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1.
Nature ; 618(7963): 118-125, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37225999

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/fisiologia
2.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34244444

RESUMO

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/metabolismo
3.
Proc Natl Acad Sci U S A ; 116(9): 3805-3810, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808766

RESUMO

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/fisiologia
4.
J Neurogenet ; 34(1): 133-150, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31997675

RESUMO

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 (∼30% 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 , Drosophila
5.
J Neurosci ; 37(45): 10971-10982, 2017 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-28986465

RESUMO

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/fisiologia
6.
Proc Natl Acad Sci U S A ; 111(50): 18049-54, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25453076

RESUMO

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/inervação
7.
J Physiol ; 593(22): 4871-88, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26332699

RESUMO

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/metabolismo
8.
Eur J Neurosci ; 39(10): 1572-85, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24620836

RESUMO

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/metabolismo
9.
J Comput Neurosci ; 34(2): 211-29, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22878689

RESUMO

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/genética
10.
Sci Adv ; 9(7): eade7804, 2023 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-36800417

RESUMO

At presynaptic active zones (AZs), conserved scaffold protein architectures control synaptic vesicle (SV) release by defining the nanoscale distribution and density of voltage-gated Ca2+ channels (VGCCs). While AZs can potentiate SV release in the minutes range, we lack an understanding of how AZ scaffold components and VGCCs engage into potentiation. We here establish dynamic, intravital single-molecule imaging of endogenously tagged proteins at Drosophila AZs undergoing presynaptic homeostatic potentiation. During potentiation, the numbers of α1 VGCC subunit Cacophony (Cac) increased per AZ, while their mobility decreased and nanoscale distribution compacted. These dynamic Cac changes depended on the interaction between Cac channel's intracellular carboxyl terminus and the membrane-close amino-terminal region of the ELKS-family protein Bruchpilot, whose distribution compacted drastically. The Cac-ELKS/Bruchpilot interaction was also needed for sustained AZ potentiation. Our single-molecule analysis illustrates how the AZ scaffold couples to VGCC nanoscale distribution and dynamics to establish a state of sustained potentiation.


Assuntos
Proteínas de Drosophila , Sinapses , Animais , Sinapses/metabolismo , Drosophila/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Drosophila/metabolismo , Transmissão Sináptica
11.
J Physiol ; 590(4): 809-25, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22183725

RESUMO

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 Genéticos
12.
PLoS Biol ; 5(4): e66, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17341135

RESUMO

Neuronal calcium acts as a charge carrier during information processing and as a ubiquitous intracellular messenger. Calcium signals are fundamental to numerous aspects of neuronal development and plasticity. Specific and independent regulation of these vital cellular processes is achieved by a rich bouquet of different calcium signaling mechanisms within the neuron, which either can operate independently or may act in concert. This study demonstrates the existence of a novel calcium signaling mechanism by simultaneous patch clamping and calcium imaging from acutely isolated central neurons. These neurons possess a membrane voltage sensor that, independent of calcium influx, causes G-protein activation, which subsequently leads to calcium release from intracellular stores via phospholipase C and inositol 1,4,5-trisphosphate receptor activation. This allows neurons to monitor activity by intracellular calcium release without relying on calcium as the input signal and opens up new insights into intracellular signaling, developmental regulation, and information processing in neuronal compartments lacking calcium channels.


Assuntos
Sinalização do Cálcio , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Neurônios/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Canais de Cálcio/metabolismo , Ativação Enzimática , Gafanhotos , Microscopia de Fluorescência , Neurônios/enzimologia , Técnicas de Patch-Clamp
13.
Sci Rep ; 10(1): 13670, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792569

RESUMO

Voltage gated calcium channels (VGCCs) regulate neuronal excitability and translate activity into calcium dependent signaling. The α1 subunit of high voltage activated (HVA) VGCCs associates with α2δ accessory subunits, which may affect calcium channel biophysical properties, cell surface expression, localization and transport and are thus important players in calcium-dependent signaling. In vertebrates, the functions of the different combinations of the four α2δ and the seven HVA α1 subunits are incompletely understood, in particular with respect to partially redundant or separate functions in neurons. This study capitalizes on the relatively simpler situation in the Drosophila genetic model containing two neuronal putative α2δ subunits, straightjacket and CG4587, and one Cav1 and Cav2 homolog each, both with well-described functions in different compartments of identified motoneurons. Straightjacket is required for normal Cav1 and Cav2 current amplitudes and correct Cav2 channel function in all neuronal compartments. By contrast, CG4587 does not affect Cav1 or Cav2 current amplitudes or presynaptic function, but is required for correct Cav2 channel allocation to the axonal versus the dendritic domain. We suggest that the two different putative α2δ subunits are required in the same neurons to regulate different functions of VGCCs.


