneous cell population. However, GCs were shown to express diverse neurochemical markers. In this study we investigated more particularly into two of those markers, showed to be expressed by GCs: the Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) and Calretinin (CR). Hence, such a heterogeneity in the phenotype of OB interneurons could also underlie a functional heterogeneity of the different GC subpopulation, each one contributing in a unique way to olfactory processing and thus olfactory behavior. In the first part of this work, we investigated the functional role of CaMKIIα- expressing cells and compared it to the general population of GCs. Interestingly we revealed that CaMKIIα-positive GCs are more prone to activation following specific behavioral tasks, likely due to a decreased level of inhibition as compared to their negative counterparts. Moreover, the specific inhibition of this GC subpopulation let to alteration of animals’ fine discrimination abilities. In the second part of our work, when focusing this time on the subpopulation of CR-expressing GCs, taking this time also into account the developmental period at which they were generated (i.e early- versus adult-born cells), we showed that adultborn CR-expressing and non-expressing GCs, but also early-born CR-expressing GCs display different electrophysiological characteristics. Moreover, as for CaMKIIα- positive GCs, CR-positive GCs present a higher level of activation following specific olfactory tasks and are also important for a proper ability to perform olfactory discrimination tasks

Electrophysiological analysis of voltage-dependent currents in two types of periglomerular cells in the mouse olfactory bulb

openPeriglomerular cells represent an important class of interneurons of the olfactory bulb, contributing to the processing of olfactory information coming from the olfactory epithelium. Despite their involvement in the glomerular circuitry, a larger number of data is required to clarify their function. To further complicate matters, several types of periglomerular cells are described in literature. The main goal of my thesis is to provide new information about two types of periglomerular cells: dopaminergic and calretinin-positive neurons. They belong to different classes of periglomerular cells, suggesting distinct roles in modulating olfactory stimuli. Using patch clamp technique, in both whole cell and perforated patch configuration, properties of these periglomerular neurons have been illustrated. Electrophysiological experiments have been performed in two transgenic mouse strains, each one specific for a distinct periglomerular class. The first part of my thesis is focused on the investigation of the properties of h-current in the dopaminergic periglomerular neurons. Most of the experiments have been carried out in perforated patch, in order to minimize the disturbance of the intracellular fluid. Biophysical properties of h-current have been described, calculating the values of the main kinetic parameters. In addition, the influence of both thermic conditions and intracellular levels of cAMP on this conductance has been analyzed. The blockage of h-current suppresses the spontaneous firing of these neurons through its effect on the resting membrane potential. Finally, only noradrenaline have been observed to act on Ih, decreasing its amplitude. The second part of the thesis regards the calretinin-positive periglomerular cells. In whole cell configuration and under voltage clamp conditions, activation protocols have been applied to the cell membrane, in order to elicit a complete set of depolarization-evoked currents. The three main conductances (a fast transient sodium current, an A-type potassium current, and an L-type calcium current) have been further isolated and analyzed, determining their voltage dependence and kinetic properties. As a whole, all the data collected contribute to a better understanding of the functional role of the periglomerular cells in the glomerular network. The characterization of h-current in the dopaminergic cells enlarges the previous body of knowledge regarding this class of interneurons. On the other hand, the description of the properties of calretinin-positive cells represents their first electrophysiological analysis in literature.SCIENZE BIOMEDICHEopenFOGLI ISEPPE, Ale

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

Selective Gene Expression by Postnatal Electroporation during Olfactory Interneuron Neurogenesis

Neurogenesis persists in the olfactory system throughout life. The mechanisms of how new neurons are generated, how they integrate into circuits, and their role in coding remain mysteries. Here we report a technique that will greatly facilitate research into these questions. We found that electroporation can be used to robustly and selectively label progenitors in the Subventicular Zone. The approach was performed postnatally, without surgery, and with near 100% success rates. Labeling was found in all classes of interneurons in the olfactory bulb, persisted to adulthood and had no adverse effects. The broad utility of electroporation was demonstrated by encoding a calcium sensor and markers of intracellular organelles. The approach was found to be effective in wildtype and transgenic mice as well as rats. Given its versatility, robustness, and both time and cost effectiveness, this method offers a powerful new way to use genetic manipulation to understand adult neurogenesis

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