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Understanding how the brain encodes behaviour is the ultimate goal of neuroscience and the ability to objectively and reproducibly describe and quantify behaviour is a necessary milestone on this path. Recent technological progresses in machine learning and computational power have boosted the development and adoption of systems leveraging on high-resolution video recording to track an animal pose and describe behaviour in all four dimensions. However, the high temporal and spatial resolution that these systems offer must come as a compromise with their throughput and accessibility. Here, we describe coccinella, an open-source reductionist framework combining high-throughput analysis of behaviour using real-time tracking on a distributed mesh of microcomputers (ethoscopes) with resource-lean statistical learning (HCTSA/Catch22). Coccinella is a reductionist system, yet outperforms state-of-the-art alternatives when exploring the pharmacobehaviour in Drosophila melanogaster.
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Escarabajos , Neurociencias , Animales , Drosophila melanogaster , Encéfalo , Aprendizaje AutomáticoRESUMEN
The Drosophila connectome project aims to map the synaptic connectivity of entire larval and adult fly neural networks, which is essential for understanding nervous system development and function. So far, the project has produced an impressive amount of electron microscopy data that has facilitated reconstructions of specific synapses, including many in the larval locomotor circuit. While this breakthrough represents a technical tour-de-force, the data remain under-utilised, partly due to a lack of functional validation of reconstructions. Attempts to validate connectivity posited by the connectome project, have mostly relied on behavioural assays and/or GRASP or GCaMP imaging. While these techniques are useful, they have limited spatial or temporal resolution. Electrophysiological assays of synaptic connectivity overcome these limitations. Here, we combine patch clamp recordings with optogenetic stimulation in male and female larvae, to test synaptic connectivity proposed by connectome reconstructions. Specifically, we use multiple driver lines to confirm that several connections between premotor interneurons and the anterior corner cell (aCC) motoneuron are, as the connectome project suggests, monosynaptic. In contrast, our results also show that conclusions based on GRASP imaging may provide false positive results regarding connectivity between cells. We also present a novel imaging tool, based on the same technology as our electrophysiology, as a favourable alternative to GRASP. Finally, of eight Gal4 lines tested, five are reliably expressed in the premotors they are targeted to. Thus, our work highlights the need to confirm functional synaptic connectivity, driver line specificity, and use of appropriate genetic tools to support connectome projects.SIGNIFICANCE STATEMENTThe Drosophila connectome project aims to provide a complete description of connectivity between neurons in an organism that presents experimental advantages over other models. It has reconstructed over 80 percent of the fly larva's synaptic connections by manual identification of anatomical landmarks present in serial section transmission electron microscopy (ssTEM) volumes of the larval CNS. We use a highly reliable electrophysiological approach to verify these connections, so provide useful insight into the accuracy of work based on ssTEM. We also present a novel imaging tool for validating excitatory monosynaptic connections between cells, and show that several genetic driver lines designed to target neurons of the larval connectome exhibit non-specific and/or unreliable expression.
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Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.
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Insecticidas , Receptores Nicotínicos , Animales , Bungarotoxinas/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Insecticidas/toxicidad , Neurotoxinas , Péptidos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMEN
Dravet syndrome is a severe rare epileptic disease caused by mutations in the SCN1A gene coding for the Nav1.1 protein, a voltage-gated sodium channel alpha subunit. We have made a knock-out of the paralytic gene, the single Drosophila melanogaster gene encoding this type of protein, by homologous recombination. These flies showed a heat-induced seizing phenotype, and sudden death in long term seizures. In addition to seizures, neuromuscular alterations were observed in climbing, flight, and walking tests. Moreover, they also manifested some cognitive alterations, such as anxiety and problems in learning. Electrophysiological analyses from larval motor neurons showed a decrease in cell capacitance and membrane excitability, while persistent sodium current increased. To detect alterations in metabolism, we performed an NMR metabolomic profiling of heads, which revealed higher levels in some amino acids, succinate, and lactate; and also an increase in the abundance of GABA, which is the main neurotransmitter implicated in Dravet syndrome. All these changes in the paralytic knock-out flies indicate that this is a good model for epilepsy and specifically for Dravet syndrome. This model could be a new tool to understand the pathophysiology of the disease and to find biomarkers, genetic modifiers and new treatments.