Assuntos
Canais de Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Neurônios Motores/metabolismo , Animais , Axônios/metabolismo , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Dendritos/metabolismo , Feminino , Masculino
14.
J Neurophysiol ; 102(6): 3673-88, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19828724

RESUMO

Ionic currents underlie the firing patterns, excitability, and synaptic integration of neurons. Despite complete sequence information in multiple species, our knowledge about ion channel function in central neurons remains incomplete. This study analyzes the potassium currents of an identified Drosophila flight motoneuron, MN5, in situ. MN5 exhibits four different potassium currents, two fast-activating transient ones and two sustained ones, one of each is calcium activated. Pharmacological and genetic manipulations unravel the specific contributions of Shaker and Shal to the calcium independent transient A-type potassium currents. alpha-dendrotoxin (Shaker specific) and phrixotoxin-2 (Shal specific) block different portions of the transient calcium independent A-type potassium current. Following targeted expression of a Shaker dominant negative transgene in MN5, the remaining A-type potassium current is alpha-dendrotoxin insensitive. In Shal RNAi knock down the remaining A-type potassium current is phrixotoxin-2 insensitive. Additionally, barium blocks calcium-activated potassium currents but also a large portion of phrixotoxin-2-sensitive A-type currents. Targeted knock down of Shaker or Shal channels each cause identical reduction in total potassium current amplitude as acute application of alpha-dendrotoxin or phrixotoxin-2, respectively. This shows that the knock downs do not cause upregulation of potassium channels underlying other A-type channels during development. Immunocytochemistry and targeted expression of modified GFP-tagged Shaker channels with intact targeting sequence in MN5 indicate predominant axonal localization. These data can now be used to investigate the roles of Shaker and Shal for motoneuron intrinsic properties, synaptic integration, and spiking output during behavior by targeted genetic manipulations.


Assuntos
Proteínas de Drosophila/metabolismo , Voo Animal/fisiologia , Neurônios Motores/fisiologia , Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Canais de Potássio Shal/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Biofísica , Cádmio/farmacologia , Drosophila , Proteínas de Drosophila/genética , Venenos Elapídicos/farmacologia , Estimulação Elétrica , Voo Animal/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/citologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/fisiologia , RNA Interferente Pequeno/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetraetilamônio/farmacologia , Tetrodotoxina/farmacologia
15.
Front Syst Neurosci ; 11: 68, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29021745

RESUMO

The biogenic amines octopamine (OA) and tyramine (TA) modulate insect motor behavior in an antagonistic manner. OA generally enhances locomotor behaviors such as Drosophila larval crawling and flight, whereas TA decreases locomotor activity. However, the mechanisms and cellular targets of TA modulation of locomotor activity are incompletely understood. This study combines immunocytochemistry, genetics and flight behavioral assays in the Drosophila model system to test the role of a candidate enzyme for TA catabolism, named Nazgul (Naz), in flight motor behavioral control. We hypothesize that the dehydrogenase/reductase Naz represents a critical step in TA catabolism. Immunocytochemistry reveals that Naz is localized to a subset of Repo positive glial cells with cell bodies along the motor neuropil borders and numerous positive Naz arborizations extending into the synaptic flight motor neuropil. RNAi knock down of Naz in Repo positive glial cells reduces Naz protein level below detection level by Western blotting. The resulting consequence is a reduction in flight durations, thus mimicking known motor behavioral phenotypes as resulting from increased TA levels. In accord with the interpretation that reduced TA degradation by Naz results in increased TA levels in the flight motor neuropil, the motor behavioral phenotype can be rescued by blocking TA receptors. Our findings indicate that TA modulates flight motor behavior by acting on central circuitry and that TA is normally taken up from the central motor neuropil by Repo-positive glial cells, desaminated and further degraded by Naz.

16.
Neuron ; 93(3): 632-645.e6, 2017 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-28132832

RESUMO

Brain development requires correct targeting of multiple thousand synaptic terminals onto staggeringly complex dendritic arbors. The mechanisms by which input synapse numbers are matched to dendrite size, and by which synaptic inputs from different transmitter systems are correctly partitioned onto a postsynaptic arbor, are incompletely understood. By combining quantitative neuroanatomy with targeted genetic manipulation of synaptic input to an identified Drosophila neuron, we show that synaptic inputs of two different transmitter classes locally direct dendrite growth in a competitive manner. During development, the relative amounts of GABAergic and cholinergic synaptic drive shift dendrites between different input domains of one postsynaptic neuron without affecting total arbor size. Therefore, synaptic input locally directs dendrite growth, but intra-neuronal dendrite redistributions limit morphological variability, a phenomenon also described for cortical neurons. Mechanistically, this requires local dendritic Ca2+ influx through Dα7nAChRs or through LVA channels following GABAAR-mediated depolarizations. VIDEO ABSTRACT.


