Noradrenergic modulatoins of odor learning and odor representation in the rat

How experience alters neuronal ensemble dynamics and how locus coeruleus-mediated norepinephrine release facilitates memory formation in the brain are the topics of this thesis. Here we employed a visualization technique, cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH), to assess activation patterns of neuronal ensembles in the olfactory bulb (OB) and anterior piriform cortex (aPC) to repeated odor inputs. Two associative learning models were used, early odor preference learning in rat pups and adult rat go-no-go odor discrimination learning. With catFISH of an immediate early gene, Arc, we showed that odor representation in the OB and aPC was sparse (~5-10%) and widely distributed. Odor associative learning enhanced the stability of the rewarded odor representation in the OB and aPC. The stable component, indexed by the overlap between the two ensembles activated by the rewarded odor at two time points, increased from ~25% to ~50% (p = 0.004-1.43E⁻4; Chapter 3 and 4). Adult odor discrimination learning promoted pattern separation between rewarded and unrewarded odor representations in the aPC. The overlap between rewarded and unrewarded odor representations reduced from ~25% to ~14% (p = 2.28E⁻⁵). However, learning an odor mixture as a rewarded odor increased the overlap of the component odor representations in the aPC from ~23% to ~44% (p = 0.010; Chapter 4). Blocking both α- and β-adrenoreceptors in the aPC prevented highly similar odor discrimination learning in adult rats, and reduced OB mitral and granule ensemble stability to the rewarded odor. Similar treatment in the OB only slowed odor discrimination learning. However, OB adrenoceptor blockade disrupted pattern separation and ensemble stability in the aPC when the rats demonstrated deficiency in discrimination (Chapter 5). In another project, the role of α₂-adrenoreceptors in the OB during early odor preference learning was studied. OB α2-adrenoceptor activation was necessary for odor learning in rat pups. α₂-adrenoceptor activation was additive with β-adrenoceptor mediated signalling to promote learning (Chapter 2). Together, these experiments suggest that odor representations are highly adaptive at the early stages of odor processing. The OB and aPC work in concert to support odor learning and top-down adrenergic input exerts a powerful modulation on both learning and odor representation

Aversive learning effect on odor coding in rat's piriform cortex

Olfaction, a sense for detecting and discriminating chemical molecules in the environment, is critical for animal survival, reproduction and other adaptive behaviors. The olfactory system is organized in three major stations (a sensor sheet, an initial processing and projection unit, and a central processing unit) that are shared across phyla, and has been functioning for millions of years. Since Buck and Axel identified a multigene family for coding the olfactory receptors, knowledge of the olfactory system has quickly accumulated in the last 20 years. This allows us to investigate fundamental questions in olfaction, including how odor percepts are formed, how olfactory information is used and stored, and how experiences shape olfactory perception in our daily life.Aversive events involving olfactory information are commonly experienced in nature. In the lab, aversive olfactory experiences have been shown to modify odor responses in rodents behaviorally and physiologically. Traditionally, studies regarding olfactory aversive learning were conducted by using odor-shock conditioning. Here, I explored the possibility of using 2-way active avoidance conditioning for awake unit recording in rats. The results confirmed previous findings that the rats can learn to actively avoid both auditory and olfactory cues that are associated with a dangerous event. Interestingly, the rats appeared to have rapid acquisition but poor behavioral retention. After comparing between the two paradigms, I decided to use odor-shock conditioning for chronic unit recording in awake rats.Three different odor-shock conditioning paradigms were used to investigate how aversive learning affects odor processing in the olfactory cortex. We first found that odor-evoked fear responses were training paradigm-dependent and each induced different levels of fear responses and odor generalization. In addition, we observed a decrease in spontaneous firing rate in the olfactory cortical neurons after conditioning and that was associative learning dependent. The results also suggested that generalized fear is associated with an impairment of olfactory cortical discrimination. In conclusion, changes in sensory processing are dependent on the nature of training, and can predict the behavioral outcome of the training

