Supported by APA to RJH, NHMRC program grant 400121 to SP, and NMHRC fellowship 1005050 to SP. CAR acknowledges the support of the ARC (FT0990628) and the DOWD fellowship scheme. The Florey Institute of Neuroscience and Mental Health is supported by Victorian State Government infrastructure funds.

Todos los experimentos con ratones fueron aprobados por el comité de ética animal Florey Instituto. 1. Configuración para cortar rebanadas de cerebro <ol><li> Preparar una solución de corte compuesto por (mM) 125 La colina-Cl, 2,5 KCl, CaCl2 0,4, 6 MgCl2, 1,25 NaH 2 PO 4, 26 NaHCO3, 20 D-glucosa satura con gas carbógeno (95% O 2 -5 % de CO 2) y un líquido cefalorraquídeo artificial (LCRa solución) de grabación consta de (mM) 125 NaCl, 2,5 KCl, 2 CaCl2, 2 MgCl2, 1,25 NaH 2 PO 4, 26 NaHCO 3, 10 D-glucosa, saturado con carbógeno. Coloque la solución de corte en el hielo para mantenerlo frío. </li><li> Congelar aproximadamente 400 ml de la solución de corte y mezclar junto con 100 ml de solución de corte no congelada para crear una suspensión de hielo. Burbuja con carbógeno (95% O 2 -5% de CO 2) a un flujo de aproximadamente 0,5 L / min a través de pequeños cAliber tubo o de vidrio sinterizado para producir un flujo constante pero suave de burbujas. </li><li> Preparar un vaso de precipitados de 250 ml con un inserto de malla de nylon elevada sobre la que se colocarán los cortes de cerebro. Rellenar con LCRa para cubrir la malla de aproximadamente 2 cm y burbuja con carbógeno, asegurando que las burbujas no interrumpen directamente el área del sector sostiene. También es importante que no haya burbujas de aire en la malla de nylon, por lo que si están presentes los eliminan. Esta será la cámara de retención y se mantiene a temperatura ambiente (20 - 25 ° C). </li><li> Layout los instrumentos de disección incluyendo un gran par de tijeras, un pequeño par de tijeras, pequeños y grandes espátulas micro y parejas grandes y pequeños de fórceps y relajarse en hielo. Coloque el bloque de corte de tejido vibratome sobre hielo en un cuadrado de papel de aluminio. Obtener 2 piezas de 6 cm de papel de filtro, una cuchilla de afeitar sola punta, y un vaso de precipitados de 25 ml llena con la suspensión de solución de corte también. </li><li> Llene un segundo recipiente con hielo, y poner un pedazo de tejido paper en el hielo y colocar una placa de cultivo de 12 cm en la parte superior. Llene la placa de cultivo con el corte de solución de suspensión de hielo y burbujas con carbógeno. Este es el contenedor en el que se realizará con la disección del cerebro. </li><li> Prepare el vibratome. Eliminar una hoja de afeitar de doble filo fresco (use una hoja fresca cada vez) de su envoltorio y rociar con etanol al 80% de agua desionizada a continuación. Cortar un 3 ml pipeta de transferencia de plástico en el punto donde comienza a disminuir. Esto se utiliza para transferir rodajas de cerebro. Doble un 27 G aguja en la base en aproximadamente 45 ° y se unen a una jeringa de 1 ml. Esto será utilizado para manipular rebanadas durante el corte. </li></ol> 2. Cortar las rebanadas del cerebro <ol><li> Anestesiar un ratón (P16 - P18) con un 2% de isoflurano o un método aprobado a nivel local. Después de la inducción, decapitar al animal con un gran par de tijeras y colocar la cabeza en la placa de cultivo de 12 cm que contiene burbujeado suspensión solución de corte. La suspensión debe sumergir totalmente la cabeza deenfriamiento rápido. </li><li> Mantenga la parte delantera de la cabeza con una mano el pelado de la piel y el tejido conectivo hacia adelante hacia la nariz. Utilizando las pequeñas tijeras cortar el tejido conectivo para revelar el cráneo subyacente. A continuación, retire los músculos que recubren cara dorsal del cráneo y el cuello. </li><li> Retire el cerebro del cráneo fijando primero la parte frontal