Structure and function of the moth mushroom body

The mushroom bodies are paired, high-order neuropils in the insect brain involved in complex functions such as learning and memory, sensory integration, context recognition and olfactory processing. This thesis explores the structure of the mushroom bodies in the noctuid moth Spodoptera littoralis using neuroanatomical staining methods, immunocytochemistry and electron microscopy, and investigates how the intrinsic neurons of the mushroom body, the Kenyon cells, respond to olfactory stimulation of the antennae using whole-cell patch clamp technique. The mushroom body in S. littoralis contains about 4,000 Kenyon cells, and consists of a calyx, pedunculus and two lobes, one medial and one vertical. The calyx houses dendritic branches of Kenyon cells and the pedunculus and lobes contain the axons and terminals of these neurons respectively. The calyx is doubled and concentrically divided into a broad peripheral zone, which receives input from antennal lobe projection neurons, and a narrow inner zone, which receives yet unidentified input. The lobes are parsed into three longitudinal divisions, which contain a separate subset of Kenyon cells each. The Kenyon cells are divided into three morphological classes, I-III. Class I Kenyon cells have widely branching spiny dendritic arborisations in both zones of the calyx and occupy the two most posterior subdivisions of the lobes called α/β and α´/β´. Class II Kenyon cells have narrow clawed dendritic trees in the calyx and invade the most anterior division in the lobes, called γ. Class III Kenyon cells have clawed, diffusely branching dendrites in the calyx and provide a separate system of axons and terminal branches, partly detached from the rest of the mushroom body, called the Y tract and lobelets. Kenyon cells within the classes display differential labeling with antisera against neuroactive substances. Kenyon cells make synaptic contact with one another and with other neuron types in the mushroom body. Extrinsic inhibitory and putative modulatory neurons were identified. Whole-cell patch clamp recordings revealed that Kenyon cells exhibit broadly tuned subthreshold activation by odor stimulation and a few cells responded with action potentials to specific biologically relevant odor combinations

Discrimination Training with Multimodal Stimuli Changes Activity in the Mushroom Body of the Hawkmoth Manduca sexta

The mushroom bodies of the insect brain play an important role in olfactory processing, associative learning and memory. The mushroom bodies show odor-specific spatial patterns of activity and are also influenced by visual stimuli.Functional imaging was used to investigate changes in the in vivo responses of the mushroom body of the hawkmoth Manduca sexta during multimodal discrimination training. A visual and an odour stimulus were presented either together or individually. Initially, mushroom body activation patterns were identical to the odour stimulus and the multimodal stimulus. After training, however, the mushroom body response to the rewarded multimodal stimulus was significantly lower than the response to the unrewarded unimodal odour stimulus, indicating that the coding of the stimuli had changed as a result of training. The opposite pattern was seen when only the unimodal odour stimulus was rewarded. In this case, the mushroom body was more strongly activated by the multimodal stimuli after training. When no stimuli were rewarded, the mushroom body activity decreased for both the multimodal and unimodal odour stimuli. There was no measurable response to the unimodal visual stimulus in any of the experiments. These results can be explained using a connectionist model where the mushroom body is assumed to be excited by olfactory stimulus components, and suppressed by multimodal configurations.Discrimination training with multimodal stimuli consisting of visual and odour cues leads to stimulus specific changes in the in vivo responses of the mushroom body of the hawkmoth

Characterization of the pleiotropic effects of the Drosophila gene mushroom body miniature B

The mushroom body miniature B ( mbmB) mutation has documented defects in adult mushroom body anatomy, olfactory memory and female fertility. In this thesis I report that mbmB mutants also have reduced viability and growth rate. I further demonstrate, using the mushroom body cell marker dachshund, that loss of mbmB function does not affect the early development of mushroom body neuroectoderm and neuroblasts. Additionally, I found the observed delay in growth and reduced viability were not manifested during embryonic development; Courtship behavior assays and immunohistochemical techniques were used to investigate causes of mbmB female sterility. Courtship involving mbmB females is delayed and reduced in total amount relative to that of wild type. Additional data presented herein suggest that mechanisms required during early oogenesis are not significantly impaired by loss of mbmB function. However, evidence is presented which demonstrates abnormalities in follicle-cell migration in mbmB mutants

Specific requirement of NMDA receptors for long-term memory consolidation in Drosophila ellipsoid body

In humans and many other animals, memory consolidation occurs through multiple temporal phases and usually involves more than one neuroanatomical brain system. Genetic dissection of Pavlovian olfactory learning in Drosophila melanogaster has revealed multiple memory phases, but the predominant view holds that all memory phases occur in mushroom body neurons. Here, we demonstrate an acute requirement for NMDA receptors (NMDARs) outside of the mushroom body during long-term memory (LTM) consolidation. Targeted dsRNA-mediated silencing of Nmdar1 and Nmdar2 (also known as dNR1 or dNR2, respectively) in cholinergic R4m-subtype large-field neurons of the ellipsoid body specifically disrupted LTM consolidation, but not retrieval. Similar silencing of functional NMDARs in the mushroom body disrupted an earlier memory phase, leaving LTM intact. Our results clearly establish an anatomical site outside of the mushroom body involved with LTM consolidation, thus revealing both a distributed brain system subserving olfactory memory formation and the existence of a system-level memory consolidation in Drosophila

Functional architecture of reward learning in mushroom body extrinsic neurons of larval Drosophila.

