Este estudio fue apoyado por los Institutos Nacionales de Salud (Grant 1UM1HL112856).

Todos los procedimientos y protocolos fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Illinois en Chicago. Los experimentos descritos aquí son experimentos agudos sin supervivencia, y no hubo uso de pomada para los ojos. El mantenimiento de condiciones estériles sólo se produce cuando los instrumentos quirúrgicos se lavan con etanol al 70% en DiH 2 O. Sacrificio de ratas al final del experimento se produjo a través de sobredosis de IV ketamina / xilazina. 1. Preparación de los instrumentos y productos químicos <ol><li> Preparar solución madre de 0,05 M de 5-HT HCl en PBS. Luego diluir la acción con PBS a una concentración de 5-HT final del 0,203 mM. </li><li> Diluir dronabinol en aceite de sésamo a una concentración de 20 g / l. </li><li> Cortar 20 cm de longitud de polietileno (PE) -50 tubo (ID 0,58 mm, OD 0,965 mm). En un extremo del tubo, se corta una punta de bisel con unas tijeras, y en el otro extremo cuadrado, inserte un 23 g de la aguja. Conecte este 23 g de agujas a un 1 mljeringa, y llenarlo con 0,3 ml de 50 u / ml de heparina. </li><li> Corte cuatro piezas de hilo de seda 4-0 trenzado y se puso a un lado. </li><li> Esterilizar tijeras quirúrgicas, 2 pinzas Graefe curvos, y 3 pinzas micro con 70% de etanol en DIH 2 O. </li></ol> 2. La cateterización de la vena femoral Protocolo para la cateterización se modifica desde Jespersen et al. 21 <ol><li> Anestesiar una rata Sprague-Dawley con intraperitoneal (IP) de ketamina / xilazina (100 mg / kg: 10 mg / kg). Pellizcar la punta de la rata y observar cualquier movimiento para confirmar un nivel apropiado de anestesia. Afeitar el aspecto ventral del muslo izquierdo, y el aspecto ventral del cuello. Si es necesario por el Comité de Cuidado y Uso de Animales institucional local, aplicar pomada para los ojos para evitar la sequedad. </li><li> Secure rata en una posición supina sobre una tabla quirúrgica. Utilice tijeras quirúrgicas para cortar la piel en el muslo posterior izquierdo. </li><li> Con unas pinzas, conducta dissectio romon del músculo superficial para exponer la vena femoral. Utilice las pinzas para separar la vena de la arteria femoral, y coloque 2 hilos alrededor de la vena femoral. </li><li> Utilice las pinzas Graefe curvos para pull-up de la vena para detener el flujo sanguíneo. El uso de un G aguja de la jeringa 22, pinchar la vena femoral y luego insertar el extremo biselado de la tubería de PE-50 en la vena. </li><li> Compruebe si el tubo de PE-50 se inserta correctamente por la retracción del émbolo de la jeringa. La sangre debe ser visto entrar en el tubo de PE-50. Ate 2 nudos alrededor de la vena y PE-50 tubos utilizando los 2 hilos. </li></ol> 3. 5-HT inducida por la apnea vía IV Infusion <ol><li> Coloque un calibrador de tensión piezoeléctrico alrededor de la rata para medir la respiración. </li><li> Cambiar la configuración de nivel de amplificación del amplificador electrónico a través de software de amplificador en el equipo: para amplificar (100x) y el filtro de paso de banda (1-10 Hz) las señales respiratorias obtenidas de la galga de tensión, ajuste &quot;filtro highpas&quot; en &amp; #8220; AC @ 1 HZ &quot;y&quot; filtro de paso bajo &quot;a&quot; 10 Hz &quot;, y establecer la amplificación mediante la introducción de&quot; 10 &quot;para la&quot; ganancia inicial &quot;y&quot; 100 &quot;por&quot; Total Gain &quot;.. </li><li> Cambiar la configuración de velocidad de muestreo mediante la apertura de &quot;Entrada analógica&quot; a través de un software de grabación en el equipo. Para