Con el apoyo de la Universidad de Kentucky, Departamento de Biología de la Oficina de Estudios de Grado y la Facultad de Artes y Ciencias.

<ol>
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Los datos facilitados en la película y el texto asociado han proporcionado los pasos claves a fin de registrar lo suficiente la actividad en el MRO de los cangrejos de río en el lugar. Uno de los objetivos de nuestro informe es aumentar la conciencia en el potencial de esta preparación en los estudiantes ejecutar los laboratorios de investigación para enseñar los conceptos fundamentales de la fisiología sensorial. Los preparativos son muy robustos en la viabilidad, mientras se bañaba en una solución salina mínima. El control de motores en los músculos de la MRO ha sido identificado, pero la regulación de la transmisión y el potencial de la plasticidad sináptica, así como la eficacia de la transmisión de las neuronas excitadoras e inhibidoras sigue siendo un espacio abierto para la investigación (Elekes y Florey, 1987a, b; Florey y Florey, 1955; Kuffler, 1954; Kuffler y Eyzaguirre, 1955). Esta preparación se puede utilizar para investigar una serie de condiciones experimentales, así como el área de distribución natural de locomoción dentro del animal para comprender mejor la biología de la MRO para la investigación primaria, así como con fines demostrativos. Las propiedades biofísicas de estas neuronas sensoriales en parte ha sido abordado en su naturaleza de adaptación en la actividad neuronal con un estímulo mantenido (Brown et al, 1978; Edwards et al, 1981;. Purali, 1997; Rydqvist y Purali, 1991; Rydqvist y Swerup, 1991). Sin embargo, sólo unos pocos neuromodulación dirección informes sobre estos receptores sensoriales y fibras musculares asociados (Cooper et al, 2003;. Pasztor y Macmillan, 1990). Los informes de acuerdo con sólo unos pocos de los muchos compuestos que son conocidos por estar presentes en la hemolinfa. Moduladores de muchos cócteles y de los moduladores quedan por examinar en el complejo de MRO (músculos y neuronas). Pasztor y Macmillan (1990) ha examinado los neuromoduladores 5-HT y octopamina sobre la actividad de las operaciones principales de financiación entre las distintas especies de crustáceos y señaló que existen diferencias entre las especies. Que no examinó en detalle la influencia a largo plazo de estos neuromoduladores, ni los efectos sobre la actividad en diferentes posiciones estáticas de la MRO. Este tipo de preparación puede ayudar a comprender la base de la percepción sensorial y la regulación del procesamiento neural que es importante en la rehabilitación y el tratamiento de la enfermedad para los seres humanos con la anormalidad de unidades motoras (Patel et al, 2009;. Rabin et al, 2009;. Marino et al ., 2010). Los diferentes tipos de entrada y los patrones de activación para vigilar los movimientos conjuntos en los preparativos de invertebrados accesible puede ser utilizado en la robótica / prótesis (Macmillan y Patullo, 2001). Todavía hay muchas preguntas en espera de respuestas en esta preparación que puede ser beneficioso en un número de maneras.

<ol>
<li><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML856/" target="_blank">PowerLab 26T (AD Instruments)</a> </li>
<li><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">LabChart 7 (ADI Instruments, Colorado Springs, CO, USA)</a> </li>
</ol>

muscle receptor organs in the crayfish abdomen: a student laboratory exercise in proprioception

El propósito principal de este experimento es demostrar la información las neuronas sensoriales primarias de transporte de los movimientos y posiciones conjuntas en la información propioceptiva de un animal. Un objetivo adicional de este experimento es conocer la anatomía de la preparación de tinción, la disección y la visualización de las neuronas sensoriales y las estructuras bajo un microscopio de disección. Esto se realiza mediante el uso de equipos neurofisiológicos básicos para registrar la actividad eléctrica de un órgano receptor de las articulaciones y las técnicas de tinción. El receptor de los músculos de órganos (MRO) del sistema en el cangrejo de río es análoga a la de los husos musculares intrafusales en los mamíferos, lo que ayuda a servir como modelo comparativo que se encuentra más accesible para los registros electrofisiológicos. Además, se trata de identificar las neuronas sensoriales de los preparativos. La preparación es viable en una solución salina mínimo de horas que se presta para los ejercicios de laboratorio de los estudiantes. El MRO es también susceptible a la neuromodulación, que anima a las preguntas intrigantes en los sitios de acción moduladora y la integración de señales dinámicas de los movimientos y la posición estática con una ganancia que se puede cambiar en el sistema.

Watch this Scientific Journal Video about Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception at JoVE.com

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

El propósito principal de este experimento es entender cómo las neuronas sensoriales primarias transmitir la información de los movimientos y posiciones conjuntas en la información propioceptiva de un animal. Un objetivo adicional de este informe es presentar la anatomía de la preparación mediante la disección y la visualización de las neuronas bajo un microscopio de disección.

Los órganos receptores de los músculos en el abdomen del cangrejo de río: Un ejercicio de laboratorio los estudiantes en la propiocepción

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

muscle-receptor-organs-crayfish-abdomen-student-laboratory-exercise

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Neuroscience

22.1K vistas.  University of Kentucky. El propósito principal de este experimento es entender cómo las neuronas sensoriales primarias transmitir la información de los movimientos y posiciones conjuntas en la información propioceptiva de un animal. Un objetivo adicional de este informe es presentar la anatomía de la preparación mediante la disección y la visualización de las neuronas bajo un microscopio de disección.

Video: Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Grabación de las respuestas de comportamiento a la reflexión en el cangrejo de río

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Investigación

Neurociencias

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

Un procedimiento para el estudio de la reposición de estímulo eléctrico plantar inducido por la cocaína Buscando en ratas de laboratorio

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

Las grabaciones fisiológicas de NMJs de salida de alta y baja en el músculo extensor de piernas cangrejo de río

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

Los potenciales de membrana, las respuestas sinápticas, circuitos neuronales, neuromodulación y la histología muscular utilizando el cangrejo de río: Los ejercicios de laboratorio para estudiantes

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

La experimentación fisiológica con el intestino grueso cangrejo de río: Un ejercicio de laboratorio los estudiantes

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Formación de imágenes pHluorin-etiquetados Inserción del receptor a la membrana plasmática en neuronas primarias de ratón en cultivo

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

Propiocepción y la tensión en los receptores de cangrejo Limbs: Ejercicios de Laboratorio Estudiante

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

Medición oral de ácidos grasos Umbrales, Percepción de grasa, alimentos grasos gusto, y papilas Densidad en Humanos

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Todo el montaje de Immunolabeling olfativa del receptor de las neuronas en el<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

Adaptación de la tecnología de matriz de microelectrodos para el estudio de la neurotoxicidad inducida por la anestesia en el cerebro intacto cochinillo

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...