RESUMO
Insect asynchronous flight is one of the most prevalent forms of animal locomotion used by more than 600,000 species. Despite profound insights into the motor patterns1, biomechanics2,3 and aerodynamics underlying asynchronous flight4,5, the architecture and function of the central-pattern-generating (CPG) neural network remain unclear. Here, on the basis of an experiment-theory approach including electrophysiology, optophysiology, Drosophila genetics and mathematical modelling, we identify a miniaturized circuit solution with unexpected properties. The CPG network consists of motoneurons interconnected by electrical synapses that, in contrast to doctrine, produce network activity splayed out in time instead of synchronized across neurons. Experimental and mathematical evidence support a generic mechanism for network desynchronization that relies on weak electrical synapses and specific excitability dynamics of the coupled neurons. In small networks, electrical synapses can synchronize or desynchronize network activity, depending on the neuron-intrinsic dynamics and ion channel composition. In the asynchronous flight CPG, this mechanism translates unpatterned premotor input into stereotyped neuronal firing with fixed sequences of cell activation that ensure stable wingbeat power and, as we show, is conserved across multiple species. Our findings prove a wider functional versatility of electrical synapses in the dynamic control of neural circuits and highlight the relevance of detecting electrical synapses in connectomics.
Assuntos
Drosophila melanogaster , Sinapses Elétricas , Voo Animal , Junções Comunicantes , Vias Neurais , Animais , Sinapses Elétricas/fisiologia , Fenômenos Eletrofisiológicos , Voo Animal/fisiologia , Junções Comunicantes/metabolismo , Neurônios Motores/fisiologia , Drosophila melanogaster/fisiologiaRESUMO
Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca2+ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)-triggered presynaptic Ca2+ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Cav2 homolog, Dmca1A or cacophony, and the Cav1 homolog, Dmca1D) but with spatial and functional separation. Cav2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Cav1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca2+ influx but has a small impact on release. Instead, L-type calcium currents through Cav1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca2+ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Cav1-mediated Ca2+ influx. We show that the plasma membrane Ca2+ ATPase (PMCA) resides in between active zones and isolates Cav2-triggered release from Cav1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Cav2 may also contribute to. As L-type Cav1 channels also localize next to PQ-type Cav2 channels within axon terminals of some central mammalian synapses, we propose that Cav2, Cav1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Endocitose , Exocitose , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Vesículas Sinápticas/metabolismo , Potenciais de Ação , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Neurônios Motores/metabolismo , Terminações Pré-Sinápticas , Probabilidade , Receptores de Glutamato/metabolismoRESUMO
Adrenergic signaling profoundly modulates animal behavior. For example, the invertebrate counterpart of norepinephrine, octopamine, and its biological precursor and functional antagonist, tyramine, adjust motor behavior to different nutritional states. In Drosophila larvae, food deprivation increases locomotor speed via octopamine-mediated structural plasticity of neuromuscular synapses, whereas tyramine reduces locomotor speed, but the underlying cellular and molecular mechanisms remain unknown. We show that tyramine is released into the CNS to reduce motoneuron intrinsic excitability and responses to excitatory cholinergic input, both by tyraminehonoka receptor activation and by downstream decrease of L-type calcium current. This central effect of tyramine on motoneurons is required for the adaptive reduction of locomotor activity after feeding. Similarly, peripheral octopamine action on motoneurons has been reported to be required for increasing locomotion upon starvation. We further show that the level of tyramine-ß-hydroxylase (TBH), the enzyme that converts tyramine into octopamine in aminergic neurons, is increased by food deprivation, thus selecting between antagonistic amine actions on motoneurons. Therefore, octopamine and tyramine provide global but distinctly different mechanisms to regulate motoneuron excitability and behavioral plasticity, and their antagonistic actions are balanced within a dynamic range by nutritional effects on TBH.
