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1.
J Neurosci ; 40(9): 1819-1833, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-31964717

RESUMO

Dendritic arborization is highly regulated and requires tight control of dendritic growth, branching, cytoskeletal dynamics, and ion channel expression to ensure proper function. Abnormal dendritic development can result in altered network connectivity, which has been linked to neurodevelopmental disorders, including autism spectrum disorders (ASDs). How neuronal growth control programs tune dendritic arborization to ensure function is still not fully understood. Using Drosophila dendritic arborization (da) neurons as a model, we identified the conserved Ste20-like kinase Tao as a negative regulator of dendritic arborization. We show that Tao kinase activity regulates cytoskeletal dynamics and sensory channel localization required for proper sensory function in both male and female flies. We further provide evidence for functional conservation of Tao kinase, showing that its ASD-linked human ortholog, Tao kinase 2 (Taok2), could replace Drosophila Tao and rescue dendritic branching, dynamic microtubule alterations, and behavioral defects. However, several ASD-linked Taok2 variants displayed impaired rescue activity, suggesting that Tao/Taok2 mutations can disrupt sensory neuron development and function. Consistently, we show that Tao kinase activity is required in developing and as well as adult stages for maintaining normal dendritic arborization and sensory function to regulate escape and social behavior. Our data suggest an important role for Tao kinase signaling in cytoskeletal organization to maintain proper dendritic arborization and sensory function, providing a strong link between developmental sensory aberrations and behavioral abnormalities relevant for Taok2-dependent ASDs.SIGNIFICANCE STATEMENT Autism spectrum disorders (ASDs) are linked to abnormal dendritic arbors. However, the mechanisms of how dendritic arbors develop to promote functional and proper behavior are unclear. We identified Drosophila Tao kinase, the ortholog of the ASD risk gene Taok2, as a regulator of dendritic arborization in sensory neurons. We show that Tao kinase regulates cytoskeletal dynamics, controls sensory ion channel localization, and is required to maintain somatosensory function in vivo Interestingly, ASD-linked human Taok2 mutations rendered it nonfunctional, whereas its WT form could restore neuronal morphology and function in Drosophila lacking endogenous Tao. Our findings provide evidence for a conserved role of Tao kinase in dendritic development and function of sensory neurons, suggesting that aberrant sensory function might be a common feature of ASDs.


Assuntos
Citoesqueleto/fisiologia , Dendritos/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Sensação/fisiologia , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Citoesqueleto/ultraestrutura , Dendritos/ultraestrutura , Drosophila , Reação de Fuga , Feminino , Humanos , Masculino , Mecanorreceptores/fisiologia , Mutação/genética , Comportamento Social
2.
Commun Biol ; 5(1): 687, 2022 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-35810216

RESUMO

Optogenetic silencing allows to reveal the necessity of selected neuronal populations for various neurophysiological functions. These range from synaptic transmission and coordinated neuronal network activity to control of specific behaviors. An ideal single-component optogenetic silencing tool should be switchable between active and inactive states with precise timing while preserving its activity in the absence of light until switched to an inactive state. Although bistable anion-conducting channelrhodopsins (ACRs) were previously engineered to reach this goal, their conducting state lifetime was limited to only a few minutes and some ACRs were not fully switchable. Here we report Aion, a bistable ACR displaying a long-lasting open state with a spontaneous closing time constant close to 15 min. Moreover, Aion can be switched between the open and closed state with millisecond precision using blue and orange light, respectively. The long conducting state enables overnight silencing of neurons with minimal light exposure. We further generated trafficking-optimized versions of Aion, which show enhanced membrane localization and allow precisely timed, long-lasting all-optical control of nociceptive responses in larvae of Drosophila melanogaster. Thus, Aion is an optogenetic silencing tool for inhibition of neuronal activity over many hours which can be switched between an active and inactive state with millisecond precision.


