ABSTRACT
PAR3/INSC/LGN form an evolutionarily conserved complex required for asymmetric cell division in the developing brain, but its post-developmental function and disease relevance in the peripheral nervous system (PNS) remains unknown. We mapped a new locus for axonal Charcot-Marie-Tooth disease (CMT2) and identified a missense mutation c.209 T > G (p.Met70Arg) in the INSC gene. Modeling the INSCM70R variant in Drosophila, we showed that it caused proprioceptive defects in adult flies, leading to gait defects resembling those in CMT2 patients. Cellularly, PAR3/INSC/LGN dysfunction caused tubulin aggregation and necrotic neurodegeneration, with microtubule-stabilizing agents rescuing both morphological and functional defects of the INSCM70R mutation in the PNS. Our findings underscore the critical role of the PAR3/INSC/LGN machinery in the adult PNS and highlight a potential therapeutic target for INSC-associated CMT2.
Subject(s)
Adaptor Proteins, Signal Transducing , Charcot-Marie-Tooth Disease , Cytoskeletal Proteins , Mutation, Missense , Animals , Humans , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Charcot-Marie-Tooth Disease/genetics , Charcot-Marie-Tooth Disease/pathology , Disease Models, Animal , Drosophila/genetics , Nuclear Proteins , Peripheral Nervous System Diseases/genetics , Peripheral Nervous System Diseases/pathology , Tubulin/genetics , Tubulin/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolismABSTRACT
Olfaction is a vital sense that insects use to forage and interact with each other. When an insect smells an odor, its nervous system processes the odor information and transforms it into an appropriate behavioral decision. Olfactory processing and transformation are not label-lined, but instead are modulated by internal states. The vinegar fly, Drosophila melanogaster, has become a primary model organism for studying this modulation. It has been observed that internal state modulates olfactory behaviors in multiple sites of the fly brain. In this review article, I focus on the mushroom body, a computational center in the fly brain, and discuss how the dopamine system in this brain region mediates internal-state signals and shapes olfactory responses in adult flies.
ABSTRACT
The mushroom body (MB) is a computational center in the Drosophila brain. The intricate neural circuits of the mushroom body enable it to store associative memories and process sensory and internal state information. The mushroom body is composed of diverse types of neurons that are precisely assembled during development. Tremendous efforts have been made to unravel the molecular and cellular mechanisms that build the mushroom body. However, we are still at the beginning of this challenging quest, with many key aspects of mushroom body assembly remaining unexplored. In this review, I provide an in-depth overview of our current understanding of mushroom body development and pertinent knowledge gaps.
ABSTRACT
Calcium-dependent nuclear import of LexA (CaLexA) and transcriptional reporter of intracellular calcium (TRIC) are transcription-based genetically encoded calcium indicators (transcriptional GECIs). When expressed in neurons, CaLexA and TRIC report neuronal activity by converting intracellular calcium levels into transcription activities and, subsequently, reporter gene expression. CaLexA and TRIC have been used successfully in many studies to label neurons associated with particular behaviors, regulated by internal states, or evoked by specific sensory inputs. This protocol details procedures for generating flies expressing genetic components of CaLexA and TRIC in specific neurons, performing immunostaining to label CaLexA and TRIC signals, and quantifying the signals using an open-source imaging analysis software.