Assuntos
Oxigenases de Função Mista/genética , Neurônios Motores/metabolismo , Octopamina/genética , Receptores de Amina Biogênica/genética , Tiramina/metabolismo , Animais , Comportamento Animal/fisiologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Privação de Alimentos/fisiologia , Larva/metabolismo , Larva/fisiologia , Locomoção/genética , Locomoção/fisiologia , Oxigenases de Função Mista/metabolismo , Neurônios Motores/fisiologia , Estado Nutricional/genética , Estado Nutricional/fisiologia , Octopamina/metabolismo , Receptores de Amina Biogênica/metabolismo , Sinapses/metabolismo , Sinapses/fisiologiaRESUMO
Neuronal excitability is determined by the combination of different ion channels and their sub-neuronal localization. This study utilizes protein trap fly strains with endogenously tagged channels to analyze the spatial expression patterns of the four Shaker-related voltage-gated potassium channels, Kv1-4, in the larval, pupal, and adult Drosophila ventral nerve cord. We find that all four channels (Shaker, Kv1; Shab, Kv2; Shaw, Kv3; and Shal, Kv4) each show different spatial expression patterns in the Drosophila ventral nerve cord and are predominantly targeted to different sub-neuronal compartments. Shaker is abundantly expressed in axons, Shab also localizes to axons but mostly in commissures, Shaw expression is restricted to distinct parts of neuropils, and Shal is found somatodendritically, but also in axons of identified motoneurons. During early pupal life expression of all four Shaker-related channels is markedly decreased with an almost complete shutdown of expression at early pupal stage 5 (â¼30% through metamorphosis). Re-expression of Kv1-4 channels at pupal stage 6 starts with abundant channel localization in neuronal somata, followed by channel targeting to the respective sub-neuronal compartments until late pupal life. The developmental time course of tagged Kv1-4 channel expression corresponds with previously published data on developmental changes in single neuron physiology, thus indicating that protein trap fly strains are a useful tool to analyze developmental regulation of potassium channel expression. Finally, we take advantage of the large diameter of the giant fiber (GF) interneuron to map channel expression onto the axon and axon terminals of an identified interneuron. Shaker, Shaw, and Shal but not Shab channels localize to the non-myelinated GF axonal membrane and axon terminals. This study constitutes a first step toward systematically analyzing sub-neuronal potassium channel localization in Drosophila. Functional implications as well as similarities and differences to Kv1-4 channel localization in mammalian neurons are discussed.
Assuntos
Metamorfose Biológica/fisiologia , Neurogênese/fisiologia , Neurônios/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , DrosophilaRESUMO
Behaviorally adequate neuronal firing patterns are critically dependent on the specific types of ion channel expressed and on their subcellular localization. This study combines in situ electrophysiology with genetic and pharmacological intervention in larval Drosophila melanogaster of both sexes to address localization and function of L-type like calcium channels in motoneurons. We demonstrate that Dmca1D (Cav1 homolog) L-type like calcium channels localize to both the somatodendritic and the axonal compartment of larval crawling motoneurons. In situ patch-clamp recordings in genetic mosaics reveal that Dmca1D channels increase burst duration and maximum intraburst firing frequencies during crawling-like motor patterns in semi-intact animals. Genetic and acute pharmacological manipulations suggest that prolonged burst durations are caused by dendritically localized Dmca1D channels, which activate upon cholinergic synaptic input and amplify EPSPs, thus indicating a conserved function of dendritic L-type channels from Drosophila to vertebrates. By contrast, maximum intraburst firing rates require axonal calcium influx through Dmca1D channels, likely to enhance sodium channel de-inactivation via a fast afterhyperpolarization through BK channel activation. Therefore, in unmyelinated Drosophila motoneurons different functions of axonal and dendritic L-type like calcium channels likely operate synergistically to maximize firing output during locomotion.SIGNIFICANCE STATEMENT Nervous system function depends on the specific excitabilities of different types of neurons. Excitability is largely shaped by different combinations of voltage-dependent ion channels. Despite a high degree of conservation, the huge diversity of ion channel types and their differential localization pose challenges in assigning distinct functions to specific channels across species. We find a conserved role, from fruit flies to mammals, for L-type calcium channels in augmenting motoneuron excitability. As in spinal cord, dendritic L-type channels amplify excitatory synaptic input. In contrast to spinal motoneurons, axonal L-type channels enhance firing rates in unmyelinated Drosophila motoraxons. Therefore, enhancing motoneuron excitability by L-type channels seems an old strategy, but localization and interactions with other channels are tuned to species-specific requirements.
