Dissertationen zum Thema „Olfactory circuit“

In Drosophila the male sex pheromone cis-vaccenyl acetate (cVA) elicits different behaviours in males and females: while males are repelled by cVA, it stimulates female receptivity. However, olfactory receptor neurons and their second-order partners, the olfactory projection neurons, do not show significantly different physiological responses to cVA. Using in vivo whole-cell electrophysiology I have identified two distinct clusters of third-order olfactory neurons that are pheromone-responsive only in males or females, respectively. These clusters are present in both sexes and share a common input pathway, but sex-specific wiring reroutes pheromone information. To my knowledge this is the first functional characterisation of a bidirectional circuit switch in any organism, and provides a simple mechanism for sex-specific activation of conserved motor programmes. Investigating the genetic logic of this switch, I found that the action of the fruitless transcription factor is both necessary and sufficient for the sex-appropriate wiring of these third-order olfactory neurons. Critically, expression of the male form of fruitless in females is also sufficient to masculinise the pheromone responses of both classes of neurons. Somewhat surprisingly, even selective genetic masculinisation of third-order neurons is sufficient to masculinise their morphology and pheromone responses; thus a complex neural circuit can be functionally rewired by the cell-autonomous action of a switch gene.

This thesis focuses on several aspects of olfactory processing in Drosophila. In chapter I and II, I will discuss how odorants are encoded in the brain. In both insects and mammals, olfactory receptor neurons (ORNs) expressing the same odorant receptor gene converge onto the same glomerulus. This topographical organization segregates incoming odor information into combinatorial maps. One prominent theory suggests that insects and mammals discriminate odors based on these distinct combinatorial spatial codes. I tested the combinatorial coding hypothesis by engineering flies that have only one class of functional ORNs and therefore cannot support combinatorial maps. These files can be taught to discriminate between two odorants that activate the single functional class of ORN and identify an odorant across a range of concentrations, demonstrating that a combinatorial code is not required to support learned odor discrimination. In addition, these data suggest that odorant identity can be encoded as temporal patterns of ORN activity. Behaviors are influenced by motivational states of the animal. Chapter III of this thesis focuses on understanding how motivational states control behavior. Appetitive memory in Drosophilaprovides an excellent system for such studies because the motivational state of hunger promotes reliance on learned appetitive cues whereas satiety suppresses it. We found that activation of neuropeptide F (dNPF) neurons in fed flies releases appetitive memory performance from satiety-mediated suppression. Through a GAL4 screen, we identified six dopaminergic neurons that are a substrate for dNPF regulation. In satiated flies, these neurons inhibit mushroom body output, thereby suppressing appetitive memory performance. Hunger promotes dNPF release, which blocks the inhibitory dopaminergic neurons. The motivational drive of hunger thus affects behavior through a hierarchical inhibitory control mechanism: satiety inhibits memory performance through a subset of dopaminergic neurons, and hunger promotes appetitive memory retrieval via dNPF-mediated disinhibition of these neurons. The aforementioned studies utilize sophisticated genetic tools for Drosophila. In chapter IV, I will talk about two new genetic tools. We developed a new technique to restrict gene expression to different subsets of mushroom body neurons with unprecedented precision. We also adapted the light-activated adenylyl cyclase (PAC) from Euglena gracilis as a light-inducable cAMP system for Drosophila. This system can be used to induce cAMP synthesis in targeted neurons in live, behaving preparations.

Alors que les signaux biochimiques impliqués dans la croissance axonale et la migration neuronale sont largement étudiés, la contribution des signaux mécaniques dans la formation des circuits neuronaux reste peu explorée in vivo. Nous cherchons à étudier comment les forces mécaniques contribuent à la formation du circuit olfactif du poisson-zèbre. Ce circuit se développe durant la morphogénèse de la placode olfactive (PO), par le mouvement passif des corps cellulaires qui s’éloignent de l’extrémité de leurs axones. Mes travaux de thèse s’intéressent à la contribution mécanique de l’œil, qui se forme sous la PO par des mouvements d’évagination et d’invagination, à cette migration passive des neurones et à l’extension de leurs axones. L'analyse quantitative des mouvements cellulaires a tout d’abord révélé que les mouvements des cellules de la PO et de l’œil sont corrélés. Chez des embryons dans lesquels l’œil ne se développe pas, les mouvements des cellules de la PO sont affectés, ce qui produit des PO plus fines et des axones plus courts, et la tension mécanique dans la direction d’élongation des axones dans la PO est réduite. Enfin, la matrice extracellulaire s’accumule à l’interface oeil/PO et sa dégradation enzymatique réduit la corrélation entre les mouvements des cellules de la PO et de l’œil. Ces résultats suggèrent que l’œil en formation exerce des forces de traction sur la PO, transmises par la matrice, entrainant le mouvement des neurones et l’extension des axones. Ce travail apporte un éclairage nouveau sur le rôle des forces mécaniques échangées entre les neurones en développement et les tissus environnants dans la formation des circuits neuronaux in vivo<br>Whereas the biochemical signals guiding axon growth and neuronal migration are extensively studied, the contribution of mechanical cues in neuronal circuit formation is still poorly explored in vivo. We aim at investigating how mechanical forces influence the construction of the zebrafish olfactory circuit. This circuit forms during the morphogenesis of the olfactory placode (OP) by the passive displacement of neuronal cell bodies away from the tip of their axons. My PhD work focuses on the mechanical contribution of the adjacent eye tissue, which develops underneath the OP through extensive evagination and invagination movements, to this passive neuronal migration and to their associated axon elongation. Quantitative live cell imaging analysis during OP morphogenesis first revealed that OP and eye cells undergo correlated movements. In embryos lacking eyes, the movements of OP cell bodies are affected, resulting in thinner placodes and shorter axons, and the mechanical stress along the direction of axon elongation within the OP is reduced. Finally, extracellular matrix was observed to accumulate at the eye/OP interface, and its enzymatic degradation decreased the correlation between OP and eye cell movements. Altogether, these results suggest that the developing eye exerts traction forces on the OP through extracellular matrix, mediating proper neuronal movements and axon extension. This work sheds new light on the role of mechanical forces exchanged between developing neurons and surrounding tissues in the sculpting of neuronal circuits in vivo

Drosophila is a simple and genetically tractable model system for studying neural circuits. This dissertation consists of two studies, with the broad goal of understanding sensory processing in neural circuits using Drosophila as a model system.

Les cellules periglomerulaires du bulbe olfactif conforment une population hétérogène avec des propriétés moléculaires, synaptiques, morphologiques et biophysiques diverses toujours étudiés de façon indépendante. Toutefois, cette diversité suggère que des groupes différents des cellules periglomerulaires pourraient avoir des rôles différents. Dans la première partie de ma thèse je cherche à associer, pour la première fois, différents marqueurs de la diversité des neurones periglomerulaires de façon à aider à comprendre les potentielles implications fonctionnelles que les cellules periglomerulaires pourraient avoir dans le traitement de l’information olfactive. Les cellules periglomerulaires reçoivent des courants inhibiteurs postsynaptiques mais les circuits responsables de cette inhibition restent méconnus. À l’aide des enregistrements électrophysiologiques dans des tranches aiguës horizontales de bulbe olfactif de souris et des techniques d’optogénétique je montre que des projections centrifuges GABAergiques en provenance du télencéphale basal modulent fortement l’inhibition des cellules periglomerulaires de type 2 ainsi que des cellules granulaires et des cellules à axone courtes<br>In the olfactory bulb periglomerular cells form a heterogeneous population with diverse molecular, synaptic, morphological and biophysical properties that have always been considered independently and never explored together. However, such diversity suggests different functional implications. On the first part of this thesis, I aim to associate, for the first time, different markers of periglomerular diversity together to put in perspective the functional implications that differebt subgroups of these cells could exert in odor processing. Periglomerular cells receive inhibitory postsynaptic currents but the circuits mediating this inhibition remain poorly understood. Using a combination of patch-clamp recordings in mouse horizontal olfactory bulb slices and optogenetics I demonstrate that centrifugal GABAergic projections from the basal forebrain strongly mediate inhibition of type 2 periglomerular cells but also granule cells and deep short axon cells