digitalizar (500 Hz de frecuencia de muestreo), utilizando un convertidor de analógico a digital, &quot;muestra&quot; en &quot;1000 Hz&quot; y &quot;saltar&quot; todos los demás puntos de grabación por &quot;1&quot; (frecuencia de muestreo efectiva 500 Hz), y luego grabar la señal usando software de grabación. </li><li> Retire la jeringa 1 ml del catéter e insertar una jeringa de vidrio de precisión 500 l lleno de 0,203 mM de 5-HT en el catéter. </li><li> Coloque la 500 l jeringa de vidrio de precisión en una bomba de infusión. Infundir 12,5 mg / kg por 350 l / kg de solución de 5-HT a una velocidad de 63 ml / hr. Realizar múltiples infusiones y observar la apnea en ratas, que se ve unasa hacer una pausa en la respiración (≥ 2,5 seg) en las señales respiratorias registradas en el monitor de la computadora. </li><li> Antes de proceder con la cirugía de cuello, monitorear el patrón respiratorio y compruebe si hay dolor reflejo del pellizco del dedo del pie en la rata. Si la respiración es irregular, o si hay un dolor reflejo del pellizco del dedo del pie, administrar IP ketamina / xilazina (100 mg / kg: 5 mg / kg) y luego vuelve a confirmar un nivel adecuado de anestesia. </li></ol> 4. Cuello Cirugía para exposicion nodose Ganglia <ol><li> Secure rata en una posición supina sobre una tabla quirúrgica. Haga un corte longitudinal de la línea media con unas tijeras quirúrgicas en el cuello. </li><li> Utilizando disección roma en el músculo cutáneo del cuello (utilizando 2 abrazaderas micro mantener este músculo clara de la zona quirúrgica), exponer los músculos esternohioideo y omohyoideus. Separar estos músculos para exponer la arteria carótida interna y una de sus ramas, la arteria pterigopalatino. </li><li> Observe el nervio vago, ya que corre a lo largo de la arteria carótida interna y luego a lo largo ª<html> e arteria pterigopalatino. Observe cómo el nervio vago y la arteria pterigopalatino, junto con el nervio glosofaríngeo (IX nervio craneal) y el nervio espinal accesorio (XI par craneal), entran en el foramen lacerado posterior en la base del cráneo. </li><li> Observe que el ganglio nudoso se muestra como una inflamación del nervio vago derecho antes de que entre el foramen lacerado posterior. Observe también que las ramas faríngeas y los nervios de la laringe salen las caras anterior y posterior, respectivamente, del ganglio nudoso. 5 </li><li> Usando las pinzas Graefe, separar el nervio vago de las arterias, y colocar un trozo de hilo alrededor del nervio vago. Coloque una abrazadera en el hilo para aplicar una ligera tensión sobre el nervio vago, y limpiar el ganglio nodoso de cualquier tejido conectivo para proporcionar menos resistencia al inyectar. </li><li> Repita la apnea inducida por 5-HT como se indica en la sección 3 para confirmar que no hubo ningún daño a los ganglios nudosos. </li></ol>itle &quot;&gt; 5. Intranodose Ganglia Inyección de dronabinol <ol><li> Llene una jeringa de vidrio de precisión impermeable al gas 10 l fijada con un 28 g de media pulgada aguja de la jeringa a la medida con un 35 ° biselados punta con 5 l de dronabinol en aceite de sésamo. </li><li> Coloque una abrazadera micro en la rosca para aplicar una ligera tensión sobre el nervio vago, y perforar el ganglio teniendo cuidado de no perforar nodose a través de él. Presionar la jeringa lentamente (≥ 60 seg) para inyectar todo el contenido de la jeringa. Tenga en cuenta que algunos de los contenido de la jeringa se saldrá por el ganglio. </li><li> Apnea Repita 5-HT inducida como se indica en la sección 3. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