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The emergence of coordinated network function during nervous system development is often associated with critical periods. These phases are sensitive to activity perturbations during, but not outside, of the critical period, that can lead to permanently altered network function for reasons that are not well understood. In particular, the mechanisms that transduce neuronal activity to regulating changes in neuronal physiology or structure are not known. Here, we take advantage of a recently identified invertebrate model for studying critical periods, the Drosophila larval locomotor system. Manipulation of neuronal activity during this critical period is sufficient to increase synaptic excitation and to permanently leave the locomotor network prone to induced seizures. Using genetics and pharmacological manipulations, we identify nitric oxide (NO)-signaling as a key mediator of activity. Transiently increasing or decreasing NO-signaling during the critical period mimics the effects of activity manipulations, causing the same lasting changes in synaptic transmission and susceptibility to seizure induction. Moreover, the effects of increased activity on the developing network are suppressed by concomitant reduction in NO-signaling and enhanced by additional NO-signaling. These data identify NO signaling as a downstream effector, providing new mechanistic insight into how activity during a critical period tunes a developing network.
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Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica , Neuronas Motoras/metabolismo , Óxido Nítrico/metabolismo , Transmisión Sináptica , Animales , Electrofisiología , Femenino , Ratones , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Optogenética , Transducción de SeñalRESUMEN
Reactive oxygen species (ROS) have been extensively studied as damaging agents associated with ageing and neurodegenerative conditions. Their role in the nervous system under non-pathological conditions has remained poorly understood. Working with the Drosophila larval locomotor network, we show that in neurons ROS act as obligate signals required for neuronal activity-dependent structural plasticity, of both pre- and postsynaptic terminals. ROS signaling is also necessary for maintaining evoked synaptic transmission at the neuromuscular junction, and for activity-regulated homeostatic adjustment of motor network output, as measured by larval crawling behavior. We identified the highly conserved Parkinson's disease-linked protein DJ-1ß as a redox sensor in neurons where it regulates structural plasticity, in part via modulation of the PTEN-PI3Kinase pathway. This study provides a new conceptual framework of neuronal ROS as second messengers required for neuronal plasticity and for network tuning, whose dysregulation in the ageing brain and under neurodegenerative conditions may contribute to synaptic dysfunction.
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Drosophila melanogaster/metabolismo , Neuronas Motoras/metabolismo , Plasticidad Neuronal , Especies Reactivas de Oxígeno/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Larva/genética , Larva/metabolismo , Microscopía Electrónica de Transmisión , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Proteína Desglicasa DJ-1 , Transducción de Señal , Transmisión SinápticaRESUMEN
Mutations in the Golgi SNARE (SNAP [soluble NSF attachment protein] receptor) protein Membrin (encoded by the GOSR2 gene) cause progressive myoclonus epilepsy (PME). Membrin is a ubiquitous and essential protein mediating ER-to-Golgi membrane fusion. Thus, it is unclear how mutations in Membrin result in a disorder restricted to the nervous system. Here, we use a multi-layered strategy to elucidate the consequences of Membrin mutations from protein to neuron. We show that the pathogenic mutations cause partial reductions in SNARE-mediated membrane fusion. Importantly, these alterations were sufficient to profoundly impair dendritic growth in Drosophila models of GOSR2-PME. Furthermore, we show that Membrin mutations cause fragmentation of the presynaptic cytoskeleton coupled with transsynaptic instability and hyperactive neurotransmission. Our study highlights how dendritic growth is vulnerable even to subtle secretory pathway deficits, uncovers a role for Membrin in synaptic function, and provides a comprehensive explanatory basis for genotype-phenotype relationships in GOSR2-PME.