del cráneo con las grandes pinzas y luego hacer dos cortes laterales a través del hueso de cada lado del agujero occipital utilizando las tijeras pequeñas (Figura 1, cortes etiquetados A1 y ​​A2). <ol><li> Haga otro corte entre los ojos (justo anterior del bregma) (Figura 1, corte la etiqueta B) luego cuidadosamente cortado a lo largo de la sutura sagital anterior y reflejar las secciones del cráneo corte para revelar el cerebro (Figura 1, corte C marcado). </li><li> Utilice la espátula para sacar el cerebro y el lugar en una placa de cultivo. Tenga en cuenta el crannervios ial en la cara inferior del cerebro que tendrá que ser cortadas, esto se puede hacer con la espátula micro tamaño más pequeño. Usando la espátula más grandes transfieren el cerebro para el vaso de precipitados de 25 ml lleno con la suspensión de solución de corte. </li></ol></li><li> Preparando el hemisferio cerebral para cortar. <ol><li> Coloque un pedazo de papel de filtro 6 cm en la parte inferior de una placa de cultivo fresco luego rellenar con lechada fresca solución de corte y de la burbuja. Utilice la espátula grande para colocar el cerebro, con el lado ventral hacia abajo, sobre el papel de filtro. Tome una hoja de afeitar nueva y limpia solo filo y cortar el cerebro para eliminar el cerebelo, a continuación, hacer un corte a lo largo de la línea media para separar el cerebro en dos hemisferios. </li></ol></li><li> Preparación del bloque de tejido vibratome. <ol><li> Tome el bloque de tejido vibratome fría, secar la superficie y colocar una gota de pegamento de cianoacrilato en el medio y extender uniformemente con el tamaño aproximado del cerebro. Utilice la pequeña espátula para manipular uno de lahemisferios cerebrales sobre la espátula grande micro por lo que el lado medial del cerebro está en el suelo. </li><li> Toque el borde de la espátula en la interfaz del cerebro en la segunda pieza de papel de filtro para eliminar la mayor cantidad de la solución posible. Deslice el cerebro de los más grandes espátula utilizando la espátula pequeña para guiarla en la cola. </li><li> Asegure el bloque de corte en la cámara vibratome y llenar la cámara con lechada de solución de corte y de la burbuja, lo que garantiza que el cerebro está completamente inmerso. Gire el bloque de corte por lo que la parte ventral del cerebro se enfrenta a la hoja. </li></ol></li><li> Cortando el cerebro. Nota: Cada vibratome es única así que siga las instrucciones del fabricante. <ol><li> Ajuste el grosor de corte de 450 m, y garantizar la cuchilla está vibrando mientras se mueve a través del cerebro a una velocidad de aproximadamente 0,3 mm / s. Cortar por completo a través del cerebro desde la parte ventral a la superficie cortical, esto producirá cerebro rebanadas sagital enteros que pueden Be utilizado para registros electrofisiológicos. Rebanadas serán producidos lateralmente a medialmente y típicamente 3 - 4 rebanadas se pueden cortar de cada hemisferio. </li><li> Si la base de los ascensores rebanada utilizar la inclinación 27 G aguja suave presión el corte hacia abajo. Como se corta cada rebanada, utilice la pipeta de transferencia para moverlo a la plataforma en la cámara de contención en el que son viables para un máximo de 8 horas. </li></ol></li></ol> 3. extracelulares Electrofisiología Grabaciones <ol><li> Montaje de la rebanada en la cámara de grabación. <ol><li> Usando la pipeta de transferencia, coloque una rebanada de cerebro en una cámara de grabación sumergida perfundido con LCRa que fluye a 1 - 2 ml / min y se calienta a 32 ° C. El LCRa utilizado para grabaciones difiere de la de la cámara de retención como la concentración de Mg 2+ se incrementa de 2 mM a 4 mM. </li><li> Asegure la rebanada con un &quot;arpa&quot; (acero inoxidable semi circular con cadenas de nylon estirados a través de 2 - 3 mm de separación). Lugarel