For all animals it is highly advantageous to associate an environmental sensory stimulus with a reinforcing experience. During associative learning, the neural representation of the sensory stimulus (conditioned stimulus; CS) converges in time and location with that of the reinforcer (unconditioned stimulus; US). The CS is then affiliated with a predictive value, altering the animal’s response towards it in following exposures. In my PhD thesis I made use of olfactory aversive conditioning in Drosophila to ask where these two different stimuli are represented and how they are processed in the nervous system to allow association. In the first part of my thesis, I investigated the presentation of the odor stimulus (CS) and its underlying neuronal pathway. CS-US association is possible even when the US is presented after the physical sensory stimulus is gone ('trace conditioning'). I compared such association of temporally non-overlapping stimuli to learning of overlapping stimuli ('delay conditioning'). I found that flies associate an odor trace with electric shock reinforcement even when they were separated with a 15 s gap. Memories after trace and delay conditioning have striking similarities: both reached the same asymptotic learning level, although at different rates, and both memories have similar decay kinetics and highly correlated generalization profiles across odors. Altogether, these results point at a common odor percept which is probably kept in the nervous system throughout and following odor presentation. In search of the physiological correlate of the odor trace, we used in vivo calcium imaging to characterize the odor-evoked activity of the olfactory receptor neurons (ORNs) in the antennal lobe (in collaboration with Alja Luedke, Konstanz University). After the offset of odor presentation, ORNs showed odor-specific response patterns that lasted for a few seconds and were fundamentally different from the response patterns during odor stimulation. Weak correlation between the behavioral odor generalization profile in trace conditioning and the physiological odor similarity profiles in the antennal lobe suggest that the odor trace used for associative learning may be encoded downstream of the ORNs. In the second part of the thesis I investigated the presentation of different aversive stimuli (USs) and their underlying neuronal pathways. I established an odor-temperature conditioning assay, comparable to the commonly used odor-shock conditioning, and compared the neural pathways mediating both memory types. I described a specific sensory pathway for increased temperature as an aversive reinforcement: the thermal sensors AC neurons, expressing dTrpA1 receptors. Despite the separate sensory pathways for odor-temperature and odor-shock conditioning, both converge to one central pathway: the dopamine neurons, generally signaling reinforcement in the fly brain. Although a common population of dopamine neurons mediates both reinforcement types, the population mediating temperature reinforcement is smaller, and probably included within the population of dopamine neurons mediating shock reinforcement. I conclude that dopamine neurons integrate different noxious signals into a general aversive reinforcement pathway. Altogether, my results contribute to our understanding of aversive olfactory conditioning, demonstrating previously undescribed behavioral abilities of flies and their neuronal representations.

The mammalian olfactory system is strikingly shallow. While peripheral input in other sensory systems is sequentially processed by brainstem, midbrain, and thalamic nuclei before reaching primary sensory and associational cortices, olfactory input is processed by only a single region of the brain – the main olfactory bulb – before reaching higher cortical areas. A tremendous amount of neural processing is thus compressed within the main olfactory bulb, making this region of the brain uniquely well suited for investigating fundamental principles of neural processing. Currently, the identity and functional roles of multiple cell types and circuits within the main olfactory bulb remain almost entirely unknown, significantly limiting our understanding of olfaction. Herein, I describe a set of studies addressing this broad gap in knowledge. In Chapter 1, I introduce the known cellular and circuit components of the main olfactory bulb. In Chapter 2, I examine the complexity in biophysical cell-to-cell differences among mitral cells, a class of principal neurons in the main olfactory bulb, and quantify how this within-class diversity regulates mitral cell synchrony. In Chapter 3, I systematically explore synaptic and intrinsic biophysical properties to functionally establish mitral cells and tufted cells as two distinct classes of principal neurons in the main olfactory bulb. In Chapter 4, I reveal that disinhibitory circuitry mediated by a largely uncharacterized class of interneurons is widespread throughout the main olfactory bulb and critically involved in regulating the sensory-evoked activity of inhibitory granule cells. In Chapter 4 Appendix, I provide the first quantitative evidence for the morphological and functional subdivision of granule cells into two distinct classes that separately interact with mitral cells and tufted cells. In Chapter 5 and Chapter 5 Appendix, I molecularly identify a novel class of deep short-axon cells and show that this class of interneurons integrates centrifugal cholinergic input with broadly tuned sensory input and provides highly divergent synaptic output to dynamically regulate the balance of activity between mitral cells and tufted cells. Finally, in Chapters 6 and 7, I present general conclusions from these studies and provide a reappraisal of inhibitory circuitry within the main olfactory bulb.

Different neurons and glial cells in the Drosophila olfactory circuitry have distinct functions in olfaction. The mechanisms to generate most of diverse neurons and glial cells in the olfactory circuitry remain unclear due to the incomprehensive study of cell lineages. To facilitate the analyses of cell lineages and neural diversity, two independent binary transcription systems were introduced into Drosophila to drive two different transgenes in different cells. A technique called ‘dual-expression-control MARCM’ (mosaic analysis with a repressible cell marker) was created by incorporating a GAL80-suppresible transcription factor LexA::GAD (GAL4 activation domain) into the MARCM. This technique allows the induction of UAS- and lexAop- transgenes in different patterns among the GAL80-minus cells. Dual-expression-control MARCM with a ubiquitous driver tubP-LexA::GAD and various subtype-specific GAL4s which express in antennal lobe neurons (ALNs) allowed us to characterize diverse ALNs and their lineage relationships. Genetic studies showed that ALN cell fates are determined by spatial identities rooted in their precursor cells and temporal identities based on their birth timings within the lineage, and then finalized through cell-cell interactions mediated by Notch signaling. Glial cell lineage analyses by MARCM and dual-expression-control MARCM show that diverse post-embryonic born glial cells are lineage specified and independent of neuronal lineage. Specified glial lineages expand their glial population by symmetrical division and do not further diversify glial cells. Construction of a GAL4-insensitive transcription factor LexA::VP16 (VP16 acidic activation domain) allows the independent induction of lexAop transgenes in the entire mushroom body (MB) and labeling of individual MB neurons by MARCM in the same organism. A computer algorithm is developed to perform morphometric analysis to assist the study of MB neuron diversity.

Comment les odeurs contrôlent-elles le comportement animal ? Dans ma thèse, j'ai utilisé des manipulations optogénétiques et chimiogénétiques in vivo de l'activité neurale combinées à des analyses comportementales pour explorer l'organisation de circuits cérébraux impliqués dans des comportements olfactifs chez la souris. J'ai mis au point un test de conditionnement aversif olfactif indépendant de l'intensité des odeurs. J'ai démontré que les souris pouvaient généraliser une réponse aversive en présentant différentes concentrations d'odeurs. J’ai ensuite testé si les souris pouvaient apprendre cette tâche en inactivant les interneurones exprimant la parvalbumine dans le cortex olfactif (piriforme). J'ai trouvé que l’inactivation des cellules PV, n'était pas suffisante pour abolir l'aversion aux odeurs acquise, ce qui suggère que des composants de circuits neuronaux supplémentaires contribuent à la perception de l'odeur indépendamment de sa concentration. Ensuite, j'ai tenté de comprendre la constitution relative des différentes voies neurales du piriforme dans ce comportement d’aversion apprise. À l'aide d'outils génétiques et viraux, j'ai ciblé des sous-populations distinctes de neurones piriformes, et j'ai constaté que l'activité neurale induite par la lumière dans les cellules du piriforme projetant vers le bulbe olfactif et vers le cortex préfrontal, mais pas dans les cellules du piriforme projetant vers l’amygdale corticale et vers le cortex entorhinal latéral était suffisante pour supporter le conditionnement aversif. Ces résultats contribuent à mieux comprendre les propriétés fonctionnelles des circuits neuronaux corticaux pour l'olfaction<br>How do odors control animal behavior ? In my thesis, I have used in vivo optogenetic and chemogenetic manipulations of neural activity combined with behavioral analyses to explore the organization of brain circuits involved in olfactory behaviors in mice. In the first part of the thesis, I established an odor intensity-independent olfactory conditioning task. I demonstrated that mice were able to generalize a learned escape behavior across a range of different odor concentrations. I then tested if by silencing Parvalbumin-expressing interneurons in the olfactory (piriform) cortex, a candidate cell population for mediating odor concentration invariance, mice would fail to learn the task. I found that silencing PV cells was not sufficient to abolish learned aversion, suggesting that additional neural circuit components contribute to concentration-invariant odor perception. Next, I asked whether different piriform neural output pathways differed in their ability to support learned aversion. Using viral-genetic tools, I targeted distinct subpopulations of piriform neurons and I found that light-induced neural activity in only piriform principle cells could drive a behavioral response. Furthermore, I tested the sufficiency of subpopulations of piriform projection neurons to drive learned aversion. I found that photostimulation of olfactory bulb- and prefrontal cortex-projecting piriform neurons was sufficient to support aversive conditioning, but not the photostimulation of cortical amygdala- and lateral entorhinal cortex-projecting piriform neurons. Together, these results provide new insights into the functional properties of cortical neural circuits for olfaction