Subject(s)
Calcium , Diptera , Animals , Calcium/metabolism , Diptera/metabolism , Neurons/metabolism , Central Nervous System/metabolism , Active Transport, Cell NucleusABSTRACT
Knowing which neurons are active during behavior is a crucial step toward understanding how nervous systems work. Neuronal activation is generally accompanied by an increase in intracellular calcium levels. Therefore, intracellular calcium levels are widely used as a proxy for neuronal activity. Many types of synthetic components and bioluminescent or fluorescent proteins that report transient and long-term changes in intracellular calcium levels have been developed over the past 60 years. Calcium indicators that enable imaging of the dynamic activity of a large ensemble of neurons in behaving animals have revolutionized the field of neuroscience. Among these, transcription-based genetically encoded calcium indicators (transcriptional GECIs) have proven easy to use and do not depend on sophisticated imaging systems, offering unique advantages over other types of calcium indicators. Here, we describe the two currently available fly transcriptional GECIs-calcium-dependent nuclear import of LexA (CaLexA) and transcriptional reporter of intracellular calcium (TRIC)-and review studies that have used them. In the accompanying protocol, we present step-by-step details for generating CaLexA- and TRIC-ready flies and for imaging CaLexA and TRIC signals in dissected brains after experimental manipulations of intact free-moving flies.
Subject(s)
Calcium , Drosophila , Animals , Drosophila/genetics , Calcium/metabolism , Neurons/metabolism , Indicators and Reagents , Calcium Signaling/physiologyABSTRACT
SignificanceThe adult Drosophila mushroom body (MB) is one of the most extensively studied neural circuits. However, how its circuit organization is established during development is unclear. In this study, we provide an initial characterization of the assembly process of the extrinsic neurons (dopaminergic neurons and MB output neurons) that target the vertical MB lobes. We probe the cellular mechanisms guiding the neurite targeting of these extrinsic neurons and demonstrate that Semaphorin 1a is required in several MB output neurons for their dendritic innervations to three specific MB lobe zones. Our study reveals several intriguing molecular and cellular principles governing assembly of the MB circuit.
Subject(s)
Mushroom Bodies , Semaphorins , Animals , Dopaminergic Neurons , Drosophila/physiology , Mushroom Bodies/physiology , Neurites , Semaphorins/geneticsABSTRACT
Intrinsic mechanisms such as temporal series of transcription factors orchestrate neurogenesis from a limited number of neural progenitors in the brain. Extrinsic regulations, however, remain largely unexplored. Here we describe a two-step glia-derived signal that regulates neurogenesis in the Drosophila mushroom body (MB). In a temporal manner, glial-specific ubiquitin ligase dSmurf activates non-cell-autonomous Hedgehog signaling propagation by targeting the receptor Patched to suppress and promote the exit of MB neuroblast (NB) proliferation, thereby specifying the correct α/ß cell number without affecting differentiation. Independent of NB proliferation, dSmurf also stabilizes the expression of the cell-adhesion molecule Fasciclin II (FasII) via its WW domains and regulates FasII homophilic interaction between glia and MB axons to refine α/ß-lobe integrity. Our findings provide insights into how extrinsic glia-to-neuron communication coordinates with NB proliferation capacity to regulate MB neurogenesis; glial proteostasis is likely a generalized mechanism in orchestrating neurogenesis.
Subject(s)
Cell Communication , Cell Proliferation , Mushroom Bodies/embryology , Neurogenesis , Neuroglia/metabolism , Animals , Drosophila Proteins/metabolism , Drosophila melanogasterABSTRACT
Motivational states modulate how animals value sensory stimuli and engage in goal-directed behaviors. The motivational states of thirst and hunger are represented in the brain by shared and unique neuromodulatory systems. However, it is unclear how such systems interact to coordinate the expression of appropriate state-specific behavior. We show that the activity of two brain neurons expressing leucokinin neuropeptide is elevated in thirsty and hungry flies, and that leucokinin release is necessary for state-dependent expression of water- and sugar-seeking memories. Leucokinin inhibits two types of mushroom-body-innervating dopaminergic neurons (DANs) to promote thirst-specific water memory expression, whereas it activates other mushroom-body-innervating DANs to facilitate hunger-dependent sugar memory expression. Selection of hunger- or thirst-appropriate memory emerges from competition between leucokinin and other neuromodulatory hunger signals at the level of the DANs. Therefore, coordinated modulation of the dopaminergic system allows flies to prioritize the expression of the relevant state-dependent motivated behavior.