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Dendritas/metabolismo , Mutación , Epilepsias Mioclónicas Progresivas/genética , Proteínas Qb-SNARE/genética , Vías Secretoras/genética , Sinapsis/metabolismo , Animales , Dendritas/ultraestructura , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Estudios de Asociación Genética , Aparato de Golgi/metabolismo , Humanos , Masculino , Fusión de Membrana , Epilepsias Mioclónicas Progresivas/metabolismo , Epilepsias Mioclónicas Progresivas/patología , Fenotipo , Cultivo Primario de Células , Proteínas Qb-SNARE/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sinapsis/patología , Adulto JovenRESUMEN
Epilepsy is a significant disorder for which approximately one-third of patients do not respond to drug treatments. Next-generation drugs, which interact with novel targets, are required to provide a better clinical outcome for these individuals. To identify potential novel targets for antiepileptic drug (AED) design, we used RNA sequencing to identify changes in gene transcription in two seizure models of the fruit fly Drosophila melanogaster The first model compared gene transcription between wild type (WT) and bangsenseless1 (parabss), a gain-of-function mutant in the sole fly voltage-gated sodium channel (paralytic). The second model compared WT with WT fed the proconvulsant picrotoxin (PTX). We identified 743 genes (FDR≤1%) with significant altered expression levels that are common to both seizure models. Of these, 339 are consistently upregulated and 397 downregulated. We identify pumilio (pum) to be downregulated in both seizure models. Pum is a known homeostatic regulator of action potential firing in both flies and mammals, achieving control of neuronal firing through binding to, and regulating translation of, the mRNA transcripts of voltage-gated sodium channels (Nav). We show that maintaining expression of pum in the CNS of parabss flies is potently anticonvulsive, whereas its reduction through RNAi-mediated knockdown is proconvulsive. Using a cell-based luciferase reporter screen, we screened a repurposed chemical library and identified 12 compounds sufficient to increase activity of pum Of these compounds, we focus on avobenzone, which significantly rescues seizure behaviour in parabss flies. The mode of action of avobenzone includes potentiation of pum expression and mirrors the ability of this homeostatic regulator to reduce the persistent voltage-gated Na+ current (INaP) in an identified neuron. This study reports a novel approach to suppress seizure and highlights the mechanisms of neuronal homeostasis as potential targets for next-generation AEDs.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Homeostasis , Neuronas Motoras/patología , Propiofenonas/uso terapéutico , Proteínas de Unión al ARN/genética , Convulsiones/tratamiento farmacológico , Convulsiones/genética , Animales , Anticonvulsivantes , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efectos de los fármacos , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Epilepsia/patología , Predisposición Genética a la Enfermedad , Homeostasis/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Propiofenonas/farmacología , Proteínas de Unión al ARN/metabolismo , Convulsiones/patología , Análisis de Secuencia de ARN , Transcripción Genética/efectos de los fármacos , Transgenes , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Stability of neural circuits is reliant on homeostatic mechanisms that return neuron activity towards pre-determined and physiologically appropriate levels. Without these mechanisms, changes due to synaptic plasticity, ageing and disease may push neural circuits towards instability. Whilst widely documented, understanding of how and when neurons determine an appropriate activity level, the so-called set-point, remains unknown. Genetically tractable model systems have greatly contributed to our understanding of neuronal homeostasis and continue to offer attractive models to explore these additional questions. This review focuses on the development of Drosophila motoneurons including defining an embryonic critical period during which these neurons encode their set-points to enable homeostatic regulation of activity.
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Drosophila/fisiología , Homeostasis/fisiología , Animales , Drosophila/embriología , Modelos Animales , Neuronas Motoras/fisiologíaRESUMEN
Many animals are able to sense the Earth's geomagnetic field to enable behaviors such as migration. It is proposed that the magnitude and direction of the geomagnetic field modulates the activity of cryptochrome (CRY) by influencing photochemical radical pair intermediates within the protein. However, this proposal will remain theoretical until a CRY-dependent effect on a receptor neuron is shown to be modified by an external magnetic field (MF). It is established that blue-light (BL) photoactivation of CRY is sufficient to depolarize and activate Drosophila neurons. Here, we show that this CRY-dependent effect is significantly potentiated in the presence of an applied MF (100 mT). We use electrophysiological recordings from larval identified motoneurons, in which CRY is ectopically expressed, to show that BL-dependent depolarization of membrane potential and increased input resistance are markedly potentiated by an MF. Analysis of membrane excitability shows that these effects of MF exposure evoke increased action potential firing. Almost nothing is known about the mechanism by which a magnetically induced change in CRY activity might produce a behavioral response. We further report that specific structural changes to the protein alter the impact of the MF in ways that are strikingly similar to those from recent behavioral studies into the magnetic sense of Drosophila These observations provide the first direct experimental evidence to support the hypothesis that MF modulation of CRY activity is capable of influencing neuron activity to allow animal magnetoreception. SIGNIFICANCE STATEMENT: The biophysical mechanism of animal magnetoreception is still unclear. The photoreceptor protein cryptochrome has risen to prominence as a candidate magnetoreceptor molecule based on multiple reports derived from behavioral studies. However, the role of cryptochrome as a magnetoreceptor remains controversial primarily because of a lack of direct experimental evidence linking magnetic field (MF) exposure to a change in neuronal activity. Here, we show that exposure to an MF (100 mT) is sufficient to potentiate the ability of light-activated cryptochrome to increase neuronal action potential firing. Our results provide critical missing evidence to show that the activity of cryptochrome is sensitive to an external MF that is capable of modifying animal behavior.