Assuntos
Drosophila melanogaster , Optogenética , Animais , Ânions/metabolismo , Channelrhodopsins/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Neurônios/fisiologia
3.
Curr Biol ; 32(1): 149-163.e8, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-34798050

RESUMO

Animals display selective escape behaviors when faced with environmental threats. Selection of the appropriate response by the underlying neuronal network is key to maximizing chances of survival, yet the underlying network mechanisms are so far not fully understood. Using synapse-level reconstruction of the Drosophila larval network paired with physiological and behavioral readouts, we uncovered a circuit that gates selective escape behavior for noxious light through acute and input-specific neuropeptide action. Sensory neurons required for avoidance of noxious light and escape in response to harsh touch, each converge on discrete domains of neuromodulatory hub neurons. We show that acute release of hub neuron-derived insulin-like peptide 7 (Ilp7) and cognate relaxin family receptor (Lgr4) signaling in downstream neurons are required for noxious light avoidance, but not harsh touch responses. Our work highlights a role for compartmentalized circuit organization and neuropeptide release from regulatory hubs, acting as central circuit elements gating escape responses.


Assuntos
Proteínas de Drosophila , Neuropeptídeos , Animais , Drosophila/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Larva/fisiologia , Neuropeptídeos/genética , Nociceptores/fisiologia , Células Receptoras Sensoriais/fisiologia
4.
Nat Commun ; 12(1): 4527, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312384

RESUMO

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.


Assuntos
Membrana Celular/fisiologia , Opsinas/metabolismo , Optogenética/métodos , Células Piramidais/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Furões/genética , Furões/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Opsinas/genética , Técnicas de Patch-Clamp/métodos , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos Wistar , Reprodutibilidade dos Testes
5.
Nat Commun ; 10(1): 3506, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31383864

RESUMO

Sensory circuits are typically established during early development, yet how circuit specificity and function are maintained during organismal growth has not been elucidated. To gain insight we quantitatively investigated synaptic growth and connectivity in the Drosophila nociceptive network during larval development. We show that connectivity between primary nociceptors and their downstream neurons scales with animal size. We further identified the conserved Ste20-like kinase Tao as a negative regulator of synaptic growth required for maintenance of circuit specificity and connectivity. Loss of Tao kinase resulted in exuberant postsynaptic specializations and aberrant connectivity during larval growth. Using functional imaging and behavioral analysis we show that loss of Tao-induced ectopic synapses with inappropriate partner neurons are functional and alter behavioral responses in a connection-specific manner. Our data show that fine-tuning of synaptic growth by Tao kinase is required for maintaining specificity and behavioral output of the neuronal network during animal growth.


Assuntos
Comunicação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Rede Nervosa/metabolismo , Nociceptores/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Encéfalo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Técnicas de Silenciamento de Genes , Larva/metabolismo , Modelos Animais , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Sinapses/metabolismo
6.
Bio Protoc ; 8(4): e2736, 2018 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-34179264

RESUMO

Drosophila melanogaster larvae have been extensively used as a model to study the molecular and cellular basis of nociception. The larval nociceptors, class IV dendritic arborization (C4da) neurons, line the body wall of the animal and respond to various stimuli including noxious heat and touch. Activation of C4da neurons results in a stereotyped escape behavior, characterized by a 360° rolling response along the body axis followed by locomotion speedup. The genetic accessibility of Drosophila has allowed the identification of mechanosensory channels and circuit elements required for nociceptive responses, making it a useful and straightforward readout to understand the cellular and molecular basis of nociceptive function and behavior. We have optimized the protocol to assay mechanonociceptive behavior in Drosophila larvae.

7.
Bio Protoc ; 8(4): e2737, 2018 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-34179265

RESUMO

Thermo-nociception, the detection and behavioral response to noxious temperatures, is a highly conserved action to avoid injury and ensure survival. Basic molecular mechanisms of thermal responses have been elucidated in several model organisms and are of clinical relevance as thermal hypersensitivity (thermos-allodynia) is common in neuropathic pain syndromes. Drosophila larvae show stereotyped escape behavior upon noxious heat stimulation, which can be easily quantified and coupled with molecular genetic approaches. It has been successfully used to elucidate key molecular components and circuits involved in thermo-nociceptive responses. We provide a detailed and updated protocol of this previously described method ( Tracey et al., 2003 ) to apply a defined local heat stimulus to larvae using a fast-regulating hot probe.