Assuntos
Axônios/fisiologia , Canais de Cálcio/fisiologia , Células Dendríticas/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Locomoção/fisiologia , Neurônios Motores/fisiologia , Animais , Canais de Cálcio/genética , Proteínas de Drosophila/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Larva/fisiologia , Canais de Sódio/efeitos dos fármacos , Sinapses/fisiologiaRESUMO
Dendrites are highly complex 3D structures that define neuronal morphology and connectivity and are the predominant sites for synaptic input. Defects in dendritic structure are highly consistent correlates of brain diseases. However, the precise consequences of dendritic structure defects for neuronal function and behavioral performance remain unknown. Here we probe dendritic function by using genetic tools to selectively abolish dendrites in identified Drosophila wing motoneurons without affecting other neuronal properties. We find that these motoneuron dendrites are unexpectedly dispensable for synaptic targeting, qualitatively normal neuronal activity patterns during behavior, and basic behavioral performance. However, significant performance deficits in sophisticated motor behaviors, such as flight altitude control and switching between discrete courtship song elements, scale with the degree of dendritic defect. To our knowledge, our observations provide the first direct evidence that complex dendrite architecture is critically required for fine-tuning and adaptability within robust, evolutionarily constrained behavioral programs that are vital for mating success and survival. We speculate that the observed scaling of performance deficits with the degree of structural defect is consistent with gradual increases in intellectual disability during continuously advancing structural deficiencies in progressive neurological disorders.
Assuntos
Comportamento Animal/fisiologia , Dendritos/fisiologia , Drosophila melanogaster/fisiologia , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Animais , Voo Animal/fisiologia , Imuno-Histoquímica , Microscopia Confocal , Técnicas de Patch-Clamp , Estatísticas não Paramétricas , Asas de Animais/inervaçãoRESUMO
Using a Drosophila model of MECP2 gain-of-function, we identified memory associated KIBRA as a target of MECP2 in regulating dendritic growth. We found that expression of human MECP2 increased kibra expression in Drosophila, and targeted RNAi knockdown of kibra in identified neurons fully rescued dendritic defects as induced by MECP2 gain-of-function. Validation in mouse confirmed that Kibra is similarly regulated by Mecp2 in a mammalian system. We found that Mecp2 gain-of-function in cultured mouse cortical neurons caused dendritic impairments and increased Kibra levels. Accordingly, Mecp2 loss-of-function in vivo led to decreased Kibra levels in hippocampus, cortex, and cerebellum. Together, our results functionally link two neuronal genes of high interest in human health and disease and highlight the translational utility of the Drosophila model for understanding MECP2 function.
Assuntos
Córtex Cerebral/patologia , Hipocampo/patologia , Memória/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , Neurônios/metabolismo , Animais , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Hipocampo/metabolismo , Humanos , CamundongosRESUMO
Neural activity has profound effects on the development of dendritic structure. Mechanisms that link neural activity to nuclear gene expression include activity-regulated factors, such as CREB, Crest or Mef2, as well as activity-regulated immediate-early genes, such as fos and jun. This study investigates the role of the transcriptional regulator AP-1, a Fos-Jun heterodimer, in activity-dependent dendritic structure development. We combine genetic manipulation, imaging and quantitative dendritic architecture analysis in a Drosophila single neuron model, the individually identified motoneuron MN5. First, Dα7 nicotinic acetylcholine receptors (nAChRs) and AP-1 are required for normal MN5 dendritic growth. Second, AP-1 functions downstream of activity during MN5 dendritic growth. Third, using a newly engineered AP-1 reporter we demonstrate that AP-1 transcriptional activity is downstream of Dα7 nAChRs and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Fourth, AP-1 can have opposite effects on dendritic development, depending on the timing of activation. Enhancing excitability or AP-1 activity after MN5 cholinergic synapses and primary dendrites have formed causes dendritic branching, whereas premature AP-1 expression or induced activity prior to excitatory synapse formation disrupts dendritic growth. Finally, AP-1 transcriptional activity and dendritic growth are affected by MN5 firing only during development but not in the adult. Our results highlight the importance of timing in the growth and plasticity of neuronal dendrites by defining a developmental period of activity-dependent AP-1 induction that is temporally locked to cholinergic synapse formation and dendritic refinement, thus significantly refining prior models derived from chronic expression studies.