Los pasos críticos para el éxito de la inyección de neuroquímicos en los ganglios nodose son: 1) la identificación y la limpieza del tejido conectivo de los ganglios nodose; 2) confirmar la integridad de los ganglios nodose antes de la inyección; 3) y usando una aguja de pequeño calibre para inyectar delicadamente en, pero no completamente a través de la perforación, los ganglios nudosos. El nervio vago inerva muchos órganos en el cuello y el abdomen, y transmite información impor...

Afferent signaling via the vagus nerve (cranial nerve X) transmits important general visceral information to the central nervous system (CNS) from baro-, chemo-, hepatic osmo-, cardiac, pulmonary, and gastric receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract (NTS) of the medulla. The cell bodies of the pseudounipolar neurons of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority is found in the former. Nodose ganglion cells contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that when activated, can modify afferent vagus nerve activity.1 Numerous innervations of the afferent vagus nerves coupled with the diversity of receptors located on the nodose ganglia illustrate the biological importance of this cranial nerve, and systemic drugs that do not cross into the CNS targeted at receptors on nodose ganglia can be used to treat various diseases, such as sleep apnea, gastroesophageal reflux disease, or chronic cough.2-4
The ease of access to the nodose ganglia lends itself to experimental manipulation by midline longitudinal incision made at the neck. The vagus nerve emerges from the posterior lacerated foramen at the base of the skull, and immediately displays a swelling of the nerve that is the nodose ganglion. The nodose ganglion is easily recognizable due to two nerve branches that arise from it: anteriorly the pharyngeal branch; and posteriorly superior laryngeal branch.5 Previous experimental manipulations of the nodose ganglia involved electrophysiological recordings,6 injections of immunohistochemical or immunofluorescent compounds for nerve tracings,7-10 superfusion or injections of neuroexcitotoxins,11-13 injections of adeno-associated virus to knockdown receptors,14,15 and injections of receptor-specific neurochemicals to change the activity of the vagus nerve.16,17
Systemic injections of neurochemicals are problematic in that systemic treatment affects both peripheral and central nervous systems. Thus, systemic treatment does not isolate the effect of neurochemicals on afferent vagal nerve activity. This protocol describes a method using readily available equipment of intranodose injections in the Sprague-Dawley rat that modulates vagus nerve activity without affecting the central nervous system. Stimulation of serotonin type 3 (5-HT3) receptors on nodose ganglia by intravenous (IV) infusion of serotonin (5-HT) induces the Bezold-Jarisch reflex, a vagal response trifecta of bradycardia, hypotension, and apnea, which can be abolished by supranodose vagatomy.11,17-19 Apnea is easily measured by placing a respiratory transducer around the abdomen of the rat.17,18 Cannabinoids decrease 5-HT-induced current in nodose ganglia cells,20 and intranodose ganglia injections of dronabinol attenuate 5-HT-induced apnea.

<table border="0" cellpadding="0" cellspacing="0" width="660">
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Name of Material/ Equipment</td>
			<td style="width:155px;">Company</td>
			<td style="width:141px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">5-HT HCl</td>
			<td style="width:155px;">MP Biomedicals</td>
			<td style="width:141px;">215376591</td>
			<td style="width:175px;">12.5 &#181;g/kg per 350 &#181;l/kg</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Dronabinol (Marinol) 10 mg Capsules (80 &#181;g/&#181;l)</td>
			<td style="width:155px;">AbbVie</td>
			<td style="width:141px;">NDC 0051-0023-21</td>
			<td style="width:175px;">Dilute with sesame oil to 20 &#181;g/&#181;l</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Sesame Oil</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td style="width:141px;">S3547</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Intramedic Polyethylene-50</td>
			<td style="width:155px;">BD</td>
			<td style="width:141px;">427411</td>
			<td style="width:175px;">Ordered from VWR (Cat. # 63019-047)</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Graefe Forceps</td>
			<td style="width:155px;">Roboz</td>
			<td style="width:141px;">RS-5138</td>
			<td style="width:175px;">Two are needed</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Johns Hopkins Bulldog Clamp</td>
			<td style="width:155px;">Roboz</td>
			<td style="width:141px;">RS-7441</td>
			<td style="width:175px;">Three are needed</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Piezoelectric Strain Gauge</td>
			<td style="width:155px;">Ambu</td>
			<td style="width:141px;">813255-100</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Data Acquisition USB Subsystems</td>
			<td rowspan="2" style="width:155px;">DataWave Technologies</td>
			<td style="width:141px;">NA</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Sciworks Experimenter Software</td>
			<td style="width:141px;">NA</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">CyberAmp</td>
			<td style="width:155px;">Axon Instruments</td>
			<td style="width:141px;">NA</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Syringe, 500 &#181;L, Model 1750 TLL</td>
			<td rowspan="3" style="width:155px;">Hamilton Company</td>
			<td style="width:141px;">81220</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Syringe, 10 &#181;L, Model 1801 RN</td>
			<td style="width:141px;">7659-01</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:191px;">Needle, 28 gauge, Small Hub RN</td>
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a method of nodose ganglia injection in sprague-dawley rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