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Criptocromos/efectos de la radiación , Luz , Campos Magnéticos , Neuronas/efectos de los fármacos , Potenciales de Acción/efectos de la radiación , Animales , Drosophila melanogaster , Larva , Potenciales de la Membrana/efectos de la radiación , Neuronas Motoras/efectos de la radiaciónRESUMEN
Maturation of neural circuits requires activity-dependent processes that underpin the emergence of appropriate behavior in the adult. It has been proposed that disruption of these events, during specific critical periods when they exert maximal influence, may lead to neurodevelopmental diseases, including epilepsy. However, complexity of neurocircuitry, coupled with the lack of information on network formation in mammals, makes it difficult to directly investigate this hypothesis. Alternative models, including the fruit fly Drosophila melanogaster, show remarkable similarities between experimental seizure-like activity and clinical phenotypes. In particular, a group of flies, termed bang-sensitive (bs) mutants have been extensively used to investigate the pathophysiological mechanisms underlying seizure. Seizure phenotype can be measured in larval stages using an electroshock assay, and this behavior in bs mutants is dramatically reduced following ingestion of typical anti-epileptic drugs (AEDs;). In this study we describe a critical period of embryonic development in Drosophila during which manipulation of neural activity is sufficient to significantly influence seizure behavior at postembryonic stages. We show that inhibition of elevated activity, characteristic of bs seizure models, during the critical period is sufficient to suppress seizure. By contrast, increasing neuronal excitation during the same period in wild-type (WT) is sufficient to permanently induce a seizure behavior. Further, we show that induction of seizure in WT correlates with functional alteration of motoneuron inputs that is a characteristic of bs mutants. Induction of seizure is rescued by prior administration of AEDs, opening a new perspective for early drug intervention in the treatment of genetic epilepsy.
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Neuronas Motoras/fisiología , Convulsiones/genética , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Desarrollo Embrionario/genética , Epilepsia/genética , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Estudios de Asociación Genética , Mutación , Fenómenos Fisiológicos del Sistema Nervioso/genética , Optogenética/métodos , Embarazo , Efectos Tardíos de la Exposición Prenatal/genéticaRESUMEN
Synapsins (Syns) are an evolutionarily conserved family of synaptic vesicle-associated proteins related to fine tuning of synaptic transmission. Studies with mammals have partially clarified the different roles of Syns; however, the presence of different genes and isoforms and the development of compensatory mechanisms hinder accurate data interpretation. Here, we use a simple in vitro monosynaptic Helix neuron connection, reproducing an in vivo physiological connection as a reliable experimental model to investigate the effects of Syn knockdown. Cells overexpressing an antisense construct against Helix Syn showed a time-dependent decrease of Syn immunostaining, confirming protein loss. At the morphological level, Syn-silenced cells showed a reduction in neurite linear outgrowth and branching and in the size and number of synaptic varicosities. Functionally, Syn-silenced cells presented a reduced ability to form synaptic connections; however, functional chemical synapses showed similar basal excitatory postsynaptic potentials and similar short-term plasticity paradigms. In addition, Syn-silenced cells presented faster neurotransmitter release and decreased postsynaptic response toward the end of long tetanic presynaptic stimulations, probably related to an impairment of the synaptic vesicle trafficking resulting from a different vesicle handling, with an increased readily releasable pool and a compromised reserve pool.