Assuntos
Acetilcolina/metabolismo , Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Plasticidade Neuronal , Receptores de GABA-A/metabolismo , Receptores Nicotínicos/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Dendritos/fisiologia , Drosophila , Neurônios/metabolismo , Neurônios/fisiologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Sinapses/fisiologia
17.
J Vis Exp ; (68)2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23092999

RESUMO

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker(1,2). Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain(3,4) and ventral nerve cord of embryonic(5,6), larval(7,8,9,10), and adult Drosophila(11,12,13,14). A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN5(15)), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.


Assuntos
Drosophila melanogaster/fisiologia , Neurônios Motores/fisiologia , Técnicas de Patch-Clamp/métodos , Animais , Dissecação/métodos , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Neurônios Motores/citologia
18.
PLoS One ; 7(2): e31835, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22363746

RESUMO

Methyl-CpG-binding protein 2 (MECP2) is a multi-functional regulator of gene expression. In humans loss of MECP2 function causes classic Rett syndrome, but gain of MECP2 function also causes mental retardation. Although mouse models provide valuable insight into Mecp2 gain and loss of function, the identification of MECP2 genetic targets and interactors remains time intensive and complicated. This study takes a step toward utilizing Drosophila as a model to identify genetic targets and cellular consequences of MECP2 gain-of function mutations in neurons, the principle cell type affected in patients with Rett-related mental retardation. We show that heterologous expression of human MECP2 in Drosophila motoneurons causes distinct defects in dendritic structure and motor behavior, as reported with MECP2 gain of function in humans and mice. Multiple lines of evidence suggest that these defects arise from specific MECP2 function. First, neurons with MECP2-induced dendrite loss show normal membrane currents. Second, dendritic phenotypes require an intact methyl-CpG-binding domain. Third, dendritic defects are amended by reducing the dose of the chromatin remodeling protein, osa, indicating that MECP2 may act via chromatin remodeling in Drosophila. MECP2-induced motoneuron dendritic defects cause specific motor behavior defects that are easy to score in genetic screening. In sum, our data show that some aspects of MECP2 function can be studied in the Drosophila model, thus expanding the repertoire of genetic reagents that can be used to unravel specific neural functions of MECP2. However, additional genes and signaling pathways identified through such approaches in Drosophila will require careful validation in the mouse model.


Assuntos
Drosophila melanogaster/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Neurônios/metabolismo , Animais , Comportamento Animal , Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Fenômenos Eletrofisiológicos , Humanos , Proteína 2 de Ligação a Metil-CpG/química , Camundongos , Modelos Animais , Atividade Motora , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Neurônios/citologia , Estrutura Terciária de Proteína
19.
J Neurophysiol ; 100(5): 2525-36, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18715893

RESUMO

Dendrites are the fundamental determinant of neuronal wiring. Consequently dendritic defects are associated with numerous neurological diseases and mental retardation. Neuronal activity can have profound effects on dendritic structure, but the mechanisms controlling distinct aspects of dendritic architecture are not fully understood. We use the Drosophila genetic model system to test the effects of altered intrinsic excitability on postembryonic dendritic architecture development. Targeted dominant negative knock-downs of potassium channel subunits allow for selectively increasing the intrinsic excitability of a selected subset of motoneurons, whereas targeted expression of a genetically modified noninactivating potassium channel decrease intrinsic excitability in vivo. Both manipulations cause significant dendritic overgrowth, but by different mechanisms. Increased excitability causes increased dendritic branch formation, whereas decreased excitability causes increased dendritic branch elongation. Therefore dendritic branching and branch elongation are controlled by separate mechanisms that can be addressed selectively in vivo by different manipulations of neuronal intrinsic excitability.


Assuntos
Dendritos/fisiologia , Neurônios Motores/classificação , Neurônios Motores/citologia , Análise de Variância , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Comportamento Animal , Antígenos CD8/genética , Antígenos CD8/metabolismo , Dendritos/efeitos dos fármacos , Dendritos/efeitos da radiação , Relação Dose-Resposta à Radiação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Estimulação Elétrica , Feminino , Gânglios dos Invertebrados/citologia , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Locomoção/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Atividade Motora/genética , Neurônios Motores/fisiologia , Técnicas de Patch-Clamp , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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