La Figura 1 representa la grabación de respiración muestra en las ratas que tenían infusión de 5-HT para inducir apnea antes y después de las inyecciones de los ganglios intranodose de dronabinol. 5-HT activa 5-HT 3 receptores en los ganglios nodose que contribuyen al reflejo de Bezold-Jarisch de bradicardia, hipotensión y apnea. 11,17-19 inyecciones ganglios Intranodose de dronabinol activan los receptores CB inhibitorios, o modula alostéricamente 5-HT 3 receptor...

Watch this Scientific Journal Video about A Method of Nodose Ganglia Injection in Sprague-Dawley Rat at JoVE.com

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

Un método de nodose Ganglios Inyección en rata Sprague-Dawley

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

a-method-of-nodose-ganglia-injection-in-sprague-dawley-rat

Research

JoVE Journal

Neuroscience

18.5K vistas.  University of Illinois at Chicago. Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

Video: A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Gen de entrega de cerebro de rata postnatal por la inyección no ventricular plásmido y electroporación

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Investigación

Neurociencias

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Un método general para la evaluación de incubación de sacarosa en ratas Deseo

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

Técnica mínimamente invasiva para la inyección en nervio óptico de la rata

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

Modelo de una rata de la fatiga Central mediante la método de plataforma múltiple

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

Optimización del Microdissection de la captura de láser para el aislamiento de los ganglios entéricos de tejido humano fresco congelado

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Inyección directa de un Vector lentivirales destaca múltiples caminos del Motor en la médula espinal de rata

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Generación de neuroesferas a partir de neuronas de hipocampo primario mixto y cortical esinas aisladas del e14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine &#946;-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

This paper describes the isolation and culturing of embryonic rat sympathetic neurons from the superior cervical ganglia. It also provides detailed protocols for immunocytochemical staining and for preparing neuronal extracts for mass spectrometric analysis.

Cultivar neuronas simpáticas de ratas de Ganglia cervical superior embrionario para análisis morfológico y proteómico

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

Un método de limpieza de tejido hidrófobo para el tejido cerebral de rata

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors are involved in the course of the disease, resulting in great limitations in clinical research. Therefore, the establishment of stable CVST animal models that can standardize a variety of uncontrollable confounding factors have helped to circumvent shortcomings in clinical research. In recent decades, a variety of CVST animal models have been constructed, but the results based on these models have been inconsistent and incomplete. Hence, in order to further explore the pathophysiological mechanisms of CVST, it is necessary to establish a novel and highly compatible animal model, which has important practical value and scientific significance for the diagnosis and treatment of CVST. In the present study, a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis was established via a thread-embolization method, and the stability and reliability of the model were verified. Additionally, we evaluated changes in cerebral venous blood flow in rats after the formation of CVST. Collectively, the SD-rat SSS-thrombosis model represents a novel CVST animal model that is easily established, minimizes trauma, yields good stability, and allows for accurately controlling ischemic timing and location.

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

Establecimiento De Un Modelo De Rata De La Oclusión Superior Sagital-Seno A Través De Un Método De Hilo-Embolia

The mechanisms contributing to the natural onset of cerebral venous sinus thrombosis (CVST) are mostly unknown, and a variety of uncontrollable factors ...