Con el apoyo de USDA-NIFA/AFRI 2010-65105-20582, NSF 0918177, e incorporación Bedoukian investigación.

<html> 1. Los insectos <ol><li> La naranja ombligo, Amyelois transitella (Walker) (Lepidoptera: Pyralidae) colonia en nuestro laboratorio se originó a partir de las polillas recogida de los almendros en Fresno, CA. Nuestra colonia de insectos se mantiene en el medio ambiente cámaras (Percival Inc, IA) a 28 ± 2 ° C, 75 ± 10% de humedad relativa, y en 16:8 h (luz: oscuridad) photo régimen. </li><li> Polillas emergentes se recogen diariamente, sexo, y se transfieren a cajas de plástico (669 ml caja de almuerzo, 13 x 13 cm, la altura, 4,5 cm, Rubbermaid), siempre con un máximo de 10 capas de toallas de papel empapado de agua (Sed ultra absorbentes, 27,9 x 27,9 cm ; Safeway). Cubiertas de la caja están perforadas para permitir la circulación del aire. Esta disposición proporciona ~ 100% RH. </li><li> Para las mediciones electrofisiológicas usamos 2-4 días polillas de edad después de la emergencia, que se mantienen en condiciones de humedad alta. </li></ol> 2. Preparación electrofisiológicos <ol><li> Polillas (hombres o mujeres) se presiona suavemente en una punta de la pipeta (200 l, EE.UU. Scientific Inc) que se corta de la parte superior de tener un diámetro de la punta grande (aprox. 2 mm). Una polilla se empuja suavemente a través del otro extremo con un trozo de papel de seda humidificado hasta las antenas y parte de la cabeza sobresalga de la punta. </li><li> Cabeza expuesta es inmovilizado por cubrirse la cabeza con una arcilla que no reseca (Claytoon, Van Aken Internacional, CA), dejando una pequeña parte de uno de los ojos y dos antenas expuestos. Esta preparación se coloca sobre una plataforma de la Micromanipulador EAG MP-12 (Syntech, Alemania) entre las dos pinzas de soldadura. </li><li> Usamos electrodo de vidrio a partir de 1,0 mm de borosilicato tubo capilar con filamento (WPI Inc, FL) y sacó en un modelo P-97 micropipeta extractor (Sutter Instruments, CA) para obtener microelectrodos de menos de un micrómetro de punta. Los electrodos son de nuevo lleno de sensillum linfáticos del timbre (Kaissling y Thorson, 1980). Un electrodo empala a la superficie expuesta del ojo bajo el microscopio (Leica MZ75) y sirve de referencia, mientras que el electrodo de registro acomoda las dos antenas. Para facilitar el contacto, distal segmentos antenales 1-2 se cortan antes de ser insertado en el electrodo de registro. Para las señales de las grabaciones de sensilla individual, modificar ligeramente la configuración en la que el electrodo de registro se clavado bajo el microscopio (Olympus BX51WI, 800x aumentos). Los potenciales de acción se registran mediante la inserción del electrodo de vidrio en la base de un sensillum. Las señales son amplificadas por una impedancia alta pre-amplificador (Syntech, Alemania) y se introduce en una UB-IDAC para ser analizados fuera de línea con AUTOSPIKE software (Syntech, Alemania). Señales de corriente alterna son de paso de banda filtrada entre 100 -10 000 Hz y los potenciales de acción son extraídos por ordenador mediante