Assuntos
Dendritos/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Neurônios Colinérgicos/metabolismo , Dendritos/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Ativação Enzimática , Genes Reporter , Imuno-Histoquímica/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Transdução de Sinais , Transmissão Sináptica , Fatores de Tempo , Fator de Transcrição AP-1/genética , Ativação TranscricionalRESUMO
Down syndrome cell adhesion molecule, Dscam, serves diverse neurodevelopmental functions, including axon guidance and synaptic adhesion, as well as self-recognition and self-avoidance, depending on the neuron type, brain region, or species under investigation. In Drosophila, the extensive molecular diversity that results from alternative splicing of Dscam1 into >38,000 isoforms provides neurons with a unique molecular code for self-recognition in the nervous system. Each neuron produces only a small subset of Dscam1 isoforms, and distinct Dscam1 isoforms mediate homophilic interactions, which in turn, result in repulsion and even spacing of self-processes, while allowing contact with neighboring cells. While these mechanisms have been shown to underlie mushroom body development and spacing of mechanosensory neuron dendrites, here we report that Dscam1 plays no role in adult Drosophila motoneuron dendrite spacing, but is required for motoneuron dendritic growth. Targeted expression of Dscam-RNAi in an identified flight motoneuron did not impact dendrite spacing, but instead produced overgrowth. Increasing the knockdown strength severely reduced dendritic growth and branching. Similarly, Dscam mutant motoneurons in an otherwise control background (MARCM) were completely devoid of mature dendrites. These data suggest that Dscam1 is required cell autonomously for normal adult motoneuron dendrite growth in Drosophila. This demonstrates a previously unreported role of Drosophila Dscam1 in central neuron development, and expands the current understanding that Dscam1 operates as a cell adhesion molecule that mediates homophilic repulsion.
Assuntos
Processamento Alternativo/genética , Moléculas de Adesão Celular/metabolismo , Dendritos/fisiologia , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios Motores/citologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular/genética , Dendritos/genética , Drosophila , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Neurônios Motores/fisiologia , Fibras Musculares Esqueléticas/citologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA/fisiologiaRESUMO
KEY POINTS: We combine in situ electrophysiology with genetic manipulation in Drosophila larvae aiming to investigate the role of fast calcium-activated potassium currents for motoneurone firing patterns during locomotion. We first demonstrate that slowpoke channels underlie fast calcium-activated potassium currents in these motoneurones. By conducting recordings in semi-intact animals that produce crawling-like movements, we show that slowpoke channels are required specifically in motoneurones for maximum firing rates during locomotion. Such enhancement of maximum firing rates occurs because slowpoke channels prevent depolarization block by limiting the amplitude of motoneurone depolarization in response to synaptic drive. In addition, slowpoke channels mediate a fast afterhyperpolarization that ensures the efficient recovery of sodium channels from inactivation during high frequency firing. The results of the present study provide new insights into the mechanisms by which outward conductances facilitate neuronal excitability and also provide direct confirmation of the functional relevance of precisely regulated slowpoke channel properties in motor control. ABSTRACT: A large number of voltage-gated ion channels, their interactions with accessory subunits, and their post-transcriptional modifications generate an immense functional diversity of neurones. Therefore, a key challenge is to understand the genetic basis and precise function of specific ionic conductances for neuronal firing properties in the context of behaviour. The present study identifies slowpoke (slo) as exclusively mediating fast activating, fast inactivating BK current (ICF ) in larval Drosophila crawling motoneurones. Combining in vivo patch clamp recordings during larval crawling with pharmacology and targeted genetic manipulations reveals that ICF acts specifically in motoneurones to sculpt their firing patterns in response to a given input from the central pattern generating (CPG) networks. First, ICF curtails motoneurone postsynaptic depolarizations during rhythmical CPG drive. Second, ICF is activated during the rising phase of the action potential and mediates a fast afterhyperpolarization. Consequently, ICF is required for maximal intraburst firing rates during locomotion, probably by allowing recovery from inactivation of fast sodium channels and decreased potassium channel activation. This contrasts the common view that outward conductances oppose excitability but is in accordance with reports on transient BK and Kv3 channel function in multiple types of vertebrate neurones. Therefore, our finding that ICF enhances firing rates specifically during bursting patterns relevant to behaviour is probably of relevance to all brains.
