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Missing information on patients and their medication is a leading cause of medication errors and preventable harm. The TOP Projects uses pharmacy claim data and electronic decision support to improve quality and safety of care on hospital admission. In a survey 100â% of responding hospitals in Germany consider this approach helpful and important to improve availability of necessary information and medication safety and to reduce workload.
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Hospitales , Errores de Medicación , Alemania , Hospitalización , Humanos , Errores de Medicación/prevención & control , Carga de TrabajoRESUMEN
Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold transmembrane voltage in vivo. To perform high-speed voltage imaging of many neurons simultaneously over a large anatomical area, widefield microscopy remains an essential tool. However, the lack of optical sectioning makes widefield microscopy prone to background cross-contamination. We implemented a digital-micromirror-device-based targeted illumination strategy to restrict illumination to the cells of interest and quantified the resulting improvement both theoretically and experimentally with SomArchon expressing neurons. We found that targeted illumination increased SomArchon signal contrast, decreased photobleaching, and reduced background cross-contamination. With the use of a high-speed, large-area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in behaving mice. Thus, the targeted illumination strategy described here offers a simple solution for widefield voltage imaging of many neurons over a large field of view in behaving animals.
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A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here we describe a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and is compatible with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, and number of neurons observable several-fold over previously published fully genetically encoded reagents1-8. Under conventional one-photon microscopy, SomArchon enables the routine population analysis of around 13 neurons at once, in multiple brain regions (cortex, hippocampus, and striatum) of head-fixed, awake, behaving mice. Using SomArchon, we detected both positive and negative responses of striatal neurons during movement, as previously reported by electrophysiology but not easily detected using modern calcium imaging techniques9-11, highlighting the power of voltage imaging to reveal bidirectional modulation. We also examined how spikes relate to the subthreshold theta oscillations of individual hippocampal neurons, with SomArchon showing that the spikes of individual neurons are more phase-locked to their own subthreshold theta oscillations than to local field potential theta oscillations. Thus, SomArchon reports both spikes and subthreshold voltage dynamics in awake, behaving mice.
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Biomarcadores Ambientales , Hipocampo/citología , Neuronas/fisiología , Imagen Óptica/métodos , Vigilia/fisiología , Potenciales de Acción/fisiología , Animales , Biomarcadores Ambientales/genética , Hipocampo/diagnóstico por imagen , Ratones , OptogenéticaRESUMEN
Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.
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Gryllidae , Microinyecciones/instrumentación , Microinyecciones/métodos , Óvulo , Animales , Sistemas CRISPR-Cas , AgujasRESUMEN
The thalamic parafascicular nucleus (PF), an excitatory input to the basal ganglia, is targeted with deep-brain stimulation to alleviate a range of neuropsychiatric symptoms. Furthermore, PF lesions disrupt the execution of correct motor actions in uncertain environments. Nevertheless, the circuitry of the PF and its contribution to action selection are poorly understood. We find that, in mice, PF has the highest density of striatum-projecting neurons among all sub-cortical structures. This projection arises from transcriptionally and physiologically distinct classes of PF neurons that are also reciprocally connected with functionally distinct cortical regions, differentially innervate striatal neurons, and are not synaptically connected in PF. Thus, mouse PF contains heterogeneous neurons that are organized into parallel and independent associative, limbic, and somatosensory circuits. Furthermore, these subcircuits share motifs of cortical-PF-cortical and cortical-PF-striatum organization that allow each PF subregion, via its precise connectivity with cortex, to coordinate diverse inputs to striatum.
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Corteza Cerebral/citología , Cuerpo Estriado/citología , Núcleos Talámicos Intralaminares/citología , Neuronas/citología , Animales , Corteza Cerebral/fisiología , Cuerpo Estriado/fisiología , Perfilación de la Expresión Génica , Núcleos Talámicos Intralaminares/fisiología , Ratones , Vías Nerviosas , Técnicas de Trazados de Vías Neuroanatómicas , Neuronas/metabolismo , Neuronas/fisiología , Técnicas de Placa-Clamp , Análisis de la Célula Individual , Tálamo/citología , Tálamo/fisiologíaRESUMEN
In the version of this article originally published, the bottom of Figure 4f,g was partially truncated in the PDF. The error has been corrected in the PDF version of this article.