un algoritmo AUTOSPIKE que distingue a su punto máximo y el mínimo amplitudes del ruido. Las respuestas de las neuronas individuales se calculan como el aumento (o disminución) en la frecuencia del potencial de acción (picos por segundo) a través de la frecuencia espontánea. </li></ol> 3. Los estímulos químicos y protocolo de estimulación <ol><li> Productos químicos de alta pureza se utilizan. Un componente importante de la mezcla de feromona femenina de la naranja ombligo, (Z, Z) -11,13-hexadecadienal se obtuvo de Bedoukian Research Inc, CT. Productos químicos se diluyen, w / v, con vidrio destilada hexano para que las soluciones de archivo de 10 diluciones mg / l, y se hacen decimal. Una alícuota (10 l) de una sustancia química estímulo disuelto en hexano se carga en una tira de papel de filtro Whatman (8x40 mm), se evapora el disolvente agitando suavemente durante 10 s en una campana de humos y la tira se coloca en una de polipropileno de 5 ml jeringa (jeringas BD, NJ) de varios volúmenes que son expulsados. Hexano solo y una jeringa vacía servir como control. </li><li> La preparación se lleva a cabo en una corriente de aire húmedo emitido por el controlador de estímulo Syntech (CS-55 modelo; Syntech, Alemania) a 20 ml / seg a la que un pulso de estímulo de 4 ml / s, se añade a 500 ms. Las señales se registraron durante 10 s, a partir de 2 s antes de la aparición del pulso de estímulo. Preparación de las antenas se estimula con 500 ms de pulso durante el cual ca. 2 ml de aire purificado a partir de una jeringa de polipropileno de 5 ml que contienen el estímulo añadido en el flujo principal de aire. A diferencia de al menos 1 minuto o más, a raíz de las respuestas de alta, se permite entre los estímulos. </li></ol> 4. Resultados representante Las antenas de las polillas restringido (Figura 1) estimuladas con componentes de feromona femenina, (Z, Z) -11,13-hexadecadienal generar respuestas sólidas dependiente de la dosis (Figura 2). Este componente de feromonas también provoca grandes dosis de excitación depende de las respuestas a largo sensilla trichodea (Figura 3) como se muestra en los rastros de las grabaciones de una sola unidad (Figura 3B). <img src="/files/ftp_upload/2489/2489fig1.jpg" alt="Figura 1" /> Figura 1. Vivo naranja ombligo polilla restringido y la antena expuestos load/2489/2489fig2.jpg &quot;alt =&quot; Figura 2 &quot;/&gt; Figura 2. Respuestas dosis-dependiente EAG provocado en respuesta a (Z, Z) -11,13-hexadecadienal, un componente importante de feromonas femeninas. <img src="/files/ftp_upload/2489/2489fig3.jpg" alt="Figura 3" /> Figura 3. Trichoid sensilla en los hombres la naranja ombligo polilla responde a (Z, Z) -11,13-hexadecadienal en una forma dependiente de dosis. (A) de la antena macho naranja ombligo polilla es de varios segmentos y cada segmento está adornado con una gran cantidad de pelo como las estructuras, sensilla. Una micrografía electrónica de barrido, que muestra los detalles en el recuadro (barras de escala son 200 y 50 M, respectivamente). (B) extracelular sola unidad-las grabaciones de una sensillum trichoid.