Subject(s)
Dopaminergic Neurons/physiology , Drosophila , Hunger/physiology , Memory/physiology , Neuropeptides/physiology , Thirst/physiology , Animals , Animals, Inbred Strains , Behavior, Animal/physiology , Cues , Female , Food Deprivation/physiology , Male , Mushroom Bodies/physiology , Neural Inhibition/physiology , Neurons/metabolism , Neuropeptides/metabolism , Water , Water Deprivation/physiologyABSTRACT
Hunger is a motivational state that drives eating and food-seeking behaviour. In a psychological sense, hunger sets the goal that guides an animal in the pursuit of food. The biological basis underlying this purposive, goal-directed nature of hunger has been under intense investigation. With its rich behavioural repertoire and genetically tractable nervous system, the fruit fly Drosophila melanogaster has emerged as an excellent model system for studying the neural basis of hunger and hunger-driven behaviour. Here, we review our current understanding of how hunger is sensed, encoded and translated into foraging and feeding behaviours in the fruit fly.
Subject(s)
Drosophila melanogaster/physiology , Feeding Behavior/physiology , Hunger/physiology , Neurons/physiology , Animals , Brain/physiology , Eating/physiology , Models, Biological , Nerve Net/physiology , Olfactory Pathways/physiologyABSTRACT
The fruit fly can evaluate its energy state and decide whether to pursue food-related cues. Here, we reveal that the mushroom body (MB) integrates hunger and satiety signals to control food-seeking behavior. We have discovered five pathways in the MB essential for hungry flies to locate and approach food. Blocking the MB-intrinsic Kenyon cells (KCs) and the MB output neurons (MBONs) in these pathways impairs food-seeking behavior. Starvation bi-directionally modulates MBON responses to a food odor, suggesting that hunger and satiety controls occur at the KC-to-MBON synapses. These controls are mediated by six types of dopaminergic neurons (DANs). By manipulating these DANs, we could inhibit food-seeking behavior in hungry flies or promote food seeking in fed flies. Finally, we show that the DANs potentially receive multiple inputs of hunger and satiety signals. This work demonstrates an information-rich central circuit in the fly brain that controls hunger-driven food-seeking behavior.
Subject(s)
Appetitive Behavior/physiology , Drosophila melanogaster/physiology , Feeding Behavior/physiology , Hunger/physiology , Mushroom Bodies/physiology , Satiety Response/physiology , Animals , Brain/metabolism , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/physiology , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Food , Gene Expression , Instinct , Mushroom Bodies/metabolism , StarvationABSTRACT
Animals constantly assess the reliability of learned information to optimize their behaviour. On retrieval, consolidated long-term memory can be neutralized by extinction if the learned prediction was inaccurate. Alternatively, retrieved memory can be maintained, following a period of reconsolidation during which it is labile. Although extinction and reconsolidation provide opportunities to alleviate problematic human memories, we lack a detailed mechanistic understanding of memory updating. Here we identify neural operations underpinning the re-evaluation of memory in Drosophila. Reactivation of reward-reinforced olfactory memory can lead to either extinction or reconsolidation, depending on prediction accuracy. Each process recruits activity in specific parts of the mushroom body output network and distinct subsets of reinforcing dopaminergic neurons. Memory extinction requires output neurons with dendrites in the α and α' lobes of the mushroom body, which drive negatively reinforcing dopaminergic neurons that innervate neighbouring zones. The aversive valence of these new extinction memories neutralizes previously learned odour preference. Memory reconsolidation requires the γ2α'1 mushroom body output neurons. This pathway recruits negatively reinforcing dopaminergic neurons innervating the same compartment and re-engages positively reinforcing dopaminergic neurons to reconsolidate the original reward memory. These data establish that recurrent and hierarchical connectivity between mushroom body output neurons and dopaminergic neurons enables memory re-evaluation driven by reward-prediction error.