The conventional Von Neumann architecture imposes strict constraints on the development of intelligent adaptive systems. The requirements of substantial computing power to process and analyse complex data make such an approach impractical to be used in implementing smart systems. Neuromorphic engineering has produced promising results in applications such as electronic sensing, networking architectures and complex data processing. This interdisciplinary field takes inspiration from neurobiological architecture and emulates these characteristics using analogue Very Large Scale Integration (VLSI). The unconventional approach of exploiting the non-linear current characteristics of transistors has aided in the development of low-power adaptive systems that can be implemented in intelligent systems. The neuromorphic approach is widely applied in electronic sensing, particularly in vision, auditory, tactile and olfactory sensors. While conventional sensors generate a huge amount of redundant output data, neuromorphic sensors implement the biological concept of spike-based output to generate sparse output data that corresponds to a certain sensing event. The operation principle applied in these sensors supports reduced power consumption with operating efficiency comparable to conventional sensors. Although neuromorphic sensors such as Dynamic Vision Sensor (DVS), Dynamic and Active pixel Vision Sensor (DAVIS) and AEREAR2 are steadily expanding their scope of application in real-world systems, the lack of spike-based data processing algorithms and complex interfacing methods restricts its applications in low-cost standalone autonomous systems. This research addresses the issue of interfacing between neuromorphic sensors and digital neuromorphic circuits. Current interfacing methods of these sensors are dependent on computers for output data processing. This approach restricts the portability of these sensors, limits their application in a standalone system and increases the overall cost of such systems. The proposed methodology simplifies the interfacing of these sensors with digital neuromorphic processors by utilizing AER communication protocols and neuromorphic hardware developed under the Convolution AER Vision Architecture for Real-time (CAVIAR) project. The proposed interface is simulated using a JAVA model that emulates a typical spikebased output of a neuromorphic sensor, in this case an olfactory sensor, and functions that process this data based on supervised learning. The successful implementation of this simulation suggests that the methodology is a practical solution and can be implemented in hardware. The JAVA simulation is compared to a similar model developed in Nengo, a standard large-scale neural simulation tool. The successful completion of this research contributes towards expanding the scope of application of neuromorphic sensors in standalone intelligent systems. The easy interfacing method proposed in this thesis promotes the portability of these sensors by eliminating the dependency on computers for output data processing. The inclusion of neuromorphic Field Programmable Gate Array (FPGA) board allows reconfiguration and deployment of learning algorithms to implement adaptable systems. These low-power systems can be widely applied in biosecurity and environmental monitoring. With this thesis, we suggest directions for future research in neuromorphic standalone systems based on neuromorphic olfaction.

The mammalian central nervous system relies on precise synaptic connections to function correctly. The development of precise neuronal circuitry is regulated by axon guidance molecules as well as by specific pattern of activity between the pre and the post synaptic elements. In this thesis I focused on activity dependent mechanisms, and we analyzed the role of spontaneous afferent activity in the topographic organization of the olfactory bulb. To address this point we analyzed the intrabulbar connections between isofunctional glomeruli in a line of mice genetically modified to have very little spontaneous afferent activity due to the overexpression of the inward rectifying potassium channel Kir2.1 in the olfactory sensory neurons (Yu et al., 2004). Since previous studies were limited to adults (Bellusio et al., 2002; Lodovichi et al., 2003), we first defined the development of the intrabulbar projections between isofunctional glomeruli at early stages of development in control mice. Targeting focal tracer injections to the glomerular layer, we found that the intrabulbar projection is present as early as P7 and it is targeted between homologous glomeruli. However, at this early stage of development, the projection is not confined exclusively to the homologous glomerulus but larger. We found that the connections undergo a refinement process between P15 and P30, when they reach the mature organization of a point to point projection. We then analyzed the formation and the specific targeting of the intrabulbar link in animals with reduced spontaneous activity, i.e. Kir 2.1 mice. We found that the connections are preserved in these mice, but are not exclusively confined to the homologous glomeruli. The link remains larger than in control mice at all the ages tested, from postnatal day 30 to 70, due to the lack of developmental refinement. We then assess the effects of the unrefined connectivity of the bulbar circuits on olfactory behaviour using a classical behavioral test designed to assess the ability to discriminate between two different odorants. We found that Kir 2.1 mice were hampered in discriminating odorants that elicit similar spatial patterns of activated glomeruli (functional maps), such enantiomers, while retaining the ability to discriminate odorants that activate very distinct spatial pattern of glomeruli . Spontaneous activity is thought to play a prominent role in circuit formation at very early stages of development. Once sensory systems become responsive to sensory stimuli, evoked activity contributes to the stabilization and further refinement of neuronal connections. It has been clearly demonstrated that sensory experience often modulates the development of neuronal circuitry within a defined period of time (critical period) in which the brain is particularly plastic. Whether spontaneous activity can modulate synaptic connections in adult life, remains unknown. We addressed this topic in the olfactory system and we studied whether manipulation of afferent spontaneous activity in adulthood could affect the already established and refined synaptic contacts, i.e. the intrabulbar connections. Taking advantage of the inducible nature of the Kir 2.1 construct, we allow the expression of the Kir 2.1 channels only in adulthood (P30-P60), for 30 days. We found that the expression of the Kir 2.1 channel in adults was able to induce a regression of the intrabulbar link to an unrefined and enlarged status.<br>Il corretto funzionamento del sistema nervoso centrale dei mammiferi si basa sulla specificità delle connessioni sinaptiche. Lo sviluppo di un circuito neurale è regolato sia da molecole specifiche, dette axon guidance molecules che da precisi schemi di attività elettrica che si stabiliscono tra i neuroni pre e post-sinaptici. In questo lavoro afferente spontanea nell' organizzazione topografica del bulbo olfattivo. Per raggiungere tale obiettivo abbiamo analizzato la connessione intrabulbare tra glomeruli isofunzionali (o omologhi) in una linea di topi geneticamente modificati, nei quali l' attività afferente spontanea è estremamente ridotta a causa dell'over-espressione di un canale potassio inward rectifier (Kir2.1) nei neuroni sensoriali olfattivi (Yu et al., 2004). Poichè studi precedenti avevano analizzato la connessione intrabulbare solo in animali adulti (Belluscio et al. 2002; Lodovichi et al., 2003), abbiamo dapprima studiato lo sviluppo della connessione intrabulbare tra glomeruli omologhi, a diversi stadi di sviluppo (P7-P70), in animali di controllo. Effettuando iniezioni di un tracciante fluorescente nello strato dei glomeruli, abbiamo trovato che la connessione intrabulbare tra glomeruli omologhi è presente già a una settimana di vita post-natale (P7). Tuttavia a questo stadio di sviluppo, la connessione non è circoscritta esclusivamente al glomerulo omologo corrispondente, ma più ampia. In questa fase il rapporto tra il diametro dell' iniezione e l' estensione della proiezione è 4:1. Abbiamo trovato che la connessione va incontro ad un processo di maturazione tra P15 e P30. In questo periodo si osserva un restringimento dell' estensione della proiezione che assume le dimensioni circoscritte al singolo glomerulo omologo, tale per cui il rapporto tra l' estensione della proiezione ed il diametro dell' iniezione è 1:1, proprio della connessione matura. Le dimensioni della connessione si mantengono stabili a tutti gli stadi esaminati, sino a P70. Abbiamo quindi analizzato il ruolo dell'attività afferente spontanea nella formazione e nel mantenimento della connessione intrabulbare, in animali con ridotta attività afferente spontanea, i topi Kir 2.1. Abbiamo visto che le connessioni intrabulbari sono ancora presenti in questi animali, sebbene non siano esclusivamente circoscritte ai glomeruli omologhi. Il rapporto tra l' estensione della proiezione ed il diametro dell' iniezione non raggiunge mai il valore di 1:1 , proprio della connessione matura, a nessuna delle età analizzate (P30-P70) in animali con ridotta attività afferente. In questi animali il processo di maturazione non sembra mai completarsi. Ci siamo poi chiesti quali fossero le ripercussioni di un' alterata circuiteria neurale nel bulbo olfattivo sul comportamento olfattivo. A tale scopo abbiamo utilizzato un test comportamentale classico elaborato per valutare la capacità di un animale di discriminare tra due differenti odori. Abbiamo osservato che gli animali con ridotta attività afferente non riuscivano a discriminare gli odori che attivano pattern spaziali di glomeruli (mappe funzionali) simili, quali gli enantiomeri, mentre mantenevano la capacità di discriminare odori in grado di attivare pattern spaziali di glomeruli molto distinti. Molti studi sulla formazione dei circuiti neurali nei sistemi sensoriali hanno dimostrato che l' attività spontanea gioca un ruolo chiave nella formazione dei circuiti in stadi molto precoci dello sviluppo. Una volta che i sistemi sensoriali diventano sensibili agli stimoli sensoriali specifici, l' attività elettrica evocata rafforza e completa la maturazione dei già stabiliti contatti sinaptici. E' stato chiaramente dimostrato che l' attività sensoriale modula lo sviluppo della circuiteria neurale all' interno di un periodo circoscritto (periodo critico), in cui il sistema nervoso centrale è estremamente plastico. Se l' attività spontanea abbia un ruolo sui circuiti neurali in età adulta rimaneva oscuro. Dato l' alto grado di plasticità del sistema olfattivo ci siamo chiesti se la manipolazione dell' attività elettrica spontanea in età adulta potesse modificare architetture neurali già consolidate, come ad esempio le connessioni intrabulbari. Sfruttando la natura inducibile del costrutto Kir 2.1, abbiamo permesso l' espressione del canale Kir 2.1 solamente in età adulta (P30-P60), per 30 giorni. Abbiamo scoperto che l' assenza di attività spontanea solo in età adulta è in grado di indurre la regressione della connessione intrabulbare ad uno stadio immaturo (la connessione risulta per tanto più¹ larga). Tutti assieme questi dati indicano che l'attività afferente spontanea gioca un ruolo critico non solo nella formazione dei circuiti nervosi del bulbo olfattivo, ma anche nel loro corretto mantenimento. Inoltre la modificata architettura della circuiteria bulbare si riflette in un comportamento olfattivo alterato.