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Neuritas/fisiología , Neurogénesis/genética , Neuronas/citología , Neurotransmisores/metabolismo , Sinapsis/fisiología , Sinapsinas/metabolismo , Potenciales de Acción/genética , Animales , Células Cultivadas , Ganglios de Invertebrados/citología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Caracoles Helix , Microinyecciones , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Serotonina/farmacología , Sinapsinas/genética , Transducción GenéticaRESUMEN
This narrative review is intended to introduce clinicians treating epilepsy and researchers familiar with mammalian models of epilepsy to experimentally tractable, non-mammalian research models used in epilepsy research, ranging from unicellular eukaryotes to more complex multicellular organisms. The review focuses on four model organisms: the social amoeba Dictyostelium discoideum, the roundworm Caenorhabditis elegans, the fruit fly Drosophila melanogaster and the zebrafish Danio rerio. We consider recent discoveries made with each model organism and discuss the importance of these advances for the understanding and treatment of epilepsy in humans. The relative ease with which mutations in genes of interest can be produced and studied quickly and cheaply in these organisms, together with their anatomical and physiological simplicity in comparison to mammalian species, are major advantages when researchers are trying to unravel complex disease mechanisms. The short generation times of most of these model organisms also mean that they lend themselves particularly conveniently to the investigation of drug effects or epileptogenic processes across the lifecourse.
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Investigación Biomédica/métodos , Investigación Biomédica/tendencias , Modelos Animales de Enfermedad , Epilepsia/etiología , Epilepsia/terapia , Animales , HumanosRESUMEN
The mechanisms that facilitate animal magnetoreception have both fascinated and confounded scientists for decades, and its precise biophysical origin remains unclear. Among the proposed primary magnetic sensors is the flavoprotein, cryptochrome, which is thought to provide geomagnetic information via a quantum effect in a light-initiated radical pair reaction. Despite recent advances in the radical pair model of magnetoreception from theoretical, molecular and animal behaviour studies, very little is known of a possible signal transduction mechanism. We report a substantial effect of magnetic field exposure on seizure response in Drosophila larvae. The effect is dependent on cryptochrome, the presence and wavelength of light and is blocked by prior ingestion of typical antiepileptic drugs. These data are consistent with a magnetically-sensitive, photochemical radical pair reaction in cryptochrome that alters levels of neuronal excitation, and represent a vital step forward in our understanding of the signal transduction mechanism involved in animal magnetoreception.
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Animales Modificados Genéticamente/metabolismo , Criptocromos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Embrión no Mamífero/patología , Proteínas del Ojo/metabolismo , Larva/citología , Campos Magnéticos , Convulsiones/patología , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Anticonvulsivantes/farmacología , Criptocromos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/efectos de la radiación , Estimulación Eléctrica , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/efectos de la radiación , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/efectos de la radiación , Proteínas del Ojo/genética , Femenino , Larva/efectos de los fármacos , Larva/efectos de la radiación , Masculino , Procesos Fotoquímicos , Convulsiones/tratamiento farmacológico , Convulsiones/radioterapia , Transducción de SeñalRESUMEN
Epileptic activity is generally induced in experimental models by local application of epileptogenic drugs, including pentylenetetrazol (PTZ), widely used on both vertebrate and invertebrate neurons. Despite the high prevalence of this neurological disorder and the extensive research on it, the cellular and molecular mechanisms underlying epileptogenesis still remain unclear. In this work, we examined PTZ-induced neuronal changes in Helix monosynaptic circuits formed in vitro, as a simpler experimental model to investigate the effects of epileptiform activity on both basal release and post-tetanic potentiation (PTP), a form of short-term plasticity. We observed a significant enhancement of basal synaptic strength, with kinetics resembling those of previously described use-dependent forms of plasticity, determined by changes in estimated quantal parameters, such as the readily releasable pool and the release probability. Moreover, these neurons exhibited a strong reduction in PTP expression and in its decay time constant, suggesting an impairment in the dynamic reorganization of synaptic vesicle pools following prolonged stimulation of synaptic transmission. In order to explain this imbalance, we determined whether epileptic activity is related to the phosphorylation level of synapsin, which is known to modulate synaptic plasticity. Using western blot and immunocytochemical staining we found a PTZ-dependent increase in synapsin phosphorylation at both PKA/CaMKI/IV and MAPK/Erk sites, both of which are important for modulating synaptic plasticity. Taken together, our findings suggest that prolonged epileptiform activity leads to an increase in the synapsin phosphorylation status, thereby contributing to an alteration of synaptic strength in both basal condition and tetanus-induced potentiation.