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We developed a new way to engineer complex proteins toward multidimensional specifications using a simple, yet scalable, directed evolution strategy. By robotically picking mammalian cells that were identified, under a microscope, as expressing proteins that simultaneously exhibit several specific properties, we can screen hundreds of thousands of proteins in a library in just a few hours, evaluating each along multiple performance axes. To demonstrate the power of this approach, we created a genetically encoded fluorescent voltage indicator, simultaneously optimizing its brightness and membrane localization using our microscopy-guided cell-picking strategy. We produced the high-performance opsin-based fluorescent voltage reporter Archon1 and demonstrated its utility by imaging spiking and millivolt-scale subthreshold and synaptic activity in acute mouse brain slices and in larval zebrafish in vivo. We also measured postsynaptic responses downstream of optogenetically controlled neurons in C. elegans.
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Evolución Molecular Dirigida/métodos , Proteínas Luminiscentes/química , Ingeniería de Proteínas/métodos , Robótica , Pez Cebra/embriología , Animales , Encéfalo/diagnóstico por imagen , Caenorhabditis elegans , Separación Celular , Femenino , Citometría de Flujo , Fluorescencia , Biblioteca de Genes , Genes Reporteros , Células HEK293 , Hipocampo/citología , Humanos , Masculino , Ratones , Microscopía Fluorescente , Neuronas/citología , OptogenéticaRESUMEN
The striatum, the entry nucleus of the basal ganglia, lacks laminar or columnar organization of its principal cells; nevertheless, functional data suggest that it is spatially organized. Here we examine whether the connectivity and synaptic organization of striatal GABAergic interneurons contributes to such spatial organization. Focusing on the two main classes of striatal GABAergic interneurons (fast-spiking interneurons [FSIs] and low-threshold-spiking interneurons [LTSIs]), we apply a combination of optogenetics and viral tracing approaches to dissect striatal microcircuits in mice. Our results reveal fundamental differences between the synaptic organizations of both interneuron types. FSIs target exclusively striatal projection neurons (SPNs) within close proximity and form strong synapses on the proximal somatodendritic region. In contrast, LTSIs target both SPNs and cholinergic interneurons, and synaptic connections onto SPNs are made exclusively over long distances and onto distal dendrites. These results suggest fundamentally different functions of FSIs and LTSIs in shaping striatal output.
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Cuerpo Estriado/citología , Neuronas GABAérgicas/fisiología , Interneuronas/fisiología , Sinapsis/fisiología , Animales , Ratones , Ratones Noqueados , Ratones Transgénicos , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Técnicas de Trazados de Vías Neuroanatómicas , Receptor de Adenosina A2A/genética , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/genéticaRESUMEN
Synaptic communication between neurons requires the precise localization of neurotransmitter receptors to the correct synapse type. Kainate-type glutamate receptors restrict synaptic localization that is determined by the afferent presynaptic connection. The mechanisms that govern this input-specific synaptic localization remain unclear. Here, we examine how subunit composition and specific subunit domains contribute to synaptic localization of kainate receptors. The cytoplasmic domain of the GluK2 low-affinity subunit stabilizes kainate receptors at synapses. In contrast, the extracellular domain of the GluK4/5 high-affinity subunit synergistically controls the synaptic specificity of kainate receptors through interaction with C1q-like proteins. Thus, the input-specific synaptic localization of the native kainate receptor complex involves two mechanisms that underlie specificity and stabilization of the receptor at synapses.