Mestrado em Biologia Molecular e Celular<br>Comprehending the mechanisms that make lifelong neurogenesis possible has a clear interest for the better understanding of the basic principles that govern cellular and molecular interactions in the nervous system, as well as a relevant clinical interest. The limited ability of the central nervous system to generate new neurons in order to replace those that have been lost is a formidable obstacle to recovery from neuronal damage caused by injury or neurodegenerative disease. The olfactory system (OS) is an ideal system to study the process of neuronal recovery after injury, as it is known for its lifelong capacity to replenish cells lost during natural turnover, as well as its remarkable ability to regenerate after severe lesion. The olfactory epithelium (OE) shows neurogenesis throughout life. Newly differentiated olfactory receptor neurons (ORNs) are continuously reintegrated into an existing circuitry to maintain the sense of smell. The aim of this thesis is to describe the morphological and functional alterations that occur over time in the OS of larval Xenopus laevis, after transection of the olfactory nerve (ON). Results obtained using immunohistochemistry essays, as well as sensory neuron labeling and calcium imaging techniques, indicate that ORN cell death reaches its peak 48 hours after transection, and that proliferating stem cells found in the basal cell layer of the OE are quickly upregulated after lesion. Supporting cells seem to maintain both morphological and functional integrity after transection of the ON. The OE recovers its original morphological structure 1 week after transection, at which time the first axons reach the olfactory bulb (OB) and begin the process of reinnervation. Spontaneous activity of mitral/tufted cells occurs in the OB during the first weeks after transection but no odor-induced activity is observed. After 3-4 weeks glomerular responses were observed in some animals upon application of stimulus, but the response and glomerular morphology are clearly altered as compared to control. After 6-7 weeks responses seem to have fully recovered, indicating that the OS of larval X. laevis recovers morphologically and functionally 6-7 weeks after ON transection.<br>O estudo dos mecanismos responsáveis pela neuro-regeneração tem um marcado interesse para a compreensão dos princípios básicos que governam as interações celulares e moleculares no sistema nervoso, bem como um interesse clínico relevante. A limitada capacidade do sistema nervoso central para dar origem a novos neurónios é um obstáculo formidável para a recuperação do sistema após lesão neuronal ou doença neurodegenerativa. O sistema olfativo é um sistema ideal para o estudo do processo de recuperação após lesão neuronal, pois é conhecido no mundo científico pela sua capacidade contínua e vitalícia para repor células perdidas durante a renovação celular natural, bem como a sua notável capacidade para regenerar após uma lesão grave. O epitélio olfativo apresenta a capacidade para dar origem a novos neurónios ao longo de toda a vida. Neurónios sensoriais olfativos diferenciados são continuamente reintegrados num circuito já existente, mantendo assim o sentido do olfato. O objetivo desta tese é descrever as alterações morfológicas e funcionais que ocorrem ao longo do tempo no sistema olfativo de Xenopus laevis em estado larvar, após o corte do nervo olfativo. Os resultados obtidos através do uso de ensaios de imunohistoquímica, bem como técnicas de marcação neuronal sensorial e de imagiologia de cálcio, indicam que a morte celular na população de neurónios sensoriais olfativos atinge o seu máximo 48 horas após a lesão, e que células estaminais encontradas na camada basal do epitélio olfativo são positivamente reguladas após lesão e proliferam rapidamente. Células de suporte parecem manter tanto a integridade morfológica como funcional após o corte do nervo olfativo. O epitélio olfativo recupera a sua estrutura morfológica inicial 1 semana após a lesão, momento em que os primeiros axónios atingem o bolbo olfativo e começam o processo de reintegração. Ocorre atividade espontânea das células mitrais/tufados do bolbo olfativo durante as primeiras semanas após a lesão, mas nenhuma atividade induzida por estímulo com odor foi observada. Depois de 3-4 semanas, atividade glomerular foi observada em alguns animais após a aplicação de estímulos, mas a resposta e morfologia glomerular foram claramente alteradas em relação ao controlo. Depois de 6-7 semanas as respostas parecem ter recuperado totalmente, indicando que o sistema olfativo de X. laevis em estado larvar recupera morfológica e funcionalmente 6-7 semanas após o corte do nervo olfativo.