8.
Sci Rep ; 8(1): 4765, 2018 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-29540835

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

9.
Cell Rep ; 21(12): 3346-3353, 2017 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-29262315

RESUMO

During differentiation, neurons require a high lipid supply for membrane formation as they elaborate complex dendritic morphologies. While glia-derived lipids support neuronal growth during development, the importance of cell-autonomous lipid production for dendrite formation has been unclear. Using Drosophila larva dendritic arborization (da) neurons, we show that dendrite expansion relies on cell-autonomous fatty acid production. The nociceptive class four (CIV) da neurons form particularly large space-filling dendrites. We show that dendrite formation in these CIVda neurons additionally requires functional sterol regulatory element binding protein (SREBP), a crucial regulator of fatty acid production. The dendrite simplification in srebp mutant CIVda neurons is accompanied by hypersensitivity of srebp mutant larvae to noxious stimuli. Taken together, our work reveals that cell-autonomous fatty acid production is required for proper dendritic development and establishes the role of SREBP in complex neurons for dendrite elaboration and function.


Assuntos
Dendritos/metabolismo , Crescimento Neuronal , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Dendritos/fisiologia , Drosophila , Ácidos Graxos/metabolismo , Nociceptividade , Proteínas de Ligação a Elemento Regulador de Esterol/genética
10.
Sci Rep ; 7(1): 14957, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29097684

RESUMO

Genetic engineering of natural light-gated ion channels has proven a powerful way to generate optogenetic tools for a wide variety of applications. In recent years, blue-light activated engineered anion-conducting channelrhodopsins (eACRs) have been developed, improved, and were successfully applied in vivo. We asked whether the approaches used to create eACRs can be transferred to other well-characterized cation-conducting channelrhodopsins (CCRs) to obtain eACRs with a broad spectrum of biophysical properties. We generated 22 variants using two conversion strategies applied to 11 CCRs and screened them for membrane expression, photocurrents and anion selectivity. We obtained two novel eACRs, Phobos and Aurora, with blue- and red-shifted action spectra and photocurrents similar to existing eACRs. Furthermore, step-function mutations greatly enhanced the cellular operational light sensitivity due to a slowed-down photocycle. These bi-stable eACRs can be reversibly toggled between open and closed states with brief light pulses of different wavelengths. All new eACRs reliably inhibited action potential firing in pyramidal CA1 neurons. In Drosophila larvae, eACRs conveyed robust and specific light-dependent inhibition of locomotion and nociception.


Assuntos
Potenciais de Ação , Ânions/metabolismo , Channelrhodopsins/genética , Optogenética/métodos , Engenharia de Proteínas/métodos , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Channelrhodopsins/metabolismo , Drosophila , Células HEK293 , Humanos , Cinética , Luz , Mutação , Neurônios/citologia , Neurônios/metabolismo
11.
Nat Neurosci ; 20(8): 1085-1095, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28604684

RESUMO

Nociception is an evolutionarily conserved mechanism to encode and process harmful environmental stimuli. Like most animals, Drosophila melanogaster larvae respond to a variety of nociceptive stimuli, including noxious touch and temperature, with stereotyped escape responses through activation of multimodal nociceptors. How behavioral responses to these different modalities are processed and integrated by the downstream network remains poorly understood. By combining trans-synaptic labeling, ultrastructural analysis, calcium imaging, optogenetics and behavioral analyses, we uncovered a circuit specific for mechanonociception but not thermonociception. Notably, integration of mechanosensory input from innocuous and nociceptive sensory neurons is required for robust mechanonociceptive responses. We further show that neurons integrating mechanosensory input facilitate primary nociceptive output by releasing short neuropeptide F, the Drosophila neuropeptide Y homolog. Our findings unveil how integration of somatosensory input and neuropeptide-mediated modulation can produce robust modality-specific escape behavior.


Assuntos
Comportamento Animal/fisiologia , Drosophila melanogaster/metabolismo , Nociceptores/metabolismo , Células Receptoras Sensoriais/metabolismo , Tato/fisiologia , Animais , Larva/metabolismo , Optogenética/métodos
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