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Epilepsia/inducido químicamente , Epilepsia/fisiopatología , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Pentilenotetrazol/efectos adversos , Transmisión Sináptica , Animales , Técnicas de Cultivo de Célula , Técnicas de Cocultivo , Caracoles Helix , Humanos , Fosforilación , Sinapsinas/metabolismo , Potenciales Sinápticos/efectos de los fármacosRESUMEN
BACKGROUND: A large number of invertebrate models, including the snail Helix, emerged as particularly suitable tools for investigating the formation of synapses and the specificity of neuronal connectivity. Helix neurons can be individually identified and isolated in cell culture, showing well-conserved size, position, biophysical properties, synaptic connections, and physiological functions. Although we previously showed the potential usefulness of Helix polysynaptic circuits, a full characterization of synaptic connectivity and its dynamics during network development has not been performed. RESULTS: In this paper, we systematically investigated the in vitro formation of polysynaptic circuits, among Helix B2 and the serotonergic C1 neurons, from a morphological and functional point of view. Since these cells are generally silent in culture, networks were chemically stimulated with either high extracellular potassium concentrations or, alternatively, serotonin. Potassium induced a transient depolarization of all neurons. On the other hand, we found prolonged firing activity, selectively maintained following the first serotonin application. Statistical analysis revealed no significant changes in neuronal dynamics during network development. Moreover, we demonstrated that the cell-selective effect of serotonin was also responsible for short-lasting alterations in C1 excitability, without long-term rebounds.Estimation of the functional connections by means of cross-correlation analysis revealed that networks under elevated KCl concentrations exhibited strongly correlated signals with short latencies (about 5 ms), typical of electrically coupled cells. Conversely, neurons treated with serotonin were weakly connected with longer latencies (exceeding 20 ms) between the interacting neurons. Finally, we clearly demonstrated that these two types of correlations (in terms of strength/latency) were effectively related to the presence of electrical or chemical connections, by comparing Micro-Electrode Array (MEA) signal traces with intracellularly recorded cell pairs. CONCLUSIONS: Networks treated with either potassium or serotonin were predominantly interconnected through electrical or chemical connections, respectively. Furthermore, B2 response and short-term increase in C1 excitability induced by serotonin is sufficient to trigger spontaneous activity with chemical connections, an important requisite for long-term maintenance of firing activity.
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Caracoles Helix/citología , Caracoles Helix/crecimiento & desarrollo , Red Nerviosa/crecimiento & desarrollo , Neuronas/fisiología , Sinapsis/clasificación , Sinapsis/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Caracoles Helix/efectos de los fármacos , Técnicas In Vitro , Larva , Red Nerviosa/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas/clasificación , Neuronas/citología , Neuronas/efectos de los fármacos , Cloruro de Potasio/farmacología , Serotonina/farmacología , Sinapsis/efectos de los fármacos , Factores de TiempoRESUMEN
In mammalian brain, the cellular and molecular events occurring in both synapse formation and plasticity are difficult to study due to the large number of factors involved in these processes and because the contribution of each component is not well defined. Invertebrates, such as Drosophila, Aplysia, Helix, Lymnaea, and Helisoma, have proven to be useful models for studying synaptic assembly and elementary forms of learning. Simple nervous system, cellular accessibility, and genetic simplicity are some examples of the invertebrate advantages that allowed to improve our knowledge about evolutionary neuronal conserved mechanisms. In this paper, we present an overview of progresses that elucidates cellular and molecular mechanisms underlying synaptogenesis and synapse plasticity in invertebrate varicosities and their validation in vertebrates. In particular, the role of invertebrate synapsin in the formation of presynaptic terminals and the cell-to-cell interactions that induce specific structural and functional changes in their respective targets will be analyzed.