The vomeronasal system (VNS) is a chemosensory system designed for the detection of chemical messengers dissolved in bodily secretions which help to identify kin, conspecifics, and predators in the animal‟s environment. Although a growing body of evidence over the last two decades has revealed a very important role for the VNS in many stereotypical behaviors which are essential to the animal‟s survival, our understanding of the detailed organization of the circuitry of this system, and its development, remains limited. Consequently, the goal of this dissertation is to examine the development of connectivity from the vomeronasal organ to the accessory olfactory bulb to determine the fundamental principles of adult connectivity in this circuit, and to manipulate sensory activity to understand the role that activity might play in anatomical development of vomeronasal projections to their targets in the accessory olfactory bulb. There are several anatomical challenges we faced when trying to visualize connectivity in a select population of neurons in the VNS. We utilized two tools to overcome some of these barriers. First, we took advantage of a transgenic mouse line whose vomeronasal sensory neurons (VSNs) expressing the V2r1b-receptor also expressed tau-GFP to visualize a specific population of sensory neurons in the vomeronasal organ (VNO) and their projections in to the accessory olfactory bulb (AOB). Second we developed a local electroporation technique enabling us to label specific populations of mitral cells in the AOB receiving common input. This method is described in detail in Chapter 2 where we show the versatility of this method, not only for use in the AOB, but in many other brain systems and with a diverse set of dyes and calcium indicators. We exploited these two tools to examine both the gross anatomical development of the VNO and the AOB, as well as the development of connectivity between them in Chapter 3. Our results show that the first few post-natal weeks represent a dramatic period of growth in both theVNO and the AOB. In addition, we show that axons of VSNs expressing the V2r1b-receptor undergo a striking and rapid period of refinement and coalescence during the first four post-natal days of the animal‟s life. Subsequently, mitral cell dendrites, which initially promiscuously send out multiple dendritic branches, begin to ramify and form tufts in specific glomeruli following this period of axonal refinement into well-defined glomeruli. Finally, our results support the hypothesis that mitral cells precisely project their dendrites to target glomeruli receiving input from sensory neurons expressing the same receptor type. Finally in Chapter 4, we explored the role of activity in modulating the development described in Chapter 3. We demonstrate that the duct connecting the VNO to the external world is indeed open and thus that external ligands can access the VNO and bind to VSNs, as early as postnatal day 0. Further, we demonstrate that VSNs are capable or releasing neurotransmitter onto mitral cells at these early postnatal ages. These results suggested that early activity in this system might help regulate development. To test this hypothesis we needed a ligand that would activate a known subset of VSNs. We determined the concentration of a major histocompatibility complex peptide known to activate V2r1b-receptors and found it to be more than 1,000 fold above what would likely activate these sensory neurons. We show that these peptides are capable of inducing immediate early genes in downstream mitral cells and then employed the use of two of these peptides to selectively manipulate the activity of sensory neurons expressing the V2r1b-receptor. Although strong sensory activity did not affect the number of VSNs expressing the V2r1b-receptor, it did significantly alter axonal refinement and coalescence in AOB, resulting in delayed pruning and formation of well-defined glomeruli. Taken together, the results from the chapters in this dissertation suggest that connectivity between mitral cells and sensory neurons in the vomeronasal organ is specifically targeted and that early activity may influence the development of this circuit to ensure proper connectivity providing a substrate for information processing in this system.

In the sensory systems, peripheral neurons project axons in specific loci of the brain. The spatial segregation of the sensory afferents provides topographic maps that define the quality and the location of complex sensory stimuli. Electrical activity plays a critical role in the formation of specific synaptic contacts among neurons, although the type of activity required remains a matter of significant debate. In particular the role of spontaneous activity in the formation of the topographic organization of the olfactory system remains unknown. To address this question we investigated the role of spontaneous electrical activity in circuit formation and function in the olfactory bulb. To accomplish this goal, we took advantage of a line of mice engineered to have very little afferent spontaneous activity due to the over expression of the inward rectifying potassium channel Kir2.1 in the olfactory sensory neurons (Kir2.1 mice). We analyzed the formation of the sensory map, in particular whether the convergence of olfactory sensory neurons expressing the same odorant receptor took place properly in mice with reduced afferent spontaneous activity. The conflation of sensory axons to form homogeneous glomeruli, i.e. glomeruli formed exclusively by axons expressing the same olfactory receptor, in specific loci of the olfactory bulb is a critical feature of the sensory map that in turn defines functional units, i.e. odor columns. We found that in absence of spontaneous activity, the convergence of sensory neurons to form homogenous glomeruli took place but it was coarser than in controls. In particular we observed axons mistargeting that resulted in multiple heterogeneous glomeruli that persist also in adulthood. To ascertain the role of spontaneous activity on the post synaptic elements of the olfactory bulb, we analyzed the mitral cells, the principal output neurons, and the granule cells, the major component of the inhibitor interneurons of the olfactory bulb. We found no difference in the developmental refinement of the apical dendrite of mitral cells in Kir2.1 and in control mice. The neurogenesis and the migration of granule cells was unaltered. However the filopodia-spine density on the dendritic tree of the granule cells was significantly reduced in Kir2.1 mice. To analyze the functional outcome of these anatomical alterations, we performed behaviour experiments. We demonstrated that Kir2.1 mice were unable to discriminate between two odors, such as couple of enantiomers, that elicit very similar spatial patterns of activated glomeruli, while retained the ability to differentiate between odorants, such as 2-methylbutyric acid and cyclobutanecarboxylic acid (2Mb and CB) that activate patterns of glomeruli spatially very distinct. Due to the high degree of plasticity of the olfactory system, we asked whether manipulation of electrical activity in adulthood could affect the already refined neural circuitry in the olfactory bulb. Taking advantage of the inducible nature of the Kir2.1 construct, we allowed the expression of the Kir2.1 channels only in adulthood, for 4 weeks. We found that the expression of the Kir2.1 channel in adults disrupted the organization of the sensory map, namely the convergence of olfactory sensory neuron axons. The absence of spontaneous afferent activity in adults induced a regression in the glomeruli organization. We found supernumerary heterogeneous glomeruli that coexist with the main homogeneous glomeruli. All together our data suggest that spontaneous activity is required for the developmental refinement and maintenance of the sensory map. Furthermore we found that the unrefined connectivity of the neural circuitry of the olfactory bulb could affect olfactory discrimination behaviour.<br>Nei sistemi sensoriali, i neuroni periferici proiettano i loro assoni in specifici loci del cervello. La segregazione spaziale delle afferenze sensoriali provvede a creare mappe topografiche che definiscono la qualità e la localizzazione di complessi stimoli sensoriali. L’attività elettrica gioca un ruolo chiave nella formazione di specifici contatti sinaptici tra i neuroni, sebbene resti ancora da definire il tipo di attività richiesta. In particolare il ruolo dell’attività elettrica spontanea nell’organizzazione topografica del sistema olfattivo, non è noto. Per rispondere a questa domanda abbiamo studiato il ruolo dell’attività elettrica spontanea nella formazione e nella funzione dei circuiti neurali nel bulbo olfattivo. Per raggiungere questo obiettivo abbiamo utilizzato una linea di topi geneticamente modificati, nei quali l’attività afferente spontanea è ridotta a causa della sovra-espressione di un canale potassio inward rectifier (Kir2.1), in tutti i neuroni olfattivi sensoriali (i topi Kir2.1). Abbiamo analizzato la formazione della mappa sensoriale, in particolare se la convergenza dei neuroni sensoriali esprimenti il medesimo recettore olfattivo avveniva correttamente nei topi Kir2.1 La convergenza dei neuroni sensoriali in specifici loci del bulbo olfattivo, che porta alla formazione di glomeruli omogenei, cioé glomeruli formati esclusivamente da assoni esprimenti lo stesso recettore olfattivo, è una caratteristica critica della mappa sensoriale. Infatti i glomeruli definiscono le unità funzionali o colonne odorose del sistema. Abbiamo trovato che in assenza di attività spontanea, gli assoni dei neuroni sensoriali non convergono a formare un unico glomerulo ma proiettano in molti siti dando luogo a ulteriori glomeruli. Questi addizionali glomeruli sono caratterizzati da una organizzazione eterogenea, risultano cioè formati da assoni di neuroni sensoriali esprimenti recettori olfattivi diversi. Per capire se l’attività afferente spontanea potesse avere un ruolo anche sulle cellule postsinaptiche del bulbo olfattivo, abbiamo analizzato le cellule mitrali, i principali neuroni di output, e le cellule dei granuli, i principali neuroni inibitori, del bulbo olfattivo. Analizzando lo sviluppo morfologico del dendrite apicale delle cellule mitrali non abbiamo trovato alcuna differenza significativa nei topi Kir2.1 rispetto ai controlli. Per quanto concerne le cellule dei granuli, studiando la neurogenesi e la migrazione delle cellule dei granuli di nuova generazione, non abbiamo riscontrato differenze significative nei topi Kir2.1 rispetto ai controlli. Tuttavia l’analisi morfologica dell’arborizzazione dendritica delle cellule dei granuli ha messo in evidenza una ridotta densità di filopodi/spine nei topi Kir2.1 rispetto ai controlli. Per analizzare le conseguenze funzionali di queste alterazioni anatomiche abbiamo eseguito specifici test comportamentali. I dati che abbiamo ottenuto indicano chiaramente che i topi Kir2.1 non erano in grado di discriminare tra due odori che attivano glomeruli che hanno distribuzione spaziale molto simile, quali gli enantiomeri. Tuttavia i topi Kir2.1 mantenevano la capacità di distinguere odori che attivano glomeruli posti in aree molto diverse del bulbo, quali l’acido 2-metilbutirrico e l’acido ciclobutancarbossilico (2Mb e CB). Dato l’elevato grado di plasticità del sistema olfattivo, ci siamo chiesti se la manipolazione dell’attività elettrica in età adulta poteva influenzare la mappa sensoriale. Sfruttando la possibilità di indurre l’espressione del gene Kir2.1 in momenti diversi della vita dell’animale, abbiamo fatto esprimere il gene Kir2.1 solo in animali adulti per 4 settimane. Abbiamo trovato che l’espressione del gene Kir2.1 in animali adulti alterava l’organizzazione della mappa sensoriale, cioé la specifica convergenza degli assoni dei neuroni sensoriali nel bulbo olfattivo. I dati ottenuti indicano che l’assenza di attività spontanea nell’età adulta causa una “regressione“ nell’organizzazione dei glomeruli. Abbiamo infatti trovato un elevato numero di glomeruli eterogenei che coesistevano coi principali glomeruli omogenei. I nostri dati suggeriscono che l’attività elettrica spontanea è richiesta per lo sviluppo e il mantenimento della mappa sensoriale. Inoltre abbiamo trovato che le alterazioni morfologiche della circuiteria neuronale nel bulbo olfattivo contribuiscono ad alterare il comportamento olfattivo.