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Subunidades de Proteína/fisiología , Receptores de Ácido Kaínico/fisiología , Sinapsis/metabolismo , Animales , Cerebelo/citología , Cerebelo/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Ratones Transgénicos , Dominios Proteicos , Estabilidad Proteica , Subunidades de Proteína/química , Transporte de Proteínas , Receptores de Ácido Kaínico/químicaRESUMEN
Although neuronal activity can be modulated using a variety of techniques, there are currently few methods for controlling neuronal connectivity. We introduce a tool (GFE3) that mediates the fast, specific and reversible elimination of inhibitory synaptic inputs onto genetically determined neurons. GFE3 is a fusion between an E3 ligase, which mediates the ubiquitination and rapid degradation of proteins, and a recombinant, antibody-like protein (FingR) that binds to gephyrin. Expression of GFE3 leads to a strong and specific reduction of gephyrin in culture or in vivo and to a substantial decrease in phasic inhibition onto cells that express GFE3. By temporarily expressing GFE3 we showed that inhibitory synapses regrow following ablation. Thus, we have created a simple, reversible method for modulating inhibitory synaptic input onto genetically determined cells.
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Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Hipocampo , Masculino , Trastornos Motores/metabolismo , Trastornos Motores/patología , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Columna Vertebral/citología , Columna Vertebral/metabolismo , Ubiquitinación , Pez CebraRESUMEN
The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway.
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Sistemas de Transporte de Aminoácidos/metabolismo , Arginina/metabolismo , Lisosomas/enzimología , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Arginina/deficiencia , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits.
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Sistemas CRISPR-Cas , Técnicas de Silenciamiento del Gen/métodos , Proteínas del Tejido Nervioso/genética , Células Piramidales/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Animales , Ratones , Proteínas del Tejido Nervioso/metabolismo , Células Piramidales/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Potenciales SinápticosRESUMEN
The motor and learning functions of the striatum are critically dependent on synaptic transmission from midbrain dopamine neurons and striatal cholinergic interneurons (CINs). Both neural populations alter their discharge in vivo in response to salient sensory stimuli, albeit in opposite directions. Whereas midbrain dopamine neurons respond to salient stimuli with a brief burst of activity, CINs exhibit a distinct pause in firing that is often followed by a period of increased excitability. Although this "pause-rebound" sensory response requires dopaminergic signaling, the precise mechanisms underlying the modulation of CIN firing by dopaminergic afferents remain unclear. Here, we show that phasic activation of nigrostriatal afferents in a mouse striatal slice preparation is sufficient to evoke a pause-rebound response in CINs. Using a combination of optogenetic, electrophysiological, and pharmacological approaches, we demonstrate that synaptically released dopamine inhibits CINs through type 2 dopamine receptors, while another unidentified transmitter mediates the delayed excitation. These findings imply that, in addition to their direct effects on striatal projection neurons, midbrain dopamine neurons indirectly modulate striatal output by dynamically controlling cholinergic tone. In addition, our data suggest that phasic dopaminergic activity may directly participate in the characteristic pause-rebound sensory response that CINs exhibit in vivo in response to salient and conditioned stimuli.
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Potenciales de Acción/fisiología , Neuronas Colinérgicas/metabolismo , Cuerpo Estriado/fisiología , Interneuronas/fisiología , Sustancia Negra/fisiología , Vías Aferentes/fisiología , Animales , Neuronas Colinérgicas/fisiología , Femenino , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones TransgénicosRESUMEN
All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.
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Mamíferos/fisiología , Neuronas/fisiología , Rodopsina/fisiología , Animales , Evolución Molecular Dirigida , Proteínas Recombinantes/metabolismo , Transmisión SinápticaRESUMEN
Activity-dependent alterations in the strength of an individual glutamatergic synapse are often accompanied by changes in the size and shape of the postsynaptic terminal. Two studies in this issue of Neuron, Meyer et al. (2014) and Bosch et al. (2014), shed new light on the mechanisms and signaling pathways underlying structural long-term potentiation.