arpa de modo que las cadenas corren paralelas CA1. Aumentar la velocidad de la perfusión a 8 - 10 ml / min a 32 ° C. </li></ol></li><li> Bajo un microscopio de disección lugar un electrodo de estimulación y un electrodo de registro (electrodo de vidrio lleno con la solución de grabación LCRa) en la superficie de la stratum radiatum de la CA1 (Figura 2A). Coloque el electrodo de estimulación primero y luego colocar el electrodo de registro. </li><li> Estimular el Schaffer colaterales con un 120 - 150 mu amplitud y 0,1 ms pulso de prueba de duración y observar el campo de post sináptica excitatoria potencial (fEPSP) forma de onda resultante para determinar la salud rebanada (Figura 2B). Los electrodos de estimulación y registro pueden necesitar ser movido en la rebanada de aproximadamente 50 - 100 micras de la superficie de la rebanada para obtener un fEPSP grabación con una pequeña volley fibra y gran amplitud. La garantía Schaffer evocó fEPSPs son estereotipadas y proporcionan un buen indicador de la salud de la rebanada. Healthy rebanadas típicamente muestran una volea fibra para fEPSP relación de amplitud inferior a 0,3. </li><li> Reposicionamiento de los electrodos. <ol><li> Usando un microscopio de disección, mover el electrodo de estimulación a la mitad de los estrato oriens y mover el electrodo de registro a la capa de células piramidales tan cerca del electrodo de registro como sea posible, como se muestra en la Figura 3A. Los electrodos de estimulación y registro pueden necesitar ser empujado en la rebanada de aproximadamente 50 - 100 micras de modo que la respuesta de amplitud fEPSP a la 120 - 150 mu pulso de prueba es de aproximadamente 1 mV. </li></ol></li><li> Generación de oscilaciones γ. <ol><li> Para generar oscilaciones γ estimulan el tejido con un tren de pulsos de 20 x 0,1 mseg entregados a 200 Hz. Este estímulo tetánica puede dar respuestas reproducibles cuando se entrega cada 5 minutos. </li></ol></li><li> Utilice los siguientes parámetros de grabación. <ol><li> Escala la ganancia de la señal de salida para que coincida con el voltaje de entrada delel convertidor de analógico a digital. Asegúrese de que la señal máxima esperada excursión utiliza un mínimo de 30% del rango de tensión de entrada. Tenga cuidado al ajustar las ganancias a alto como señales pueden recortar. El ancho de banda típica necesaria para grabaciones de campo es de 500 Hz con acoplamiento de CA a 0,1 Hz para eliminar la deriva de línea base. </li><li> Digitalizar al menos 4-5 veces más rápido que la frecuencia de corte del filtro de paso bajo para evitar el aliasing de señales. </li></ol></li><li> Análisis de los datos. <ol><li> Alto de paso de datos de filtro de 5 Hz para eliminar cualquier cambio de línea de base. Identificar los picos utilizando un umbral derivado, con una separación mínima caso de 10 ms, un tiempo de evento típico a pico y el intervalo de regresión de 7 ms, un umbral de ~ 100 mV / mseg, y un valle de separación del 100%. Calcular la latencia como el tiempo tomado para la primera espiga identificado que se produzca después del final del artefacto de estimulación. Este análisis permite la caracterización del número de picos, latencia, intervalo inter-evento, y la duración calcula comode la siguiente manera: </li><li> Calcular el número de espigas como se determina el número total de espigas por oscilación para cada barrido. </li><li> Calcular la latencia de oscilación. Para cada oscilación, restar el momento de la primera espiga identificado desde el extremo del artefacto de estimulación. </li><li> Calcule el intervalo entre eventos promedio (ISI). Determinar el período de tiempo entre múltiples picos detectados y media. </li><li> Calcular la duración de oscilación. Mida el tiempo entre el primer y último pico detectado dentro de cada oscilación. </li></ol></li></ol>