This thesis consists of three major chapters, each of which has been separately published or under the process for publication. The first chapter is about anatomical characterization of the mushroom body of adult Drosophila melanogaster. The mushroom body is the center for olfactory learning and many other functions in the insect brains. The functions of the mushroom body have been studied by utilizing the GAL4/UAS gene expression system. The present study characterized the expression patterns of the commonly used GAL4 drivers for the mushroom body intrinsic neurons, Kenyon cells. Thereby, we revealed the numerical composition of the different types of Kenyon cells and found one subtype of the Kenyon cells that have not been described. The second and third chapters together demonstrate that the multiple types of dopaminergic neurons mediate the aversive reinforcement signals to the mushroom body. They induce the parallel memory traces that constitute the different temporal domains of the aversive odor memory. In prior to these chapters, “General introduction and discussion” section reviews and discuss about the current understanding of neuronal circuit for olfactory learning in Drosophila<br>Diese Dissertation umfasst drei Kapitel. Das erste Kapitel handelt von der anatomischen Charakterisierung des Pilzkörpers in adulten Drosophila melanogaster. Der Pilzkörper ist das Zentrum für olfaktorisches Lernen und viele andere Funktionen im Insektengehirn. Diese wurden mit Hilfe des GAL4/UAS Genexpressionssystems untersucht. Die vorliegende Arbeit charakterisiert die Expressionsmuster der gewöhnlich verwendeten GAL4 Treiberlinien für die Pilzkörperintrinsischen Neurone, den Kenyonzellen. Dabei zeigten ich die zahlenmäßige Zusammensetzung der unterschiedlichen Kenyonzelltypen und fanden einen Kenyonzellsubtyp, welcher bisher noch nicht beschrieben wurde. Das zweite und dritte Kapitel zeigen, dass verschiedene Typen dopaminerger Neurone aversive Verstärkungssignale (Unkonditionierte Stimuli) zum Pilzkörper übermitteln. Sie induzieren parallele Gedächtnisspuren, welche den unterschiedlichen zeitlichen Komponenten von aversivem Duftgedächtnis zugrunde liegen. Vor diesen Kapiteln enthält der Abschnitt „General introduction and discussion” einen Überblick und eine Diskussion über das derzeitige Verständnis des neuronalen Netzwerks, welches olfaktorischem Lernen in Drosophila zugrunde liegt

The mouse olfactory system detects odorants with 1000 olfactory receptors (ORs). Olfactory sensory neurons (OSNs) express only 1 OR. OSNs expressing a common OR converge on a single glomerulus, a stereotyped location in the olfactory bulb (OB). Thus, odorants are represented by a spatial map of glomerular activation. OB odor representations are then processed by five central brain regions. One region, cortical amygdala (CoA), receives spatially patterned and stereotyped axonal input from the OB and is both necessary and sufficient for innate behavioral responses to odor. However, CoA receives input from all glomeruli and forms a representation of every odor. It is not known why all odors are represented in CoA or how some odor representations elicit behavior while others do not. One hypothesis is that only rare neurons in CoA, not activated by most odors, participate in innate signaling. Another hypothesis is that all neurons in CoA participate in innate signaling, but for many odors, opposing CoA outputs cancel out downstream. These hypotheses were addressed by single nuclei sequencing and in situ hybridization which identified and localized neuronal cell types within CoA. Cell types are topographically segregated in regions well positioned to differentially receive inputs from genetically defined subsets of glomeruli. Therefore, the connectivity between OB and CoA may instantiate a genetically wired circuit from OB to cortex for innate odor processing. A number of rare and common cell types were identified. Thus, CoA may process two types of innate signals: (1) specific innate signals, produced by few glomeruli and processed by rare CoA cell types; (2) broad innate signals, produced by many glomeruli and processed by common CoA cell types through the integration of probabilistic information about the value of odorants.

<p>Raw sensory information is usually processed and reformatted by an organism’s brain to carry out tasks like identification, discrimination, tracking and storage. The work presented in this dissertation focuses on the processing strategies of neural circuits in the early olfactory system in two insects, the locust and the fruit fly. </p> <p> Projection neurons (PNs) in the antennal lobe (AL) respond to an odor presented to the locust’s antennae by firing in slow information-carrying temporal patterns, consistent across trials. Their downstream targets, the Kenyon cells (KCs) of the mushroom body (MB), receive input from large ensembles of transiently synchronous PNs at a time. The information arrives in slices of time corresponding to cycles of oscillatory activity originating in the AL. </p> <p> In the first part of the thesis, ensemble-level analysis techniques are used to understand how the AL-MB system deals with the problem of identifying odors across different concentrations. Individual PN odor responses can vary dramatically with concentration, but invariant patterns in PN ensemble responses are shown to allow odor identity to be extracted across a wide range of intensities by the KCs. Second, the sensitivity of the early olfactory system to stimulus history is examined. The PN ensemble and the KCs are found capable of tracking an odor in most conditions where it is pulsed or overlapping with another, but they occasionally fail (are masked) or reach intermediate states distinct from those seen for the odors presented alone or in a static mixture. </p> <p> The last part of the thesis focuses on the development of new recording techniques in the fruit fly, an organism with well-studied genetics and behavior. Genetically expressed fluorescent sensors of calcium offer the best available option to study ensemble activity in the fly. Here, simultaneous electrophysiology and two-photon imaging are used to estimate the correlation between G-CaMP, a popular genetically expressible calcium sensor, and electrical activity in PNs. The sensor is found to have poor temporal resolution and to miss significant spiking activity. More generally, this combination of electrophysiology and imaging enables explorations of functional connectivity and calibrated imaging of ensemble activity in the fruit fly. </p>