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Espinas Dendríticas/fisiología , Hipocampo/citología , Potenciación a Largo Plazo/fisiología , Plasticidad Neuronal/fisiología , Neuronas/citología , Neuronas/fisiología , Neuronas/ultraestructura , Sinapsis/fisiología , Animales , HumanosRESUMEN
Glutamate is the most abundant excitatory neurotransmitter in the brain, and distinct classes of glutamate receptors coordinate synaptic transmission and spike generation upon various levels of neuronal activity. However, the mechanisms remain unclear. Here, we found that loss of synaptic AMPA receptors increased kainate receptor activity in cerebellar granule cells without changing NMDA receptors. The augmentation of kainate receptor-mediated currents in the absence of AMPA receptor activity is required for spike generation and is mediated by the increased expression of the GluK5 high-affinity kainate receptor subunit. Increase in GluK5 expression is sufficient to enhance kainate receptor activity by modulating receptor channel properties, but not localization. Furthermore, we demonstrate that the combined loss of the AMPA receptor auxiliary TARPγ-2 subunit and the GluK5 subunit leads to early mouse lethality. Our findings reveal mechanisms mediated by distinct classes of postsynaptic glutamate receptors for the homeostatic maintenance of the neuronal activity.
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Potenciales Evocados/fisiología , Receptores AMPA/deficiencia , Receptores de Ácido Kaínico/fisiología , Potenciales Sinápticos/fisiología , Transmisión Sináptica/fisiología , Adaptación Fisiológica , Animales , Cerebelo/citología , Cerebelo/fisiología , Femenino , Técnicas In Vitro , Masculino , Ratones , Ratones Mutantes , Receptor Cross-Talk/fisiología , Receptores AMPA/fisiología , Receptores de Glutamato/clasificación , Receptores de Glutamato/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de SeñalRESUMEN
At excitatory synapses in the brain, glutamate released from nerve terminals binds to glutamate receptors to mediate signaling between neurons. Glutamate receptors expressed in heterologous cells show ion channel activity. Recently, native glutamate receptors were shown to contain auxiliary subunits that modulate the trafficking and/or channel properties. The AMPA receptor (AMPAR) can contain TARP and CNIHs as the auxiliary subunits, whereas kainate receptor (KAR) can contain the Neto auxiliary subunit. Each of these auxiliary subunits uniquely modulates the glutamate receptors, and determines properties of native glutamate receptors. A thorough elucidation of the properties of native glutamate receptor complexes is indispensable for the understanding of the molecular machinery that regulates glutamate receptors and excitatory synaptic transmission in the brain.
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Proteínas Nucleares/fisiología , Subunidades de Proteína/fisiología , Receptores de Glutamato/metabolismo , Animales , Modelos Biológicos , Transporte de Proteínas/fisiologíaRESUMEN
Kainate receptors are less well understood than other glutamate receptors, and synaptic kainate receptors display properties that differ from recombinant receptors. In particular, the slow decay of kainate receptor synaptic currents contrasts with the rapid deactivation and desensitization of receptors expressed in heterologous cells. We recently identified Neuropilin and Tolloid like-2 (Neto2) as a novel accessory subunit of kainate receptors and showed that Neto2 modulates the gating kinetics of GluK2 receptors. However, the kainate receptor family consists of five different subunits (GluK1-5) that can form homomeric and heteromeric receptors with different functional properties. Here, we tested whether Neto2 modulation varies with subunit composition. Rapid application techniques were used to apply glutamate to outside-out patches that contained GluK1, GluK1/5, or GluK2/5 kainate receptors. Coexpression of Neto2 slowed desensitization to varying degrees. Responses to 1 ms pulses of glutamate were also slowed by Neto2, especially for receptors containing GluK5, as were postsynaptic currents in neurons expressing recombinant kainate receptors. In addition, Neto2 markedly increased the rate at which some receptors recovered from desensitization. These results suggest that Neto2 modulates the function of most kainate receptors.
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Proteínas de la Membrana/fisiología , Isoformas de Proteínas/fisiología , Receptores de Ácido Kaínico/fisiología , Animales , Técnicas de Cultivo de Célula , Línea Celular Transformada , Ácido Glutámico/farmacología , Humanos , Potenciales de la Membrana/fisiología , Ratones , Técnicas de Placa-Clamp/métodos , Receptores de Ácido Kaínico/agonistas , Receptores de Ácido Kaínico/metabolismo , Transfección/métodosRESUMEN
Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain distribution, which has been historically defined by (3)H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.