Subject(s)
Drosophila melanogaster/physiology , Extinction, Psychological/physiology , Learning/physiology , Memory Consolidation/physiology , Reinforcement, Psychology , Animals , Dendrites , Dietary Carbohydrates , Dopaminergic Neurons/physiology , Drosophila melanogaster/cytology , Female , Male , Memory, Long-Term/physiology , Models, Animal , Mushroom Bodies/cytology , Mushroom Bodies/physiology , Odorants/analysis , Reward , Smell/physiologyABSTRACT
Remembering features of past feeding experience can refine foraging and food choice. Insects can learn to associate sensory cues with components of food, such as sugars, amino acids, water, salt, alcohol, toxins and pathogens. In the fruit fly Drosophila some food components activate unique subsets of dopaminergic neurons (DANs) that innervate distinct functional zones on the mushroom bodies (MBs). This architecture suggests that the overall dopaminergic neuron population could provide a potential cellular substrate through which the fly might learn to value a variety of food components. In addition, such an arrangement predicts that individual component memories reside in unique locations. DANs are also critical for food memory consolidation and deprivation-state dependent motivational control of the expression of food-relevant memories. Here, we review our current knowledge of how nutrient-specific memories are formed, consolidated and specifically retrieved in insects, with a particular emphasis on Drosophila.
ABSTRACT
The fruit fly Drosophila melanogaster has emerged as a popular model to investigate fundamental principles of neural circuit operation. The sophisticated genetics and small brain permit a cellular resolution understanding of innate and learned behavioural processes. Relatively recent genetic and technical advances provide the means to specifically and reproducibly manipulate the function of many fly neurons with temporal resolution. The same cellular precision can also be exploited to express genetically encoded reporters of neural activity and cell-signalling pathways. Combining these approaches in living behaving animals has great potential to generate a holistic view of behavioural control that transcends the usual molecular, cellular and systems boundaries. In this review, we discuss these approaches with particular emphasis on the pioneering studies and those involving learning and memory.
Subject(s)
Behavior, Animal/physiology , Drosophila melanogaster/physiology , Neurons/physiology , Animals , Animals, Genetically Modified , Drosophila melanogaster/genetics , Gene Expression , Genes, Insect , Hot Temperature , Learning/physiology , Light , Memory/physiology , Models, Animal , Models, Neurological , Neural Pathways/physiology , OptogeneticsABSTRACT
Dopaminergic neurons provide reward learning signals in mammals and insects [1-4]. Recent work in Drosophila has demonstrated that water-reinforcing dopaminergic neurons are different to those for nutritious sugars [5]. Here, we tested whether the sweet taste and nutrient properties of sugar reinforcement further subdivide the fly reward system. We found that dopaminergic neurons expressing the OAMB octopamine receptor [6] specifically convey the short-term reinforcing effects of sweet taste [4]. These dopaminergic neurons project to the ß'2 and γ4 regions of the mushroom body lobes. In contrast, nutrient-dependent long-term memory requires different dopaminergic neurons that project to the γ5b regions, and it can be artificially reinforced by those projecting to the ß lobe and adjacent α1 region. Surprisingly, whereas artificial implantation and expression of short-term memory occur in satiated flies, formation and expression of artificial long-term memory require flies to be hungry. These studies suggest that short-term and long-term sugar memories have different physiological constraints. They also demonstrate further functional heterogeneity within the rewarding dopaminergic neuron population.
