Nos gustaría agradecer a los miembros del Laboratorio de Santo. Este trabajo fue apoyado por el Instituto Nacional de Enfermedades Sordera y Comunicación (R01 DC005964 de LA) y NSF IGERT 0548890 (HAA). Los experimentos con animales se realizaron de acuerdo con las leyes de los Estados Unidos de Bienestar Animal y de los Institutos Nacionales de Salud directrices y fueron aprobados por el Cuidado de Animales de la Universidad de Washington y el empleo.

Parte 1. Instrumentación de grabación eléctrica <ol><li> Utilizamos una amplia multielectrodo planar (ALA Scientific Instruments) con 10 micras electrodos planos aislados de nitruro de titanio con nitruro de silicio. 60 electrodos están dispuestos en dos campos que consiste en una cuadrícula de 5x6 con el espaciamiento de centro a centro de 30 micras. (Otras configuraciones son posibles, los electrodos con una superficie de grabación de 10 micras de diámetro son mejores que los electrodos más grandes en términos de aislamiento de la actividad de neuronas individuales.) </li><li> Las señales eléctricas son amplificadas (amplificación de 1000x) con un amplificador de MEA 1060 (ALA Scientific Instruments). Las señales se adquieren a 10 kHz con una tarjeta de adquisición de datos (National Instruments) y software a la medida de adquisición de datos (también se puede comprar el software comercial). </li><li> El tejido se mantiene presionado en la MEA por una malla de nylon (53 micras), que se adjunta a una pieza de enganche personalizada que encaja perfectamente en el interior del anillo de tubo de cultivo de la MEA. </li></ol> Parte 2. Instrumentación de entrega líquido <ol><li> El tejido requiere superfusión continua, lo que nos garantiza mediante el suministro de solución de Ringer (115 de cloruro de sodio mM, 5 mM de cloruro de potasio, dos de cloruro de calcio mM, 2 de cloruro de magnesio mM, 25 de bicarbonato de sodio mM, HEPES 10 mM y 10 mM D-(+ )-glucosa) a partir de una bomba de HPLC. Los estímulos se presentan al realizar las inyecciones en una válvula de HPLC y el cambio entre flujo continuo y el lazo. Tanto la bomba de HPLC y la bomba de inyección de robot jeringas se suministran con solución de Ringer. </li><li> Solución de Ringer se mantiene en un baño de agua caliente (ISOTEMP 105) a 42 ° C durante todo el experimento y está continuamente burbujeado con 95% O 2 / 5% CO 2. Un matraz separado de Ringer sin glucosa se prepara para hacer copias de la bomba de jeringa. Solución de Ringer se entrega a los tejidos utilizando una bomba de 307 HPLC (Gilson), a razón de 3 ml / min. En un experimento típico de 5-6 horas, 1 litro de solución de Ringer cada uno es suficiente. </li><li> A lo largo del experimento, el tejido es continua superfused con 37 ° C la solución de Ringer. Una sonda de calor se coloca directamente sobre el tejido. </li><li> Los estímulos se entregan con el líquido de Gilson 215 brazo controlador robótico. Los estímulos se guardan en frascos de vidrio con tapa de goma, y ​​bajo el control del software de Gilson, el brazo robótico de entrega de los estímulos a la línea de perfusión, mientras que el caudal y presión constantes se mantienen. </li><li> El estado de la válvula de HPLC (bucle comprometido / no comprometidos) está representada por una tensión eléctrica, que es registrado por el equipo de adquisición como un canal adicional para asegurar la sincronización con la actividad neuronal. </li></ol> Parte 3. Preparación de los MEA y el dispositivo de entrega de estímulo <ol><li> Antes de realizar la disección, llenar el MEA y con BSA en dH2O durante al menos 5 minutos a temperatura ambiente. </li><li> Enjuague el MEA con calentamiento burbujea la solución de Ringer y el lugar de la MEA en el hielo. </li><li> El primer controlador de líquidos HPLC y la bomba de acuerdo a las instrucciones del fabricante. </li></ol> Parte 4. VNO Disección <ol><li> Antes de realizar la disección, llenar 2 60x15 mm placas de Petri con burbujas solución de Ringer y el lugar en el hielo. También llenar dos platos Petri Sylgard recubierto del mismo tamaño con solución de Ringer y el lugar en el hielo. Chill adicionales 50 ml de agua tibia-burbujas de timbre para su uso durante la disección. </li><li> El ratón se sacrifica por asfixia de CO 2 seguido por dislocación cervical. </li><li> Hacer incisiones bilaterales desde el lado de la boca, que corre paralela a la línea de la mandíbula, en la parte