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Los autores no tienen nada que revelar.

Un método robusto para generar CA1 oscilaciones γ en rodajas de cerebro agudas se describe. Las oscilaciones generadas surgen de un circuito local de permitir una mejor oportunidad para el control y la comprensión de la base neurofisiológica de las oscilaciones de la red 12. Receptores AMPA, receptores GABA A, I h y tipo T Ca 2 + canales son todos necesarios para oscilaciones γ en este modelo. Mientras que las oscilaciones CA1 locales descritos aquí pueden ser generados ...

Oscilaciones de la red cerebral se producen dentro de las bandas de frecuencia distintas que se correlacionan con los estados de comportamiento. En los roedores, las oscilaciones θ hipocampo - se observan (5 de 10 Hz) durante conductas exploratorias 1,2, mientras que las oscilaciones γ (20 - 80 Hz) asociado con diversos procesos cognitivos, incluyendo la percepción y la atención 3,4. Actividad de la red γ síncrono también está implicado en la patología de trastornos tales como la epilepsia y la esquizofrenia 5,6. Por ejemplo, se cree que las oscilaciones γ para corresponder a las áreas de cortical focos epilépticos 5,7,8 y podrían ser utilizados como marcadores de pharmacosensitivity o resistencia, dos áreas importantes de la investigación en la investigación de la epilepsia 9. La rebanada del hipocampo del cerebro es un modelo que ha sido ampliamente utilizado para investigar la actividad de red 10-12. Varios protocolos se han desarrollado para generar oscilaciones γ en rodajas de cerebro que normalmente involve modulación farmacológica como la baja Mg 2+, 4-aminopiridina (4AP), bicuculina y ácido kaínico 12-17. Deficiencias de oscilaciones farmacológicamente desencadenados son que ocurren al azar después de la aplicación de drogas y no se generan de forma fiable o estables en el tiempo. De disparo eléctrico oscilaciones γ superar muchos de estos problemas y también tienen la ventaja de estar temporalmente bloqueada para el evento estimulante que permite la grabación y el análisis episódica. Aquí se describe un protocolo para la generación de oscilaciones γ CA1 mediante la entrega de una estimulación tetánica a las estrato oriens en el corte de hipocampo.

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			<td>CaCl2.2H2O</td>
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			<td>MgCl2.6H2O</td>
			<td>Sigma Aldrich</td>
			<td>M2670-500G</td>
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			<td>Harvard Apparatus&#160;</td>
			<td>GC150F-10</td>
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			<td>Concentric bipolar stimulating metal electrode&#160;</td>
			<td>FHC</td>
			<td>CBBPF75</td>
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			<td>Getting Instruments</td>
			<td>Model BJN8-9V1&#160;</td>
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			<td>Model 1800 amplifier</td>
			<td>A-M systems</td>
			<td>Model 1800 amplifier</td>
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			<td>Digitizer</td>
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generation of local ca1 &#947; oscillations by tetanic stimulation

Oscilaciones de la red neuronal son características importantes de la actividad cerebral en la salud y la enfermedad y puede ser modulada por una serie de medicamentos que se utilizan clínicamente. Un protocolo se proporciona para generar un modelo para el estudio de oscilaciones γ CA1 (20 - 80 Hz). Estas oscilaciones γ son estables durante al menos 30 min y dependen de la actividad sináptica excitatoria y inhibitoria Además de la activación de las corrientes de marcapasos. Oscilaciones tetánicamente estimuladas tienen una serie de características reproducibles y fácilmente cuantificables incluyendo recuento de pico, duración de oscilación, la latencia y la frecuencia que informan sobre el estado de la red. Las ventajas de las oscilaciones estimuladas eléctricamente incluyen la estabilidad, la reproducibilidad y la adquisición episódica que permite la caracterización robusta de la función de red. Este modelo de oscilaciones γ CA1 se puede utilizar para estudiar mecanismos celulares y para investigar sistemáticamente cómo actividad de la red neuronal se altera en la enfermedad y por las drogas.Farmacología estado de enfermedad puede ser incorporado fácilmente por el uso de cortes de cerebro de modelos animales genéticamente modificados o intervencionistas para permitir la selección de fármacos que se dirigen específicamente mecanismos de la enfermedad.