Subject(s)
Dopaminergic Neurons/physiology , Drosophila melanogaster/physiology , Taste/physiology , Animals , Animals, Genetically Modified , Appetitive Behavior/physiology , Drosophila Proteins/genetics , Drosophila Proteins/physiology , Drosophila melanogaster/cytology , Drosophila melanogaster/genetics , Female , Male , Memory, Long-Term/physiology , Memory, Short-Term/physiology , Mushroom Bodies/cytology , Mushroom Bodies/physiology , Mutation , Nutritive Value , Receptors, Neurotransmitter/genetics , Receptors, Neurotransmitter/physiology , Reinforcement, Psychology , RewardABSTRACT
Drinking water is innately rewarding to thirsty animals. In addition, the consumed value can be assigned to behavioral actions and predictive sensory cues by associative learning. Here we show that thirst converts water avoidance into water-seeking in naive Drosophila melanogaster. Thirst also permitted flies to learn olfactory cues paired with water reward. Water learning required water taste and <40 water-responsive dopaminergic neurons that innervate a restricted zone of the mushroom body γ lobe. These water learning neurons are different from those that are critical for conveying the reinforcing effects of sugar. Naive water-seeking behavior in thirsty flies did not require water taste but relied on another subset of water-responsive dopaminergic neurons that target the mushroom body ß' lobe. Furthermore, these naive water-approach neurons were not required for learned water-seeking. Our results therefore demonstrate that naive water-seeking, learned water-seeking and water learning use separable neural circuitry in the brain of thirsty flies.
Subject(s)
Drosophila melanogaster/physiology , Memory/physiology , Mushroom Bodies/physiology , Reward , Thirst/physiology , Water/physiology , Animals , Conditioning, Classical/physiology , Dopaminergic Neurons/metabolism , Mushroom Bodies/innervation , Reinforcement, PsychologyABSTRACT
In Drosophila, anatomically discrete dopamine neurons that innervate distinct zones of the mushroom body (MB) assign opposing valence to odors during olfactory learning. Subsets of MB neurons have temporally unique roles in memory processing, but valence-related organization has not been demonstrated. We functionally subdivided the αß neurons, revealing a value-specific role for the â¼160 αß core (αßc) neurons. Blocking neurotransmission from αß surface (αßs) neurons revealed a requirement during retrieval of aversive and appetitive memory, whereas blocking αßc only impaired appetitive memory. The αßc were also required to express memory in a differential aversive paradigm demonstrating a role in relative valuation and approach behavior. Strikingly, both reinforcing dopamine neurons and efferent pathways differentially innervate αßc and αßs in the MB lobes. We propose that conditioned approach requires pooling synaptic outputs from across the αß ensemble but only from the αßs for conditioned aversion.
Subject(s)
Appetitive Behavior/physiology , Avoidance Learning/physiology , Dopaminergic Neurons/physiology , Memory/physiology , Mushroom Bodies/cytology , Animals , Behavior, Animal , Drosophila , Learning/physiology , Mushroom Bodies/physiology , Smell/physiologyABSTRACT
An often-overlooked aspect of neural plasticity is the plasticity of neuronal composition, in which the numbers of neurons of particular classes are altered in response to environment and experience. The Drosophila brain features several well-characterized lineages in which a single neuroblast gives rise to multiple neuronal classes in a stereotyped sequence during development. We find that in the intrinsic mushroom body neuron lineage, the numbers for each class are highly plastic, depending on the timing of temporal fate transitions and the rate of neuroblast proliferation. For example, mushroom body neuroblast cycling can continue under starvation conditions, uncoupled from temporal fate transitions that depend on extrinsic cues reflecting organismal growth and development. In contrast, the proliferation rates of antennal lobe lineages are closely associated with organismal development, and their temporal fate changes appear to be cell cycle-dependent, such that the same numbers and types of uniglomerular projection neurons innervate the antennal lobe following various perturbations. We propose that this surprising difference in plasticity for these brain lineages is adaptive, given their respective roles as parallel processors versus discrete carriers of olfactory information.