Assuntos
Potenciais de Ação , Proteínas de Drosophila/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Locomoção , Neurônios Motores/fisiologia , Animais , Geradores de Padrão Central/metabolismo , Geradores de Padrão Central/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Larva/metabolismo , Larva/fisiologia , Neurônios Motores/metabolismoRESUMO
During metamorphosis the CNS undergoes profound changes to accommodate the switch from larval to adult behaviors. In Drosophila and other holometabolous insects, adult neurons differentiate either from respecified larval neurons, newly born neurons, or are born embryonically but remain developmentally arrested until differentiation during pupal life. This study addresses the latter in the identified Drosophila flight motoneuron 5. In situ patch-clamp recordings, intracellular dye fills and immunocytochemistry address the interplay between dendritic shape, excitability and ionic current development. During pupal life, changes in excitability and spike shape correspond to a stereotyped, progressive appearance of voltage-gated ion channels. High-voltage-activated calcium current is the first current to appear at pupal stage P4, prior to the onset of dendrite growth. This is followed by voltage-gated sodium as well as transient potassium channel expression, when first dendrites grow, and sodium-dependent action potentials can be evoked by somatic current injection. Sustained potassium current appears later than transient potassium current. During the early stages of rapid dendritic growth, sodium-dependent action potentials are broadened by a calcium component. Narrowing of spike shape coincides with sequential increases in transient and sustained potassium currents during stages when dendritic growth ceases. Targeted RNAi knockdown of pupal calcium current significantly reduces dendritic growth. These data indicate that the stereotyped sequential acquisition of different voltage-gated ion channels affects spike shape and excitability such that activity-dependent calcium influx serves as a partner of genetic programs during critical stages of motoneuron dendrite growth.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Metamorfose Biológica/fisiologia , Neurônios Motores/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Sódio/metabolismo , Animais , Crescimento Celular , Dendritos/fisiologia , Drosophila melanogaster , Imuno-Histoquímica , Potenciais da Membrana/fisiologia , Microscopia Confocal , Neurônios Motores/citologia , Imagem Óptica , Técnicas de Patch-Clamp , Potássio/metabolismoRESUMO
Background: Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. Methods: We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. Results: We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Conclusions: Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.
RESUMO
Motor and cognitive aging can severely affect life quality of elderly people and burden health care systems. In search for diagnostic behavioral biomarkers, it has been suggested that walking speed can predict forms of cognitive decline, but in humans, it remains challenging to separate the effects of biological aging and lifestyle. We examined a possible association of motor and cognitive decline in Drosophila, a genetic model organism of healthy aging. Long term courtship memory is present in young male flies but absent already during mid life (4-8 weeks). By contrast, courtship learning index and short term memory (STM) are surprisingly robust and remain stable through mid (4-8 weeks) and healthy late life (>8 weeks), until courtship performance collapses suddenly at ~4.5 days prior to death. By contrast, climbing speed declines gradually during late life (>8 weeks). The collapse of courtship performance and short term memory close to the end of life occur later and progress with a different time course than the gradual late life decline in climbing speed. Thus, during healthy aging in male Drosophila, climbing and courtship motor behaviors decline differentially. Moreover, cognitive and motor performances decline at different time courses. Differential behavioral decline during aging may indicate different underlying causes, or alternatively, a common cause but different thresholds for defects in different behaviors.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Masculino , Humanos , Idoso , Drosophila melanogaster/genética , Corte , Instinto , Drosophila/genética , Envelhecimento/psicologia , Proteínas de Drosophila/genéticaRESUMO
Input-output computations of individual neurons may be affected by the three-dimensional structure of their dendrites and by the location of input synapses on specific parts of their dendrites. However, only a few examples exist of dendritic architecture which can be related to behaviorally relevant computations of a neuron. By combining genetic, immunohistochemical and confocal laser scanning methods this study estimates the location of the spike-initiating zone and the dendritic distribution patterns of putative synaptic inputs on an individually identified Drosophila flight motorneuron, MN5. MN5 is a monopolar neuron with > 4,000 dendritic branches. The site of spike initiation was estimated by mapping sodium channel immunolabel onto geometric reconstructions of MN5. Maps of putative excitatory cholinergic and of putative inhibitory GABAergic inputs on MN5 dendrites were created by charting tagged Dα7 nicotinic acetylcholine receptors and Rdl GABAA receptors onto MN5 dendritic surface reconstructions. Although these methods provide only an estimate of putative input synapse distributions, the data indicate that inhibitory and excitatory synapses were located preferentially on different dendritic domains of MN5 and, thus, computed mostly separately. Most putative inhibitory inputs were close to spike initiation, which was consistent with sharp inhibition, as predicted previously based on recordings of motoneuron firing patterns during flight. By contrast, highest densities of putative excitatory inputs at more distant dendritic regions were consistent with the prediction that, in response to different power demands during flight, tonic excitatory drive to flight motoneuron dendrites must be smoothly translated into different tonic firing frequencies.