Estimulación tetánica de los estrato oriens generado robustos y reproducibles oscilaciones γ (35,4 ± 2,2 Hz), véase la Figura 3B. Para demostrar que las oscilaciones se generaron dentro de la red local de CA1 las entradas de CA3 fueron separadas por el corte de la rebanada en la región CA2 usando un doblado 32 G aguja. Las propiedades de oscilación en las rodajas cortadas no difieren de las rodajas cortadas (p = 0,85; corte rebanadas 6,16 ± 1,1 puntos, n = 6; sin cortar rebanadas 5,89 ± 0,8 pun...

Watch this Scientific Journal Video about Generation of Local CA1 Î³ Oscillations by Tetanic Stimulation at JoVE.com

Generation of Local CA1 &amp;#947; Oscillations by Tetanic Stimulation

Las oscilaciones son propiedades fundamentales de la red y son modulados por la enfermedad y las drogas. El estudio de las oscilaciones cerebrales rebanada permite la caracterización de las redes aisladas en condiciones controladas. Los protocolos se proporcionan para la preparación de cortes de cerebro agudos para evocar oscilaciones γ CA1.

Generación de oscilaciones γ CA1 locales por tetánica Estimulación

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

generation-of-local-ca1-oscillations-by-tetanic-stimulation

Research

JoVE Journal

Neuroscience

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

8.9K vistas.  University of Melbourne.  Las oscilaciones son propiedades fundamentales de la red y son modulados por la enfermedad y las drogas. El estudio de las oscilaciones cerebrales rebanada permite la caracterización de las redes aisladas en condiciones controladas. Los protocolos se proporcionan para la preparación de cortes de cerebro agudos para evocar oscilaciones γ CA1. 

Video: Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

A Novel Approach for Documenting Phosphenes Induced by Transcranial Magnetic Stimulation

Stimulation of the human visual cortex produces a transient perception of light, known as a phosphene. Phosphenes are induced by invasive electrical stimulation of the occipital cortex, but also by non-invasive Transcranial Magnetic Stimulation (TMS)1 of the same cortical regions. The intensity at which a phosphene is induced (phosphene threshold) is a well established measure of visual cortical excitability and is used to study cortico-cortical interactions, functional organization 2, susceptibility to pathology 3,4 and visual processing 5-7. Phosphenes are typically defined by three characteristics: they are observed in the visual hemifield contralateral to stimulation; they are induced when the subject s eyes are open or closed, and their spatial location changes with the direction of gaze 2. Various methods have been used to document phosphenes, but a standardized methodology is lacking. We demonstrate a reliable procedure to obtain phosphene threshold values and introduce a novel system for the documentation and analysis of phosphenes. We developed the Laser Tracking and Painting system (LTaP), a low cost, easily built and operated system that records the location and size of perceived phosphenes in real-time. The LTaP system provides a stable and customizable environment for quantification and analysis of phosphenes.

Phosphenes are transient percepts of light that can be induced by applying Transcranial Magnetic Stimulation (TMS) to visually sensitive regions of cortex. We demonstrate a standard protocol for determining the phosphene threshold value and introduce a novel method for quantifying and analyzing perceived phosphenes.

Un nuevo enfoque para la documentación de fosfenos inducida por la estimulación magnética transcraneal

Stimulation of the human visual cortex produces a transient perception of light, known as a phosphene. Phosphenes are induced by invasive electrical ...