The brain adaptively integrates present sensory input, past experience, and options for future action. The insect mushroom body exemplifies how a central brain structure brings about such integration. Here we use a combination of systematic single-cell labeling, connectomics, transgenic silencing, and activation experiments to study the mushroom body at single-cell resolution, focusing on the behavioral architecture of its input and output neurons (MBINs and MBONs), and of the mushroom body intrinsic APL neuron. Our results reveal the identity and morphology of almost all of these 44 neurons in stage 3 Drosophila larvae. Upon an initial screen, functional analyses focusing on the mushroom body medial lobe uncover sparse and specific functions of its dopaminergic MBINs, its MBONs, and of the GABAergic APL neuron across three behavioral tasks, namely odor preference, taste preference, and associative learning between odor and taste. Our results thus provide a cellular-resolution study case of how brains organize behavior

Genetic Dissection of the Neural Circuitry Underlying Memory Stability in Drosophila: A Dissertation

Understanding how memory is formed requires looking beyond the genes involved to the neural circuitry and temporal aspects of memory. In this dissertation I have focused my investigation on Dorsal Paired Medial (DPM) neurons, two modulatory neurons essential for memory in Drosophila. DPM neurons highly express the amnesiac (amn) gene, which encodes for a putative pre-pro-neuropeptide. amn function in DPM neurons is required for memory. Here I provide evidence that DPM neurons are cholinergic and that acetylcholine (ACh) and AMN act as co-transmitters essential for DPM function. In order to investigate the temporal requirements of DPM output I blocked transmitter release during discrete intervals in the memory process using shibirets1 and tested flies for shock and sugar-reinforced memory. These experiments demonstrated that stable memory requires persistent transmitter release from DPM neurons. Furthermore these results suggest AMN and DPM neurons act as general stabilizers of mushroom body dependent memory. To further investigate the neural circuitry underlying DPM function I disrupted DPM projections onto the mushroom body lobes by ectopically expressing DScam17-2::GFP in DPM neurons. Flies with DPM neurons that predominantly project to the mushroom body α´/β´ lobes exhibit normal memory, and blocking transmitter release from the mushroom body prime lobes neurons themselves abolishes memory indicating DPM neuron-mushroom body α´/β´ neuron interaction that are critical for memory. Taken together, the experimental evidence presented here are used to provide a rudimentary model of the neural circuitry involved in memory stability, where DPM neurons form a recurrent feedback loop with the mushroom body α´/β´ lobe neurons and act to stabilize odorspecific conditioned memories at Kenyon cell synapses

Dopaminergic Modulation Shapes Sensorimotor Processing in the Drosophila Mushroom Body

To survive in a complex and dynamic environment, animals must adapt their behavior based on their current needs and prior experiences. This flexibility is often mediated by neuromodulation within neural circuits that link sensory representations to alternative behavioral responses depending on contextual cues and learned associations. In Drosophila, the mushroom body is a prominent neural structure essential for olfactory learning. Dopaminergic neurons convey salient information about reward and punishment to the mushroom body in order to adjust synaptic connectivity between Kenyon cells, the neurons representing olfactory stimuli, and the mushroom body output neurons that ultimately influence behavior. However, we still lack a mechanistic understanding of how the dopaminergic neurons represent the moment-tomoment experience of a fly and drive changes in this sensory-to-motor transformation. Furthermore, very little is known about how the output neuron pathways lead to the execution of appropriate odor-related behaviors. We took advantage of the mushroom body’s modular circuit organization to investigate how the dopaminergic neuron population encodes different contextual cues. In vivo functional imaging of the dopaminergic neurons reveals that they represent both external reinforcement stimuli, like sugar rewards or punitive electric shock, as well as the fly’s motor state, through coordinated and partially antagonistic activity patterns across the population. This multiplexing of motor and reward signals by the dopaminergic neurons parallels the dual roles of dopaminergic inputs to the vertebrate basal ganglia, thus demonstrating a conserved link between these distantly related neural circuits. We proceed to demonstrate that this dopaminergic signal in the mushroom body modifies neurotransmission with synaptic specificity and temporal precision to coordinately regulate the propagation of sensory signals through the output neurons. To explore how these output pathways ultimately influence olfactory navigation we have developed a closed loop olfactory paradigm in which we can monitor and manipulate the mushroom body output neurons as a fly navigates in a virtual olfactory environment. We have begun to probe the mushroom body circuitry in the context of olfactory navigation. These preliminary investigations have led to the identification of putative pathways for linking mushroom body output with the circuits that implement odor-tracking behavior and the characterization of the complex sensorimotor representations in the dopaminergic network. Our work reveals that the Drosophila dopaminergic system modulates mushroom body output at both acute and enduring timescales to guide immediate behaviors and learned responses