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Plasticidad Neuronal/fisiología , Terminales Presinápticos/fisiología , Sinapsis/fisiología , Sinapsinas/metabolismo , Animales , Comunicación Celular/fisiología , Invertebrados , Aprendizaje/fisiología , Neuritas/fisiologíaRESUMEN
Semiconductor nanocrystal quantum dots (QDs) possess an enormous potential of applications in nanomedicine, drug delivery and bioimaging which derives from their unique photoemission and photostability characteristics. In spite of this, however, their interactions with biological systems and impact on human health are still largely unknown. Here we used neurosecretory mouse chromaffin cells of the adrenal gland for testing the effects of CdSe-ZnS core-shell quantum dots (5-36 nM) on Ca(2+) channels functionality and Ca(2+)-dependent neurosecretion. Prolonged exposure (24 h) to commonly used concentrations of CdSe-ZnS QDs (≥16 nM) showed that the semiconductor nanocrystal is effectively internalized into the cells without affecting cell integrity (no changes of membrane resistance and cell capacitance). QDs reduced the size of Ca(2+) currents by â¼28% in a voltage-independent manner without affecting channel gating. Correspondingly, depolarization-evoked exocytosis, measured at +10 mV, where Ca(2+) currents are maximal, was reduced by 29%. CdSe-ZnS QDs reduced the size of the readily releasable pool (RRP) of secretory vesicles by 32%, the frequency of release by 33% and the overall quantity of released catecholamines by 61%, as measured by carbon fibers amperometry. In addition, the Ca(2+)-dependence of exocytosis was reduced, whereas the catecholamine content of single granules, as well as the kinetics of release, remained unaltered. These data suggest that exposure to CdSe-ZnS QDs impairs Ca(2+) influx and severely interferes with the functionality of the exocytotic machinery, compromising the overall catecholamine supply from chromaffin cells.
Asunto(s)
Compuestos de Cadmio/metabolismo , Calcio/metabolismo , Catecolaminas/metabolismo , Células Cromafines/metabolismo , Puntos Cuánticos , Compuestos de Selenio/metabolismo , Sulfuros/metabolismo , Compuestos de Zinc/metabolismo , Animales , Canales de Calcio/metabolismo , Supervivencia Celular , Células Cultivadas , Células Cromafines/citología , Exocitosis , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
MAPK/Erk is a protein kinase activated by neurotrophic factors involved in synapse formation and plasticity, which acts at both the nuclear and cytoplasmic level. Synapsin proteins are synaptic-vesicle-associated proteins that are well known to be MAPK/Erk substrates at phylogenetically conserved sites. However, the physiological role of MAPK/Erk-dependent synapsin phosphorylation in regulating synaptic formation and function is poorly understood. Here, we examined whether synapsin acts as a physiological effector of MAPK/Erk in synaptogenesis and plasticity. To this aim, we developed an in vitro model of soma-to-soma paired Helix B2 neurons, that establish bidirectional excitatory synapses. We found that the formation and activity-dependent short-term plasticity of these synapses is dependent on the MAPK/Erk pathway. To address the role of synapsin in this pathway, we generated non-phosphorylatable and pseudo-phosphorylated Helix synapsin mutants at the MAPK/Erk sites. Overexpression experiments revealed that both mutants interfere with presynaptic differentiation, synapsin clustering, and severely impair post-tetanic potentiation, a form of short-term homosynaptic plasticity. Our findings show that MAPK/Erk-dependent synapsin phosphorylation has a dual role both in the establishment of functional synaptic connections and their short-term plasticity, indicating that some of the multiple extranuclear functions of MAPK/Erk in neurons can be mediated by the same multifunctional presynaptic target.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Caracoles Helix/enzimología , Plasticidad Neuronal/fisiología , Sinapsis/enzimología , Sinapsinas/metabolismo , Secuencia de Aminoácidos , Animales , Butadienos/farmacología , Células Cultivadas , Análisis por Conglomerados , Secuencia Conservada , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Caracoles Helix/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Datos de Secuencia Molecular , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Filogenia , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Especificidad por Sustrato/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsinas/química , Factores de TiempoRESUMEN
Neuronal cell cultures on Micro-Electrode Arrays (MEAs) provide an essential experimental tool for studying the connectivity and long-term activity of complex neuronal networks. MEA studies are generally based on the analysis of mixed neuronal populations constituted by a large number of cultured cells with cell type composition and connectivity patterns which are quite unpredictable a priori. In this work, we propose a different approach which consists of assembling on MEAs neuronal circuits formed by individually identifiable C1, C3, and B2 Helix neurons. Cells were plated under conditions of controlled number and position to form neuronal networks of defined composition. We performed multi-site electrophysiological recordings, and we characterized the firing dynamics. By means of cross-correlation analysis, we studied the electrophysiological properties of MEA-coupled microcircuits and characterized their activity patterns. We showed how the synaptic connectivity, actually observed in polysynaptic circuits of C1, C3 and B2 neurons, correlates well with the expected connectivity of C1-B2, B2-B2 and B2-C3 cell pairs as previously reported in conventional electrophysiological studies in culture.