Subject(s)
Brain/physiology , Drosophila melanogaster/physiology , Mushroom Bodies/metabolism , Neuronal Plasticity , Olfactory Pathways/metabolism , Animals , Arthropod Antennae/cytology , Arthropod Antennae/metabolism , Cell Differentiation , Cell Lineage , Cell Proliferation , Drosophila Proteins/biosynthesis , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Green Fluorescent Proteins/genetics , Insulin/metabolism , Larva , Nerve Tissue Proteins/genetics , Olfactory Pathways/cytology , POU Domain Factors/genetics , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Starvation , Transcription Factors/geneticsABSTRACT
Binary cell fate decisions allow the production of distinct sister neurons from an intermediate precursor. Neurons are further diversified based on the birth order of intermediate precursors. Here we examined the interplay between binary cell fate and birth-order-dependent temporal fate in the Drosophila lateral antennal lobe (lAL) neuronal lineage. Single-cell mapping of the lAL lineage by twin-spot mosaic analysis with repressible cell markers (ts-MARCM) revealed that projection neurons (PNs) and local interneurons (LNs) are made in pairs through binary fate decisions. Forty-five types of PNs innervating distinct brain regions arise in a stereotyped sequence; however, the PNs with similar morphologies are not necessarily born in a contiguous window. The LNs are morphologically less diverse than the PNs, and the sequential morphogenetic changes in the two pairs occur independently. Sanpodo-dependent Notch activity promotes and patterns the LN fates. By contrast, Notch diversifies PN temporal fates in a Sanpodo-dispensable manner. These pleiotropic Notch actions underlie the differential temporal fate specification of twin neurons produced by common precursors within a lineage, possibly by modulating postmitotic neurons' responses to Notch-independent transcriptional cascades.
Subject(s)
Cell Lineage , Drosophila Proteins/metabolism , Drosophila/cytology , Gene Expression Regulation, Developmental , Neurons/cytology , Receptors, Notch/metabolism , Animals , Biomarkers/metabolism , Brain/cytology , Brain/embryology , Brain/metabolism , Cell Division , Drosophila/embryology , Drosophila/metabolism , Drosophila Proteins/genetics , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Genes, Insect , Immunohistochemistry , Larva/cytology , Larva/growth & development , Larva/metabolism , Mechanotransduction, Cellular , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurogenesis , Neurons/metabolism , Receptors, Notch/genetics , Single-Cell Analysis/methodsABSTRACT
Generating diverse neurons in the central nervous system involves three major steps. First, heterogeneous neural progenitors are specified by positional cues at early embryonic stages. Second, neural progenitors sequentially produce neurons or intermediate precursors that acquire different temporal identities based on their birth-order. Third, sister neurons produced during asymmetrical terminal mitoses are given distinct fates. Determining the molecular mechanisms underlying each of these three steps of cellular diversification will unravel brain development and evolution. Drosophila has a relatively simple and tractable CNS, and previous studies on Drosophila CNS development have greatly advanced our understanding of neuron fate specification. Here we review those studies and discuss how the lessons we have learned from fly teach us the process of neuronal diversification in general.
Subject(s)
Central Nervous System/anatomy & histology , Central Nervous System/embryology , Drosophila melanogaster/anatomy & histology , Drosophila melanogaster/embryology , Animals , Cell Lineage , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Morphogenesis/physiology , Neurogenesis , Neurons/cytology , Neurons/physiology , Stem Cells/cytology , Stem Cells/physiologyABSTRACT
Numb can antagonize Notch signaling to diversify the fates of sister cells. We report here that paired sister cells acquire different fates in all three Drosophila neuronal lineages that make diverse types of antennal lobe projection neurons (PNs). Only one in each pair of postmitotic neurons survives into the adult stage in both anterodorsal (ad) and ventral (v) PN lineages. Notably, Notch signaling specifies the PN fate in the vPN lineage but promotes programmed cell death in the missing siblings in the adPN lineage. In addition, Notch/Numb-mediated binary sibling fates underlie the production of PNs and local interneurons from common precursors in the lAL lineage. Furthermore, Numb is needed in the lateral but not adPN or vPN lineages to prevent the appearance of ectopic neuroblasts and to ensure proper self-renewal of neural progenitors. These lineage-specific outputs of Notch/Numb signaling show that a universal mechanism of binary fate decision can be utilized to govern diverse neural sibling differentiations.