Assuntos
Dendritos/metabolismo , Drosophila melanogaster/metabolismo , Neurônios Motores/metabolismo , Potenciais de Ação , Animais , Dendritos/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Transporte Proteico , Receptores de GABA-A/metabolismo , Receptores Nicotínicos/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Neurons show diverse firing patterns. Even neurons belonging to a single chemical or morphological class, or the same identified neuron, can display different types of electrical activity. For example, motor neuron MN5, which innervates a flight muscle of adult Drosophila, can show distinct firing patterns under the same recording conditions. We developed a two-dimensional biophysical model and show that a core complement of just two voltage-gated channels is sufficient to generate firing pattern diversity. We propose Shab and DmNa v to be two candidate genes that could encode these core currents, and find that changes in Shab channel expression in the model can reproduce activity resembling the main firing patterns observed in MN5 recordings. We use bifurcation analysis to describe the different transitions between rest and spiking states that result from variations in Shab channel expression, exposing a connection between ion channel expression, bifurcation structure, and firing patterns in models of membrane potential dynamics.
Assuntos
Potenciais de Ação/fisiologia , Canais Iônicos/metabolismo , Modelos Neurológicos , Neurônios Motores/fisiologia , Potenciais de Ação/genética , Animais , Animais Geneticamente Modificados , Biofísica , Simulação por Computador , Proteínas de Drosophila/genética , Drosophila melanogaster , Estimulação Elétrica , Proteínas de Fluorescência Verde/genética , Técnicas de Patch-Clamp , Fatores de Transcrição/genéticaRESUMO
Different blends of membrane currents underlie distinct functions of neurons in the brain. A major step towards understanding neuronal function, therefore, is to identify the genes that encode different ionic currents. This study combined in situ patch clamp recordings of somatodendritic calcium currents in an identified adult Drosophila motoneuron with targeted genetic manipulation. Voltage clamp recordings revealed transient low voltage-activated (LVA) currents with activation between 60 mV and 70 mV as well as high voltage-activated (HVA) current with an activation voltage around 30 mV. LVA could be fully inactivated by prepulses to 50 mV and was partially amiloride sensitive. Recordings from newly generated mutant flies demonstrated that DmαG (Ca(v)3 homolog) encoded the amiloride-sensitive portion of the transient LVA calcium current. We further demonstrated that the Ca(v)2 homolog, Dmca1A, mediated the amiloride-insensitive component of LVA current. This novel role of Ca(v)2 channels was substantiated by patch clamp recordings from conditional mutants, RNAi knock-downs, and following Dmca1A overexpression. In addition, we show that Dmca1A underlies the HVA somatodendritic calcium currents in vivo. Therefore, the Drosophila Ca(v)2 homolog, Dmca1A, underlies HVA and LVA somatodendritic calcium currents in the same neuron. Interestingly, DmαG is required for regulating LVA and HVA derived from Dmca1A in vivo. In summary, each vertebrate gene family for voltage-gated calcium channels is represented by a single gene in Drosophila, namely Dmca1D (Ca(v)1), Dmca1A (Ca(v)2) and DmαG (Ca(v)3), but the commonly held view that LVA calcium currents are usually mediated by Ca(v)3 rather than Ca(v)2 channels may require reconsideration.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Neurônios Motores/fisiologia , Animais , Modelos GenéticosRESUMO
Skeletal muscle innervation differs between vertebrates and insects. Insect muscle fibers exhibit graded electrical potentials and are innervated by excitatory, inhibitory, and also neuromodulatory motoneurons. The latter form a unique class of unpaired neurons with bilaterally symmetrical axons that release octopamine to alter the efficacy of synaptic transmission and regulate muscle energy metabolism by activating glycolysis. Octopaminergic neurons that innervate muscles with a high energy demand, for example, flight muscles that move the wings of a locust up and down, are active during rest but are inhibited during flight and its preparatory phase, a jump. Therefore, it is argued that these neurons are involved in providing locusts with the necessary fuel at takeoff, but then may aid the switch to lipid oxidation during flight. In general, the octopaminergic system may switch the whole organism from a tonic to a dynamic state.