KCNQ channels regulate age-related memory impairment:KCNQ regulates age-related memory

In humans KCNQ2/3 heteromeric channels form an M-current that acts as a brake on neuronal excitability, with mutations causing a form of epilepsy. The M-current has been shown to be a key regulator of neuronal plasticity underlying associative memory and ethanol response in mammals. Previous work has shown that many of the molecules and plasticity mechanisms underlying changes in alcohol behaviour and addiction are shared with those of memory. We show that the single KCNQ channel in Drosophila (dKCNQ) when mutated show decrements in associative short- and long-term memory, with KCNQ function in the mushroom body α/βneurons being required for short-term memory. Ethanol disrupts memory in wildtype flies, but not in a KCNQ null mutant background suggesting KCNQ maybe a direct target of ethanol, the blockade of which interferes with the plasticity machinery required for memory formation. We show that as in humans, Drosophila display age-related memory impairment with the KCNQ mutant memory defect mimicking the effect of age on memory. Expression of KCNQ normally decreases in aging brains and KCNQ overexpression in the mushroom body neurons of KCNQ mutants restores age-related memory impairment. Therefore KCNQ is a central plasticity molecule that regulates age dependent memory impairment

Dissecting the mechanisms of learning-by-doing in Drosophila

At the heart of learning-by-doing lies a well-known psychological phenomenon: information will be remembered better if it is actively generated rather than passively read or heard. First described in humans, this generation effect can also be observed in various animal models. However, the neurobiological mechanisms underlying the generation effect are unknown. Here we show that two reciprocal interactions between its active and passive components contribute to the generation effect in flies. One interaction consists of the active (skill-learning) component facilitating the passive (fact-learning) component. Fact-learning, on the other hand, inhibits skill-learning. Experiments with adenylyl cyclase I deficient _rutabaga_ mutant flies revealed that the fact- but not the skill-learning component requires this evolutionarily conserved learning gene. Using mushroom-body deficient transgenic flies we observed that the mushroom-bodies mediate the inhibition of skill-learning. This inhibition also enables generalization and prevents premature habit formation. Extended training in wildtype flies produced a phenocopy of mushroom-body impaired flies, such that generalization was abolished and goal-directed actions were transformed into habitual responses. Thus, our results identify various neural processes underlying learning-by-doing, delineate some of their synergisms and provide a framework for further dissecting them in a genetically tractable model system

Investigating the role of histone deacetylase HDAC4 in long-term memory formation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Manawatu, New Zealand

Listed in 2017 Dean's List of Exceptional ThesesEpigenetic mechanisms are emerging as master regulators of cognitive abilities such as learning and memory. It has been previously shown that the histone deacetylase HDAC4 plays a critical role in memory formation in both mammals and insects although the specific mechanisms through which it acts have not yet been elucidated. HDAC4 undergoes nucleocytoplasmic shuttling and, in neurons, it is largely cytoplasmic implying it may play both nuclear and non-nuclear functions. To identify upstream regulators and downstream targets of HDAC4, a genetic interaction screen was performed in the fruit fly Drosophila melanogaster, a powerful model system to study the genetic mechanisms of neurological disease. Twenty-nine genes were found to interact with HDAC4 suggesting they are part of the same molecular pathway. Functional network analysis revealed that many of the genes could be grouped into three biological categories comprising transcriptional factors, SUMOylation machinery enzymes and cytoskeletal regulators/interactors. Within the latter, Ankyrin2 was selected for further analysis as it is implicated in synaptic stability and in human intellectual disability. In addition HDAC4 harbours a conserved ankyrin binding domain. Immunohistochemical analyses showed widespread distribution of Ankyrin2 throughout the adult brain and coincident distribution with HDAC4 was observed in the axons of the mushroom body, a key structure for memory formation in flies. Both HDAC4 and Ankyrin2 were also found to regulate mushroom body development. RNAi-mediated depletion of Ankyrin2 in the adult brain impaired long-term memory in the courtship suppression assay, a model of associative memory and preliminary evidence of a physical association between HDAC4 and Ankyrin2 was also demonstrated. The genes identified in the screen provide new avenues for investigation of the mechanisms through which HDAC4 regulates memory formation and preliminary analyses suggest that interaction with the cytoskeletal adaptor Ankyrin2 may involve remodelling of the actin/spectrin cytoskeleton, phenomenon that underlies memory related processes like synaptic plasticity and neuronal excitability