<ol>
	<li>Kaissling, K. -. E., Thorson, J., Sattelle, D. B., Hall, L. M., Hildebrand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+olfactory+sensilla:+structural,+chemical+and+electrical+aspects+of+the+functional+organization.">Insect olfactory sensilla: structural, chemical and electrical aspects of the functional organization.</a> Receptors for Neurotransmitters. Hormones and Pheromones in Insects. , 261-282 (1980).</li></ol>

Este nuevo método desarrollado para la inmovilización de un ombligo en directo la polilla de la naranja para medir las señales olfativas ha demostrado ser sólido y muy fiable. Nosotros habitualmente utilizan este método para el aislamiento y la identificación de atrayentes novela a partir de sustratos huésped natural como las almendras y los pistachos.

<table border="1"><tbody><tr><td> Nombre del Material </td><td> Catálogo # / Modelo </td><td> Empresa </td><td> Comentario </td></tr><tr><td> Microscopio </td><td> BX51WI modelo </td><td> Olympus, EE.UU. </td><td></td></tr><tr><td> Microscopio estereoscópico </td><td> MZ75 </td><td> Leica Microsystems Inc. EE.UU. </td><td></td></tr><tr><td> 1,0 mm de borosilicato capilares tubo con filamento </td><td> 1B100F-3 </td><td> WPI Inc, FL </td><td></td></tr><tr><td> Micropipeta extractor </td><td> P-97 </td><td> Sutter Instruments, CA </td><td></td></tr><tr><td> Estímulo controlador </td><td> CS-55 modelo </td><td> Syntech, Alemania </td><td></td></tr><tr><td> De alta impedancia de pre-amplificadores (Sonda universal de un solo extremo) </td><td></td><td> Syntech, Alemania </td><td></td></tr><tr><td> Amplificador / sistema de adquisición de datos (USB-IDAC) </td><td></td><td> Syntech, Alemania </td><td></td></tr><tr><td> Micromanipulador EAG MP-12 </td><td></td><td> Syntech, Alemania </td><td></td></tr><tr><td> (Z, Z) -11,13-hexadecadienal </td><td></td><td> Bedoukian Research Inc, CT. </td><td></td></tr><tr><td> Papel de filtro Whatman </td><td> 1001070 </td><td> Whatman EE.UU. </td><td></td></tr><tr><td> 5 ml jeringa de polipropileno </td><td> 309633 </td><td> BD Jeringas, NJ </td><td></td></tr><tr><td> punta de la pipeta (200 l) </td><td> 1111-0806 </td><td> EE.UU. Scientific Inc </td><td></td></tr><tr><td> 669 ml caja de almuerzo, 13 x 13 cm, la altura, 4,5 cm, </td><td></td><td> Rubbermaid </td><td></td></tr><tr><td> Sed ultra absorbentes, 27,9 x 27,9 cm </td><td></td><td> Safeway </td><td></td></tr><tr><td> No secar la arcilla </td><td> 18150 </td><td> Claytoon, Van Aken Internacional, CA </td><td></td></tr><tr><td> Cámara Ambiental </td><td> I-30BLL modelo </td><td></td><td></td></tr></tbody></table>

electrophysiological measurements from a moth olfactory system

Sistemas olfativos de insectos proporcionan oportunidades únicas para el registro de olor inducida por las respuestas en las formas de electroantennograms (EAG) y las grabaciones individuales sensillum (SSR), que se resumen las respuestas de todas las neuronas del receptor odorante (ORNs), ubicado en la antena y de los alojados en individuales sensilla, respectivamente. Estos enfoques han sido explotados para obtener una mejor comprensión de la comunicación química de insectos. Los estímulos identificados se pueden utilizar como atrayentes o repelentes en las estrategias de gestión de plagas de insectos.

Watch this Scientific Journal Video about Electrophysiological Measurements from a Moth Olfactory System at JoVE.com

Electrophysiological Measurements from a Moth Olfactory System

Sistemas olfativos de insectos proporcionan oportunidades únicas para el registro de olor inducida por las respuestas en las formas de electroantennograms (EAG) y las grabaciones individuales sensillum (SSR), que se resumen las respuestas de todas las neuronas del receptor odorante (ORNs), ubicado en la antena y de los alojados en individuales sensilla, respectivamente.

Las mediciones electrofisiológicas de un sistema olfativo Polilla

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

electrophysiological-measurements-from-a-moth-olfactory-system

Research

JoVE Journal

Neuroscience

13.7K vistas.  University of California, Davis. Sistemas olfativos de insectos proporcionan oportunidades únicas para el registro de olor inducida por las respuestas en las formas de electroantennograms (EAG) y las grabaciones individuales sensillum (SSR), que se resumen las respuestas de todas las neuronas del receptor odorante (ORNs), ubicado en la antena y de los alojados en individuales sensilla, respectivamente.

Video: Electrophysiological Measurements from a Moth Olfactory System

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

Registros electrofisiológicos de la ruta de fibra gigante de D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Investigación

Neurociencias

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Aislar las células madre olfativo nasal de los roedores o los seres humanos

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Las mediciones electrofisiológicas y Análisis de la nocicepción en los bebés humanos

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Inducidas Odorante-respuestas registradas de neuronas olfativas del receptor utilizando la técnica de aspiración con una pipeta

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Apetitivo aprendizaje asociativo olfativa en<em&gt; Drosophila</em&gt; Las larvas

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

En Vivo electrofisiológicos mediciones sobre los nervios ciáticos de ratones

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Electrofisiológico Grabación desde<em&gt; Drosophila</em&gt; Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

El aislamiento de las poblaciones celulares distinto del cerebelo en desarrollo por Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

El sistema olfativo como un modelo para estudiar los patrones de crecimiento axonal y Morfología<em&gt; En Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Todo el montaje de etiquetado de los cilios en el sistema olfativo principal de ratones

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...