Simulação da preferência do odor maternal : da neuroetologia à biofísica

A tese é composta por dois trabalhos complementares. No primeiro trabalho, estudamos o aprendizado do odor materno em ratos neonatos, com foco no final do período sensível para o aprendizado precoce de preferência de odor. No segundo trabalho, estudamos as características das correntes GABAérgicas dos interneurônios do córtex piriforme anterior (CPa) e seu papel no aprendizado precoce de preferência de odor. Para ambos os estudos, foram usados dados eletrofisiológicos experimentais e modelos computacionais foram desenhados usando modelagem baseada em agentes. Os resultados são descritos como o número acumulado de spikes e número de neurônios ativos, antes e depois do condicionamento. No primeiro artigo, mostramos que as mudanças nas propriedades intrínsecas das células piramidais no CPa reduzem a disponibilidade de células piramidais que respondem à exposição do odor materno. No segundo artigo, experimentos computacionais mostraram que a entrada GABAérgica no CPa melhora a habilidade do circuito olfatório para o aprendizado do odor materno. A discussão geral apresenta uma integração dos níveis da neuroetologia à biofísica como uma perspectiva de trabalho.The thesis consists of two complementary studies. In the first study, we investigated the maternal odor learning in neonatal rats, focusing on the end of the sensitive period for early odor preference learning. In the second study, we examined the characteristics of the GABAergic currents of the interneurons of the anterior piriform cortex (aPC) and their role in early odor preference learning. For both studies, experimental electrophysiological data were used, and computational models were designed using agent-based modeling. The results are described as the cumulative number of spikes and the number of active neurons, before and after conditioning. In the first article, we showed that changes in the intrinsic properties of aPC pyramidal cells reduced the availability of the responsive pyramidal cells during maternal odor exposure. In the second article, computational experiments showed that GABAergic entry into the aPC improves the olfactory circuit's ability to learn maternal odor. The general discussion presents an integration of the levels from neuroethology to biophysics as a work perspective

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

A Computational Study Of The Influence Of Cortical Processes On The Olfactory Bulb

The olfactory bulb sits at the crossroads of input from an animal’s external and internal world. In this neural structure, chemical information from the environment interacts with contextual information emanating from higher cortical regions to shape mental representations of odor. Nevertheless, the factors influencing this interaction, and how the cortex manipulates these factors to the advantage of the animal, remain a mystery. To investigate this question, we have developed a large-scale computational model of the olfactory bulb. This model consists of a new algorithm to determine connectivity between mitral cells and granule cells, based in known anatomical constraints, combined with a dynamical systems approach utilizing the Izhikevich equations to simulate the network’s behavior. Using this model, we first examine connectivity and activity patterns of our network to demonstrate the strong relationship between structure and function in the olfactory bulb. We then further employ this model to analyze the effects of centrifugal feedback to the olfactory bulb on cortical odor representations; through this analysis, we are able to show that stochastic feedback patterns can evoke distinct trends in convergence and divergence between these representations depending on cortical excitability. Finally, we take advantage of the ease of incorporating new neurons into the model to study neurogenesis in the olfactory bulb, in particular to elucidate possible rules governing the placement of new cells. Through these experiments, our model provides new insight into the olfactory bulb and its role in the greater olfactory system

Construction of Odor Representations by Olfactory Bulb Microcircuits 7

Abstract Like other sensory systems, the olfactory system transduces specific features of the external environment and must construct an organized sensory representation from these highly fragmented inputs. As with these other systems, this representation is not accurate per se, but is constructed for utility, and emphasizes certain, presumably useful, features over others. I here describe the cellular and circuit mechanisms of the peripheral olfactory system that underlie this process of sensory construction, emphasizing the distinct architectures and properties of the two prominent computational layers in the olfactory bulb. Notably, while the olfactory system solves essentially similar conceptual problems to other sensory systems, such as contrast enhancement, activity normalization, and extending dynamic range, its peculiarities often require qualitatively different computational algorithms than are deployed in other sensory modalities. In particular, the olfactory modality is intrinsically high dimensional, and lacks a simple, externally defined basis analogous to wavelength or pitch on which elemental odor stimuli can be quantitatively compared. Accordingly, the quantitative similarities of the receptive fields of different odorant receptors (ORs) vary according to the statistics of the odor environment. To resolve these unusual challenges, the olfactory bulb appears to utilize unique nontopographical computations and intrinsic learning mechanisms to perform the necessary high-dimensional, similarity-dependent computations. In sum, the early olfactory system implements a coordinated set of early sensory transformations directly analogous to those in other sensory systems, but accomplishes these with unique circuit architectures adapted to the properties of the olfactory modality

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

The Influence of Age, Gender, and Thiol Repletion in an In Vivo Model of Lewy Body Disorders