La dépression est une maladie chronique, récurrente et potentiellement mortelle qui affecte plus de 20 % de la population à travers le monde. Les mécanismes sous-jacents de la dépression demeurent incompris et la pharmacothérapie actuelle, largement basée sur l’hypothèse monoaminergique, fait preuve d’une efficacité sous optimale et d’une latence thérapeutique élevée. Par conséquent, la recherche est amenée à élaborer de nouveaux traitements pharmacologiques. Pour détecter leur action, il est avant tout nécessaire de développer des outils expérimentaux adéquats. Dans cette optique, notre but a été de mesurer l’anhédonie, un symptôme cardinal de la dépression, chez le rat de laboratoire. L’anhédonie a été définie comme une réduction de la récompense et a été mesurée avec le test de consommation de sucrose et la technique d’autostimulation intracérébrale. En vue d’induire l’anhédonie, nous avons effectué une bulbectomie olfactive, une procédure qui entraîne divers changements biochimiques, cellulaires et comportementaux similaires à ceux de l’état dépressif et qui peuvent être renversés par un traitement antidépresseur chronique. Nos résultats montrent que la bulbectomie olfactive produit également l’anhédonie, reflétée par une réduction durable de la consommation de sucrose et par une réduction de l’efficacité de l’amphétamine dans le test d’autostimulation intracérébrale. Ces effets ont été présents jusqu’à trois à quatre semaines suivant la chirurgie. La bulbectomie olfactive a aussi été associée à une augmentation de l’élément de réponse liant l’AMPc dans le striatum, un index moléculaire associé à l’anhédonie. Ces découvertes suggèrent que l’anhédonie peut être produite et étudiée de façon fiable dans le modèle de bulbectomie olfactive et que le circuit de récompense pourrait constituer une cible cohérente pour de nouvelles drogues en vue du traitement de la dépression.<br>Depression is a chronic, recurrent and potentially deadly disorder that affects over 20 % of the population worldwide. The mechanisms underlying depression are still not understood and current pharmacotherapy, based largely on monoaminergic hypotheses, is plagued by suboptimal efficacy and delayed therapeutic latency. This has lead to a search for novel pharmacological treatments. To achieve this, it is first necessary to develop adequate experimental tools. With this in mind, we aimed to measure anhedonia, a cardinal symptom of depression, in laboratory rats. We defined anhedonia as a reduction in reward, and measured it with the sucrose intake test and in the intracranial self-stimulation paradigm. In order to induce anhedonia, we surgically removed the olfactory bulbs, a procedure that results in a host of behavioral, cellular and biochemical changes that are qualitatively similar to those observed in clinical depression. These changes are long-lasting and reversed by chronic antidepressant treatment, validating olfactory bulbectomy as an animal model of depression. Our results show that olfactory bulbectomy also produces anhedonia, reflected by a stable and long-lasting reduction in sucrose intake as well as a reduction in the rewarding effectiveness of amphetamine in the self-stimulation paradigm. These effects were present even after three to four weeks post-surgery. Olfactory bulbectomy was also associated with increased striatal cAMP response element binding, a molecular index associated with depressive-like behaviour. These findings suggest that anhedonia can be reliably produced and studied within the olfactory bulbectomy model and that reward circuitry may comprise a logical target for novel drugs to treat depression.

For many organisms the sense of smell is critical to survival. Some olfactory stimuli elicit innate responses that are mediated through hardwired circuits that have developed over long periods of evolutionary time. Most olfactory stimuli, however, have no inherent meaning. Instead, meaning must be imposed by learning during the lifetime of an organism. Despite the dominance of olfactory stimuli on animal behavior, the mechanisms by which odorants elicit learned behavioral responses remain poorly understood. All odor-evoked behaviors are initiated by the binding of an odorant to olfactory receptors located on sensory neurons in the nasal epithelium. Olfactory sensory neurons transmit this information to the olfactory bulb via spatially organized axonal projections such that individual odorants evoke a stereotyped map of bulbar activity. A subset of bulbar neurons, the mitral and tufted cells, relay olfactory information to higher brain structures that have been implicated in the generation of innate and learned behavioral responses, including the cortical amygdala and piriform cortex. Anatomical studies have demonstrated that the spatial stereotypy of the olfactory bulb is maintained in projections to the posterolateral cortical amygdala, a structure that is involved in the generation of innate odor-evoked responses. The projections of mitral and tufted cells to piriform cortex however appear to discard the spatial order of the olfactory bulb: each glomerulus sends spatially diffuse, apparently random projections across the entire cortex. This anatomy appears to constrain odor-evoked responses in piriform cortex: electrophysiological and imaging studies demonstrate that individual odorants activate sparse ensembles that are distributed across the extent of cortex, and individual piriform neurons exhibit discontinuous receptive fields such that they respond to structurally and perceptually similar and dissimilar odorants. It is therefore unlikely that olfactory representations in piriform have inherent meaning. Instead, these representations have been proposed to mediate olfactory learning. In accord with this, lesions of posterior piriform cortex prevent the expression of a previously acquired olfactory fear memory and photoactivation of a random ensemble of piriform neurons can become entrained to both appetitive and aversive outcomes. Piriform cortex therefore plays a central role in olfactory fear learning. However, how meaning is imparted on olfactory representations in piriform remains largely unknown. We developed a strategy to manipulate the neural activity of representations of conditioned and unconditioned stimuli in the basolateral amygdala (BLA), a downstream target of piriform cortex that has been implicated in the generation of learned responses. This strategy allowed us to demonstrate that distinct neural ensembles represent an appetitive and an aversive unconditioned stimulus (US) in the BLA. Moreover, the activity of these representations can elicit innate responses as well as direct Pavlovian and instrumental learning. Finally activity of an aversive US representation in the basolateral amygdala is required for learned olfactory and auditory fear responses. These data suggest that both olfactory and auditory stimuli converge on US representations in the BLA to generate learned behavioral responses. Having identified a US representation in the BLA that receives convergent olfactory information to generate learned fear responses, we were then able to step back into the olfactory system and demonstrate that the BLA receives olfactory input via the monosynaptic projection from piriform cortex. These data suggest that aversive meaning is imparted on an olfactory representation in piriform cortex via reinforcement of its projections onto a US representation in the BLA. The work described in this thesis has identified mechanisms by which sensory stimuli generate appropriate behavioral responses. Manipulations of representations of unconditioned stimuli have identified a central role for US representations in the BLA in connecting sensory stimuli to both innate and learned behavioral responses. In addition, these experiments have suggested local mechanisms by which fear learning might be implemented in the BLA. Finally, we have identified a fundamental transformation through which a disordered olfactory representation in piriform cortex acquires meaning. Strikingly this transformation appears to occur within 3 synapses of the periphery. These data, and the techniques we employ, therefore have the potential to significantly impact upon our understanding of the neural origins of motivated behavior.

The clustered protocadherins (Pcdh α, β & γ) provide individual neurons with cell surface diversity. However, the importance of Pcdh mediated diversity in neural circuit assembly and how it may promote neuronal connectivity remains largely unknown. Moreover, to date, Pcdh in vivo function has been studied at the level of individual gene clusters; whole cluster-wide function has not been addressed. Here I examine the role of all three Pcdh gene clusters in olfactory sensory neurons (OSNs); a neuronal type that expressed all three types of Pcdhs and in addition I address the role of Pcdh mediate diversity in their wiring. When OSNs share a dominant single Pcdh identity (α, β & γ) their axons fail to form distinct glomeruli, suggestive of inappropriate self-recognition of neighboring axons (loss of non-self-discrimination). By contrast, deletion of the entire α, β,γ Pcdh gene cluster, but not of each individual cluster alone, leads to loss of self-recognition and self-avoidance thus, OSN axons fail to properly arborize. I conclude that Pcdh-expression is necessary for self-recognition in OSNs, whereas its diversity allows distinction between self and non-self. Both of these functions are required for OSNs to connect and assembly into functional circuits in the olfactory bulb. My results, also reveal neuron-type specific differences in the requirement of specific Pcdh gene clusters and demonstrate significant redundancy between Pcdh isoforms in the olfactory system.