Assuntos
Insetos/fisiologia , Atividade Motora/fisiologia , Neurônios Motores/fisiologia , Músculo Esquelético/fisiologia , Animais , Humanos , Transmissão SinápticaRESUMO
Two key features endow Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) with the potential to provide a ubiquitous code for neuronal arbor self-avoidance. First, Dscam1 contains three large cassettes of alternative exons, so that stochastic alternative splicing yields 19,008 Dscam1 isoforms with different Ig ectodomains. Second, each neuron expresses a different subset of Dscam1 isoforms, and isoform-specific homophilic binding causes repulsion. This results in even spacing of self-arbors, while processes of other neurons can intermingle and share the same synaptic partners. In principle, this Dscam1 code could ensure arbor spacing of all neurons in Drosophila This model is strongly supported by studies on dendrite spacing in the peripheral nervous system and studies on axonal branch segregation during brain development. However, the situation is less clear for central neuron dendrites, the major substrate for synaptic input in the CNS. We systematically tested the role of Dscam1 for dendrite growth and spacing in eight different types of identified central neurons. Knockdown of Dscam1 causes severe dendritic clumping and length reductions in efferent glutamatergic and aminergic neurons. The primary cause for these dendritic phenotypes could be impaired self-avoidance, a growth defect, or both. In peptidergic efferent neurons, many central arbors are not formed, arguing for a growth defect. By contrast, knockdown of Dscam1 does not affect dendrite growth or spacing in any of the five different types of interneurons tested. Axon arbor patterning is not affected in any neuron type tested. We conclude that Dscam1 mediates diverse, neuron type-specific functions during central neuron arbor differentiation.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Moléculas de Adesão Celular , Dendritos , Neurônios , Isoformas de ProteínasRESUMO
Variability of synapse numbers and partners despite identical genes reveals the limits of genetic determinism. Here, we use developmental temperature as a non-genetic perturbation to study variability of brain wiring and behavior in Drosophila. Unexpectedly, slower development at lower temperatures increases axo-dendritic branching, synapse numbers, and non-canonical synaptic partnerships of various neurons, while maintaining robust ratios of canonical synapses. Using R7 photoreceptors as a model, we show that changing the relative availability of synaptic partners using a DIPγ mutant that ablates R7's preferred partner leads to temperature-dependent recruitment of non-canonical partners to reach normal synapse numbers. Hence, R7 synaptic specificity is not absolute but based on the relative availability of postsynaptic partners and presynaptic control of synapse numbers. Behaviorally, movement precision is temperature robust, while movement activity is optimized for the developmentally encountered temperature. These findings suggest genetically encoded relative and scalable synapse formation to develop functional, but not identical, brains and behaviors.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Temperatura , Adaptação Fisiológica , Animais , Axônios/metabolismo , Proteínas de Drosophila/metabolismo , Neurogênese , Células Fotorreceptoras de Invertebrados/metabolismoRESUMO
This study analyses the maturation of centrally generated flight motor patterns during metamorphosis of Manduca sexta. Bath application of the octopamine agonist chlordimeform to the isolated central nervous system of adult moths reliably induces fictive flight patterns in wing depressor and elevator motoneurons. Pattern maturation is investigated by chlordimeform application at different developmental stages. Chlordimeform also induces motor patterns in larval ganglia, which differ from fictive flight, indicating that in larvae and adults, octopamine affects different networks. First changes in motoneuron activity occur at the pupal stage P10. Rhythmic motor output is induced in depressor, but not in elevator motoneurons at P12. Adult-like fictive flight activity in motoneurons is observed at P16 and increases in speed and precision until emergence 2 days later. Pharmacological block of chloride channels with picrotoxin also induces fictive flight in adults, suggesting that the pattern-generating network can be activated by the removal of inhibition, and that proper network function does not rely on GABA(A) receptors. Our results suggest that the flight pattern-generating network becomes gradually established between P12 and P16, and is further refined until adulthood. These findings are discussed in the context of known physiological and structural CNS development during Manduca metamorphosis.