Lewy body disorders are a family of neurological brain disorders associated with olfactory, motor, and cognitive deficits and are collectively defined as α-synucleinopathies, as they are characterized by hallmark “Lewy bodies” composed of aggregated, fibrillar α-synuclein. It is not certain but often posited that fibrillar α-synuclein seeds the progressive, self-propagating spread of Lewy pathology through neuroanatomical circuitry. Furthermore, the site of disease induction is still debated. In Aim I, we developed a novel protocol for generating reproducible and robust α-synucleinopathy in the limbic temporal lobe following infusions of preformed α-synuclein iv fibrils into the mouse olfactory bulb, which is frequently the first brain region to display Lewy pathology in humans. Our tract-tracer studies revealed that all areas displaying dense Lewy-like pathology were indeed connected to the induction site. The pattern of α-synucleinopathy resembled that of Stage IIb of Beach’s unified staging theory rather than Parkinson’s disease. In Aim 2, we observed that α-synucleinopathy also remained confined to the limbic connectome regardless of gender, age, or incubation period. Lewy pathology that developed in the ventral mesencephalon was confined to the ventral tegmental area, which is also associated with limbic functions. Furthermore, our studies are the first to show that females are more resistant to the development of α-synucleinopathy and neurodegeneration than males, and that fibril infusions hastened mortality in aged mice. In Aim 3, N-acetyl cysteine protected primary neuron cultures against proteotoxicity, including that of α-synuclein fibrils, and the mechanism appeared to be partly dependent on the chaperone activity of heat shock protein 70. These findings were expanded to an in vivo study, in which mice were fed N-acetyl cysteine for 90 days after induction of olfactory α-synucleinopathy. Oral N-acetyl cysteine reduced Lewy-like pathology, but only in the anterior olfactory nucleus, the area of densest α-synucleinopathy in this model. In conclusion, we have developed a robust model of early-stage Lewy body disorders that appears to mimic the higher risk of Lewy body disorders in men and the acceleration of mortality with aging in these conditions

Mechanisms and Function of Neural Synchronization in an Insect Olfactory System

One of the fundamental questions in modem integrative neurobiology relates to the encoding of sensory information by populations of neurons, and to the significance of this activity for perception, learning, memory and behavior. Synchronization of activity across a population of neurons has been observed many times over, but has never been demonstrated to be a necessary component of this coding process. Neural synchronization has been found in many brain areas in animals across several phyla, from molluscs to mammals. Studies in mammals have correlated the degree of neural synchronization with specific behavioral or cognitive states, such as sensorimotor tasks, segmentation and binocular rivalry suggesting a functional link. In the locust olfactory system, oscillatory synchronization is a prominent feature of the odor-evoked neural activity. Stimulation of the antenna by odors evokes synchronized firing in dynamic and odor-specific ensembles of the projection neurons of the antennal lobe, the principal neurons of the first-order olfactory relay in insects. The coherent activity of these projection neurons underlies an odor-evoked oscillatory field potential which can be recorded in the mushroom body, the second-order olfactory relay to which they project. In this dissertation, we investigated two important questions raised by these findings: how are such stimulus-evoked synchronous ensembles generated, and what is their functional significance? To address these questions, we performed electrophysiological experiments and recorded odor responses from neurons of the antennal lobes and mushroom bodies of locusts, in vivo and using natural odor stimulation in an unanesthetized, semi-intact preparation. We demonstrated the critical mechanism involved in neural synchronization of the antennal lobe neurons. The synchronization of the projection neurons relies critically on fast GABA (γ-aminobutyric acid) -mediated inhibition from the local interneurons. Projection neuron synchronization could be selectively blocked by local injection of the GABA receptor antagonist, picrotoxin. Picrotoxin spared the odor-specific, slow modulation of individual projection neuron responses, but desynchronized the firing of the odor-activated projection neuron assemblies. The oscillatory activity of the local intemeurons was also blocked by picrotoxin, which indicates that such activity depends on network synaptic dynamics. We also showed that the mushroom body networks are capable of generating oscillatory behavior of a similar frequency as that of its projection neuron inputs, and that they may thus be "tuned" to accept synchronized, oscillatory inputs of that frequency range. Our understanding of this mechanism, in tum, made possible the functional investigation of neural synchronization by selective disruption of projection neuron synchronization. We studied a population of neurons downstream from the antennal lobe projection neurons, the extrinsic neurons of the β-lobe of the mushroom body (βLNs). These βLNs were chosen for investigation because they were found to be odor-responsive and because their position in the olfactory pathway makes them a suitable "read-out" of population activity in the antennal lobe. We characterized βLN odor responses before and after selective disruption of the synchronization of the projection neuron ensembles with local picrotoxin injection into the antennal lobe. We showed that the tuning of these βLN responses was altered by PN desynchronization by changing existing responses and inducing new responses. This alteration in tuning resulted in a significant loss of odor specificity in individual βLN responses, an effect that never occurred in the responses of individual, desynchronized projection neurons. We thus propose that neural synchronization is indeed important for information processing in the brain: it serves, at least in part, as a temporal substrate for the transmission of information that is contained across co-activated neurons (relational code) early in the pathway.</p