Animals have evolved sensory systems that afford innate and adaptive responses to stimuli in the environment. Innate behaviors are likely to be mediated by hardwired circuits that respond to invariant predictive cues over long periods of evolutionary time. However, most stimuli do not have innate value. Over the lifetime of an animal, learning provides a mechanism for animals to update the predictive value of cues through experience. Sensory systems must therefore generate neuronal representations that are able to acquire value through learning. A fundamental challenge in neuroscience is to understand how and where value is imposed in brain during learning. The olfactory system is an attractive sensory modality to study learning because the anatomical organization is concise in that there are relatively few synapses separating the sense organ from brain areas implicated in learning. Thus, the circuits for learned olfactory behaviors appear to be relatively shallow and therefore more experimentally accessible than other sensory systems. The goal of this thesis is to characterize the representation and function of neural circuits involved in olfactory associative learning. Odor perception is initiated by the binding of odors onto olfactory receptors expressed in the sensory epithelium. Each olfactory receptor neuron (ORN) expresses one of 1500 different receptor genes, the expression of which pushes the ORN to project with spatial specificity onto a defined loci within the olfactory bulb, the olfactory glomeruli. Therefore, each and every odor evokes a stereotyped map of glomerular activity in the bulb. The projection neurons of the olfactory bulb, mitral and tufted (M/T) cells, send axons to higher brain areas, including a significant input to the primary olfactory cortex, the piriform cortex. Axons from M/T cells project diffusely to the piriform without apparent spatial preference; as a consequence, the spatial order of the bulb is discarded in the piriform. In agreement with anatomical data, electrophysiological and optical imaging studies also demonstrate that individual odorants activate sparse subsets of neurons across the piriform without any spatial order. Moreover, individual piriform neurons exhibit discontinuous receptive fields that defy chemical or perceptual categorization. These observations suggests that piriform neurons receive random subsets of glomerular input. Therefore, odor representations in piriform are unlikely to be hardwired to drive specific behaviors. Rather, this model suggests that value must be imposed upon the piriform through learning. Indeed, the piriform has been shown to be both sufficient and necessary for aversive olfactory learning without affecting innate odor responses. However, how value is imposed on odor representations in the piriform and downstream associational areas remain largely unknown. We first developed a strategy to track neural activity in a population of neurons across multiple days in deep brain areas using 2-photon endoscopic imaging. This allowed us to assay changes in neural responses to odors during learning in piriform and in downstream associative areas. Using this technique, we first observe that piriform odor responses are unaffected by learning, so learning must therefore impose discernable changes in neural activity downstream of piriform. Piriform projects to multiple downstream areas that are implicated in appetitive associative learning, such as the orbitofrontal cortex (OFC). Imaging of neural activity in the OFC reveal that OFC neurons acquire strong responses to conditioned odors (CS+) during learning. Moreover, multiple and distinct CS+ odors activatethe same population of OFC neurons, and these responses are gated by context and internal state. Together, our imaging data shows that an external and sensory representation in the piriform is transformed into an internal and cognitive representation of value in the OFC. Moreover, we found that optogenetic silencing of the OFC impaired the ability of mice to acquire learned associations. Therefore, the robust representation of expected value of the odor cues is necessary for the formation of appetitive associations. We made an important observation: once the task has been learned with a set of odors, the OFC representation decays after learning has plateaued and remains silent even when mice encounter novel odors they haven’t previously experienced. Moreover, silencing the OFC when it was not actively engaged during the subsequent learning of new odors had no effect on learning. These sets of imaging and silencing experiments reveal that the OFC is only important during initial learning; once task structure has been acquired, it is no longer needed. Task performance after initial task acquisition must therefore be accommodated by other brain regions that can store the learned association for long durations. We therefore searched for other brain regions that held learned associations long-term. In the medial prefrontal cortex (mPFC), we observe that the learned representation persists throughout the entire course of training. Unlike the OFC, not only does this representation encode the positive expected value of CS+ odors, it also encodes the negative expected value of CS- odors in a non-overlapping ensemble of neurons. We further show through optogenetic silencing that this representation is necessary for task performance after the task structure has already been acquired. Therefore, while the OFC representation is required for initial task acquisition, the mPFC representation is required for subsequent appetitive learning and performance. Why would a learned representation vanish in the OFC and betransfered elsewhere? We hypothesize that the brain may allocate a portion of its real estate to be a cognitive playground where experimentation and hypothesis testing takes place. Once this area solves a task, it may unload what it has learned to storage units located elsewhere to free up space to learn new tasks. We further imaged another associative area, the basolateral amygdala (BLA), and found a representation of positive value that appears to be generated from a Hebbian learning mechanism. However, the silencing of this representation during learning had no effect. This suggests that while multiple and distributed brain areas encode cues that predict the reward, not all may be necessary for the learning process or for task performance. In summary, we have described a series of experiments that map the representation and function of different associational areas that underlie learning. The data and the techniques employed have the potential to significantly advance the understanding of learned behavior.

Sensory information is processed and encoded by neural networks. In order to understand how the nervous system is able to rapidly integrate and store sensory information, knowledge of the connections and properties of the neurons in these circuits is required. The fruit fly Drosophila melanogaster provides a particularly powerful species to investigate the neural circuits of the olfactory system because in addition to possessing a simple olfactory system amenable to circuit analysis, a host of genetic reagents are available, including the GAL4-UAS system for targeted gene expression. The mushroom bodies, paired structures historically implicated in olfactory learning and memory, receive olfactory information at the mushroom body calyx from second-order olfactory projection neurons (PNs). Within the calyx, individual PN axonal boutons are surrounded by dendritic arborizations from intrinsic Kenyon cells (KCs) and each tiny cluster constitutes a single microglomerulus. Cells that connect the calyx with other areas of the brain, extrinsic neurons (ENs), also contribute to microglomeruli. Most of these contain the neurotransmitter, GABA, and are presumed to be inhibitory. In this study, the synaptic characteristics, neural circuits, and plasticity of calycal cells have been investigated using a combination of serial section electron and confocal microscopy. The findings reveal several new features of the circuits in the calyx: 1) The calyx contains three ultrastructurally distinct types of PN boutons that are heterogeneous in shape and exhibit subtle differences in synaptic densities. 2) All PN boutons form both ribbon and non-ribbon synapses, and from their smaller size and fewer postsynaptic partners, non-ribbon synapses may possibly become converted to ribbon synapses after activity; the olfactory signal may then be transmitted more strongly and efficiently at ribbon synapses. 3) PN boutons with an electron-dense cytoplasm have the most ribbon synapses per unit area of membrane as well as the highest ratio of ribbon to non-ribbon synapses, and thus may be more active and efficient than other boutons. 4) KC neurites are not exclusively postsynaptic in the calyx and can form occasional ribbon synapses, the functional interpretation of which awaits identification of their postsynaptic partners and vesicle contents. 5) Each PN bouton may contribute input to a single dendritic KC claw at about three presynaptic sites. For the postsynaptic side, a single claw receives input from individual presynaptic sites that must be highly redundant. 6) There may be important processing of the olfactory signal by local circuits formed by ENs in the calyx; ENs form synaptic connections with PNs, KCs, and other ENs. 7) Extensive serial synapses link EN terminals into a network, presumed to be GABAergic and inhibitory, that extends between microglomeruli and may be autaptic. 8) The structure and synaptic connectivity of microglomeruli may undergo changes after adult emergence. 9) vGAT and GAD1-GAL4 lines drive ectopic expression of marker genes in KCs and are not reliable reporters of GABA-positive cells. 10) Previously identified calycal ENs (MB-C1, MB-C2/C3, MB-CP1) are not immunopositive for GAD1, a marker of GABA-containing cells. 11) A network of ENs expressing a GABA phenotype differently innervates anatomically and functionally discrete areas of the honeybee calyx, and in addition the density of innervation may change with alterations in age and/or experience.