posterior de la cabeza. Únete a los dos cortes a lo largo de la parte posterior del cuello y quitar la parte dorsal de la cabeza, dejando la mandíbula inferior, conectado al ratón. </li><li> Usando tijeras finas, cortadas entre el tejido y cada lado del hueso de la mandíbula superior para separar el tejido del hueso. </li><li> # 3 usando pinzas, retire el tejido del paladar para exponer la cavidad nasal. </li><li> Utilizando unas tijeras pequeñas o un bisturí, corte entre uno y otro lado de los dientes frontales superiores 2 para liberar la cápsula vomeronasal de los huesos. </li><li> # 3 usando pinzas, recoger la cápsula vomeronasal por el hueso plano y colocar en la placa de Petri en el hielo. Los pasos a través de la inmersión se debe realizar en menos de 3 minutos. </li><li> Coloque el plato en un estereomicroscopio, iluminado con la luz reflejada. Retire la cápsula ósea de un VNO a la vez usando # 3 pinzas. Utilice una mano para estabilizar la cápsula ósea sujetando el extremo caudal y la otra mano para pelar los huesos fuera de la VNO, teniendo cuidado de no dañar el VNO. </li><li> Cuando se retira el hueso, la transferencia del VNO a un plato de Petri recubiertas Sylgard. Utilice el plato que queda para el VNO otros. Interruptor de la iluminación de luz transmitida. </li><li> Utilizando micro-tijeras o pinzas de # 5, separar el VNO de los vasos sanguíneos reduciendo a lo largo del borde del VNO. En este punto, setiene la hoja neural completamente separados de los vasos sanguíneos. </li><li> Coloque el VNO con el lado interno (dendríticas). # 3 usando pinzas, anclar el VNO a la Sylgard en una esquina del tejido. Pelar el epitelio neural de la lámina basal con un pequeño fragmento de una hoja de afeitar con revestimiento de teflón celebró con una pinza. De la cuchilla en un ángulo pequeño (&lt;30 ° de la superficie de la antena) es el mejor. </li><li> Con una pipeta de vidrio, la transferencia de la pieza del epitelio neural de la antena en el AMUMA. La posición del VNO en la parte superior del campo de electrodos con el lado dendríticas arriba. Retire el exceso de solución de Ringer con una pipeta. Coloque el soporte de tejido de malla en el pozo de la matriz para mantener el VNO en su lugar, y llenar con solución de Ringer frío es. </li></ol> Parte 5. Grabación <ol><li> Coloque el MEA en un MEA 1060 amplificador y la posición de la sonda de calor directamente sobre el tejido. </li><li> Para confirmar la correcta colocación de la sonda de calefacción, estimular con solución de Ringer conteniendo 50 mM de potasio (sustituido por sodio equimolar) durante 2 segundos. Ajuste la posición de reducir al mínimo la latencia de la respuesta neuronal a la solución de potasio. </li><li> Superfuse el tejido con una solución de Ringer se calienta durante 45 minutos a 1 hora antes de la grabación para que el tejido se adapte. </li><li> Las señales eléctricas se guardan en el disco utilizando el software de grabación personalizada. </li></ol> Parte 6. Análisis de Datos <ol><li> El análisis de datos en línea se puede hacer con las grabaciones de electrodos en bruto. Por ejemplo, podemos medir los cambios en la tasa de disparo de las células registradas en respuesta a ligandos particular. Los conjuntos de datos resultantes son grandes y se puede utilizar para hacer frente a muchas preguntas diferentes, la familiaridad con un lenguaje de programación o análisis (C, Matlab, R, Python, o similar) es necesaria. </li></ol> Parte 7. Resultados representante Esta preparación suele ser lo suficientemente estable para grabar durante 6 horas, produciendo una gran cantidad de datos (6 horas de grabación de 60 electrodos). <img src="/files/ftp_upload/1845/1845fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. Una instantánea de la actividad simultánea a través de la mitad de la matriz se muestra. La ventana de tiempo se extiende por 400 ms. Por favor, <a href="https://www.jove.com/files/ftp_upload/1845/1845fig1.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 1. <img src="/files/ftp_upload/1845/1845fig2tmb.jpg" alt="Figura 2" border="0" /> Figura 2. En un experimento típico, nos estimulan con compuestos de interés en particular por 10 segundos. Este trazado, que es un ejemplo de una respuesta a 100 veces la orina diluida ratón hembra. Por favor, <a href="https://www.jove.com/files/ftp_upload/1845/1845fig2.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 2.