Oscillatory architecture of memory circuits

The coordinated activity between remote brain regions underlies cognition and memory function. Although neuronal oscillations have been proposed as a mechanistic substrate for the coordination of information transfer and memory consolidation during sleep, little is known about the mechanisms that support the widespread synchronization of brain regions and the relationship of neuronal dynamics with other bodily rhythms, such as breathing. During exploratory behavior, the hippocampus and the prefrontal cortex are organized by theta oscillations, known to support memory encoding and retrieval, while during sleep the same structures are dominated by slow oscillations that are believed to underlie the consolidation of recent experiences. The expression of conditioned fear and extinction memories relies on the coordinated activity between the mPFC and the basolateral amygdala (BLA), a neuronal structure encoding associative fear memories. However, to date, the mechanisms allowing this long-range network synchronization of neuronal activity between the mPFC and BLA during fear behavior remain virtually unknown. Using a combination of extracellular recordings and open- and closed-loop optogenetic manipulations, we investigated the oscillatory and coding mechanisms mediating the organization and coupling of the limbic circuit in the awake and asleep brain, as well as during memory encoding and retrieval. We found that freezing, a behavioral expression of fear, is tightly associated with an internally generated brain state that manifests in sustained 4Hz oscillatory dynamics in prefrontal-amygdala circuits. 4Hz oscillations accurately predict the onset and termination of the freezing state. These oscillations synchronize prefrontal-amygdala circuits and entrain neuronal activity to dynamically regulate the development of neuronal ensembles. This enables the precise timing of information transfer between the two structures and the expression of fear responses. Optogenetic induction of prefrontal 4Hz oscillations promotes freezing behavior and the formation of long-lasting fear memory, while closed-loop phase specific manipulations bidirectionally modulate fear expression. Our results unravel a physiological signature of fear memory and identify a novel internally generated brain state, characterized by 4Hz oscillations. This oscillation enables the temporal coordination and information transfer in the prefrontal-amygdala circuit via a phase-specific coding mechanism, facilitating the encoding and expression of fear memory. In the search for the origin of this oscillation, we focused our attention on breathing, the most fundamental and ubiquitous rhythmic activity in life. Using large-scale extracellular recordings from a number of structures, including the medial prefrontal cortex, hippocampus, thalamus, amygdala and nucleus accumbens in mice we identified and characterized the entrainment by breathing of a host of network dynamics across the limbic circuit. We established that fear-related 4Hz oscillations are a state-specific manifestation of this cortical entrainment by the respiratory rhythm. We characterized the translaminar and transregional profile of this entrainment and demonstrated a causal role of breathing in synchronizing neuronal activity and network dynamics between these structures in a variety of behavioral scenarios in the awake and sleep state. We further revealed a dual mechanism of respiratory entrainment, in the form of an intracerebral corollary discharge that acts jointly with an olfactory reafference to coordinate limbic network dynamics, such as hippocampal ripples and cortical UP and DOWN states, involved in memory consolidation. Respiration provides a perennial stream of rhythmic input to the brain. In addition to its role as the condicio sine qua non for life, here we provide evidence that breathing rhythm acts as a global pacemaker for the brain, providing a reference signal that enables the integration of exteroceptive and interoceptive inputs with the internally generated dynamics of the hippocampus and the neocortex. Our results highlight breathing, a perennial rhythmic input to the brain, as an oscillatory scaffold for the functional coordination of the limbic circuit, enabling the segregation and integration of information flow across neuronal networks