Investigación

Neurociencias

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

La combinación de Estimulación Magnética Transcraneal y fMRI para examinar la red en modo por defecto

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Uso del protocolo Theta-Burst para explorar Mechasnism de plasticidad en los individuos con el síndrome de X frágil y el autismo

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

El dispositivo de TMS NeuroStar: Realización de la FDA aprobó el Protocolo para el tratamiento de la depresión

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Efectos de la corriente alterna la estimulación transcraneal en la corteza de Motor primaria por Online enfoque combinado con la estimulación magnética transcraneal

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when compared to warm-blooded vertebrates. Because of this, in vitro experiments in turtles can be performed for extended periods of time to investigate the neural signals and control of their target actions. Using an isolated head preparation, we measured the kinematics of eye movements in turtles, and their modulation by electrical signals carried by cranial nerves. After the brain was removed from the skull, leaving the cranial nerves intact, the dissected head was placed in a gimbal to calibrate eye movements. Glass electrodes were attached to cranial nerves (oculomotor, trochlear, and abducens) and stimulated with currents to evoke eye movements. We monitored eye movements with an infrared video tracking system and quantified rotations of the eyes. Current pulses with a range of amplitudes, frequencies, and train durations were used to observe effects on responses. Because the preparation is separated from the brain, the efferent pathway going to muscle targets can be examined in isolation to investigate neural signaling in the absence of centrally processed sensory information.

Ocular Kinematics Measured by In Vitro Stimulation of the Cranial Nerves in the Turtle

This protocol describes how to use an in vitro isolated turtle head preparation to measure the kinematics of their eye movements. After removal of the brain from the cranium, cranial nerves can be stimulated with currents to quantify rotations of the eye and changes in pupil sizes.

Cinemática ocular midiendo In Vitro la estimulación de los nervios craneales en la tortuga

After animals are euthanized, their tissues begin to die. Turtles offer an advantage because of a longer survival time of their tissues, especially when ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic de arrastre de las oscilaciones Theta Hippocampal en comportarse ratones

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

Grabación espacial restringido a oscilaciones en el hipocampo de ratones de comportarse

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources&#8212;&#8220;Ca2+ influx&#8221; from outside the cell and &#8220;Ca2+ release&#8221; from the intracellular Ca2+ store endoplasmic reticulum (ER)&#8212;is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of &#8220;Ca2+ influx&#8221; and &#8220;Ca2+ release&#8221;. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Disección de Local Ca2 + señales en las células cultivadas por membrana dirigidos Ca2 + indicadores

Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The ...

Investigating Long-term Synaptic Plasticity in Interlamellar Hippocampus CA1 by Electrophysiological Field Recording

The study of synaptic plasticity in the hippocampus has focused on the use of the CA3-CA1 lamellar network. Less attention has been given to the longitudinal interlamellar CA1-CA1 network. Recently however, an associational connection between CA1-CA1 pyramidal neurons has been shown. Therefore, there is the need to investigate whether the longitudinal interlamellar CA1-CA1 network of the hippocampus supports synaptic plasticity.
We designed a protocol to investigate the presence or absence of long-term synaptic plasticity in the interlamellar hippocampal CA1 network using electrophysiological field recordings both in vivo and in vitro. For in vivo extracellular field recordings, the recording and stimulation electrodes were placed in a septal-temporal axis of the dorsal hippocampus at a longitudinal angle, to evoke field excitatory postsynaptic potentials. For in vitro extracellular field recordings, hippocampal longitudinal slices were cut parallel to the septal-temporal plane. Recording and stimulation electrodes were placed in the stratum oriens (S.O) and the stratum radiatum (S.R) of the hippocampus along the longitudinal axis. This enabled us to investigate the directional and layer specificity of evoked excitatory postsynaptic potentials. Already established protocols were used to induce long-term potentiation (LTP) and long-term depression (LTD) both in vivo and in vitro. Our results demonstrated that the longitudinal interlamellar CA1 network supports N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) with no directional or layer specificity. The interlamellar network, however, in contrast to the transverse lamellar network, did not present with any significant long-term depression (LTD).

We used recording and stimulation electrodes in longitudinal hippocampal brain slices and longitudinally positioned recording and stimulation electrodes in the dorsal hippocampus in vivo to evoke extracellular postsynaptic potentials and demonstrate long-term synaptic plasticity along the longitudinal interlamellar CA1.