<ol>
	<li>Dulac, C., Torelo, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+detection+of+pheromone+signals+in+mammals:+from+genes+to+behaviour.">Molecular detection of pheromone signals in mammals: from genes to behaviour.</a> Nat Rev Neurosci. 4, 551-562 (2003).</li><li>Holy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+Vomeronasal+neurons+to+natural+stimuli.">Responses of Vomeronasal neurons to natural stimuli.</a> Science. 289, 1569-1572 (2000).</li><li>Nodari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+steroids+as+natural+ligands+of+mouse+pheromone-sensing+neurons.">Sulfated steroids as natural ligands of mouse pheromone-sensing neurons.</a> J Neurosci. 28, 6407-6418 (2008).</li></ol>

Grabaciones multielectrodo conjunto nos permiten la posibilidad de monitorear la actividad simultánea de una gran población de neuronas. Esta es una herramienta útil para probar las propiedades de un sistema sensorial, en concreto el VNO. A diferencia del sistema olfatorio principal, el OVN detecta estímulos en fase líquida. Además, el VNO es un tejido heterogéneo. Hay aproximadamente 300 tipos diferentes de receptores en el ratón 1, presumiblemente con perfiles de respuesta diferente. MEA grabaciones...

multielectrode array recordings of the vomeronasal epithelium

Entender los circuitos neuronales requiere de métodos de registro de las neuronas de manera simultánea. Para<em&gt; In vitro</em&gt; Estudios, una tecnología actualmente disponible es amplia plana multielectrodo (MEA) de grabación. Aquí se documenta el uso de los acuerdos ambientales multilaterales para estudiar el ratón órgano vomeronasal (OVN), que desempeña un papel esencial en la detección de las feromonas y señales sociales a través de una diversa población de neuronas sensoriales que expresan cientos de tipos de receptores. La combinación de grabación de MEA con un controlador robótico líquido para suministrar estímulos químicos, las respuestas sensoriales de una gran y diversa población de neuronas se pueden grabar. La preparación que nos permite eliminar el neuroepitelio intacta del VNO en el ratón y estimular con una batería de productos químicos o ligandos potenciales, mientras que el control de la actividad eléctrica de las neuronas durante varias horas. Por lo tanto, esta técnica sirve como un método útil para evaluar la actividad del ligando, así como explorar las propiedades de las neuronas receptoras. Se presentan las técnicas necesarias para preparar el epitelio vomeronasal, grabación de MEA, y la estimulación química.

Watch this Scientific Journal Video about Multielectrode Array Recordings of the Vomeronasal Epithelium at JoVE.com

Multielectrode Array Recordings of the Vomeronasal Epithelium

Multielectrodo array (MEA) grabaciones de proporcionar un método para estudiar la actividad eléctrica de las grandes poblaciones de neuronas. Aquí se presentan los detalles de una preparación de MEA para grabar desde el epitelio vomeronasal del ratón al mismo tiempo estimular el tejido.

Grabaciones multielectrodo matriz del epitelio vomeronasal

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

multielectrode-array-recordings-of-the-vomeronasal-epithelium

Research

JoVE Journal

Neuroscience

12.1K vistas.  Washington University School of Medicine. Multielectrodo array (MEA) grabaciones de proporcionar un método para estudiar la actividad eléctrica de las grandes poblaciones de neuronas. Aquí se presentan los detalles de una preparación de MEA para grabar desde el epitelio vomeronasal del ratón al mismo tiempo estimular el tejido.

Video: Multielectrode Array Recordings of the Vomeronasal Epithelium

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Una sola unidad En vivo Grabaciones de la quiasma óptico de la rata

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Investigación

Neurociencias

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" ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Disección de un ojo del ratón de un monte entero del epitelio pigmentario de la retina

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Electrodos múltiples grabaciones matriz de avalanchas neuronal en cultivos organotípicos

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imágenes de detección de feromonas en una preparación de ratón Vomeronasal aguda lámina de tejido

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Las respuestas de imagen neuronal en los preparativos de la rebanada del órgano vomeronasal Expresando un sensor de calcio genéticamente codificados

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Ex vivo la Producción de la Bombilla órgano vomeronasal y accesorios olfativa Intacto

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

Análisis fisiológico en profundidad de poblaciones de células definidas en el infarto agudo del tejido Rebanadas del ratón órgano vomeronasal

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Método electrofisiológico para grabaciones de abrazadera de voltaje de células enteras<em&gt; Drosophila</em&gt; Fotorreceptores

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Grabaciones de Macropatch focal de corrientes sinápticas de la Unión Neuromuscular larvaria de Drosophila

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...