Los autores desean agradecer a Neurotar Ltd. para la asistencia técnica, la Fundación de la CIMO y FGSN de apoyo financiero.

Los procedimientos que implican sujetos animales han sido aprobados por la Junta Nacional Experimental Animal, Finlandia. 1. Preparación Animal for Imaging <ol><li> Anestesiar a un ratón mediante inyección intraperitional (IP) de ketamina (0,08 mg por peso corporal) y xilazina (0,01 mg por peso corporal). Compruebe la anestesia con la parte trasera reflejo del pellizco del dedo del pie. </li><li> Sumergir los ojos de animales en gotas para los ojos (Viscotears) para proteger los ojos de la deshidratación. </li><li> Ponga el mouse sobre una almohadilla eléctrica (Supertech) a 37 ° C para evitar la hipotermia. </li><li> Limpiar la almohadilla de la pata designado para formación de imágenes con etanol al 70%. </li><li> Añadir una gota de agua sobre la piel de la almohadilla de la pata de inmersión para el acoplamiento entre la piel y la cubierta de vidrio. </li><li> Coloque el material de embalaje de plástico debajo de la pata trasera que se coloca debajo de un anillo de metal con clase de cobertura. </li><li> Ajustar el grosor del material de embalaje de plástico para aplanar la piel. </li><li> Ponga una gota de agua sobre la cubierta glass para la inmersión entre cubreobjetos y el objetivo. </li></ol> 2. metal Fixator Preparación Anillo <ol><li> Para la estabilización de la piel utilizar el fijador de metal. Utilizamos protegido Diseño Comunidad fijador de dos hojas con un anillo de metal (cortesía de Neurotar Ltd, Finlandia). Llene la jeringa con superglue, poner la pequeña gota de pegamento a través de la aguja en el anillo y extender suavemente el pegamento para la cobertura uniforme de la superficie del anillo. Es fundamental poner la mínima cantidad de pegamento que se necesita sólo para una cobertura uniforme, debido a la cantidad excesiva de pegamento puede reducir el campo de la visión y obstruir la imagen posterior. NOTA: Como alternativa, la combinación de una barra de metal (de debajo) y portaobjetos de vidrio de microscopio estándar (como una tapa) podría ser utilizado para aplanar la piel y para asegurar el acoplamiento óptico adecuado del conjunto 14. Dos clips de papel podrían ser utilizados para fijar la pata entre el vidrio y la superficie de la barra de metal (figura 4 </strong&gt;). </li><li> Cubra el anillo con tapa deslizante microscopio (5 mm de diámetro, Microscopía Electrónica de Ciencias). </li><li> Tornillo en el fijador de anillo de la barra de metal rígido que se montaged en la platina del microscopio motorizado. </li></ol> 3. Procedimiento Imaging <ol><li> En caso de utilizar los ratones Thy1-YFP-H, elija la parte azul del espectro de fluorescencia de la lámpara para visualizar las fibras nerviosas. Encuentra el nervio de interés en el modo de epifluorescencia y se centran en ella. </li><li> Anote las coordenadas de un lugar para la imagen longitudinal. Use una almohadilla carpiano como punto de referencia. Durante las siguientes sesiones de formación de imágenes, detectar primero la almohadilla del carpo y luego encontrar las mismas fibras nerviosas grandes aguas arriba y vuelva usando las coordenadas guardadas. Es esencial para posicionar la almohadilla de la pata en la misma dirección perpendicular a la barra de metal para mantener el mismo sistema de referencia. </li><li> Activar el modo de dos fotones. Para los ratones Thy1-YFP-H, es adecuado utilizar la longitud de onda de 950 nm de radiación láserpara visualizar las fibras nerviosas. </li><li> Seleccionar la longitud de onda de emisión láser y canales (450 a 480 nm para la detección de segunda generación armónica, 520-550 nm para la obtención de imágenes de los nervios etiquetados-YFP y 580-630 nm para la detección de fluorescencia y YFP autofluorescencia del cabello), comenzar con láser de baja potencia (3-5%) para prevenir el fotodaño y blanqueo. La tensión típica en los tubos fotomultiplicadores es 600-700 V para este tipo de formación de imágenes. </li><li> Ajuste la resolución deseada (512 x 512 o 640 x 640 para mediciones de procesos rápidos, 800 x 800 o 1024 x 1024 para imágenes morfológicas), paso axial (1-3 micras), el espesor de la muestra y los intervalos de tiempo (1-10 min). El tiempo de exposición es en el orden de 1 ms por un píxel. </li><li> Primero marcar los límites superior e inferior del volumen de formación de imágenes en el software. </li><li> Adquirir la pila de imágenes de referencia para la referencia antes de la lesión. Cuando sea necesario, es posible grabar la línea de base durante varios minutos. </li><li&gt; Repetidamente (al menos cada 10-15 minutos) comprobar el ritmo respiratorio de los animales y los reflejos, cuando sea necesario aplicar una cantidad adicional de anestésicos. </li><li> Después de la formación de imágenes limpia la almohadilla con un pañuelo de papel y poner el ratón en una caja de recuperación a 36 ° C hasta que esté completamente despierto. NOTA: Por lo general, la duración de una sesión de imágenes combinadas con lesión inducida por láser (ver más abajo) no sea superior a 1 hora. Se recomienda verificar que el animal está profundamente anestesiado cada 10-15 minutos; esto no debería interferir con la formación de imágenes en sí, sino que debe ser considerado, mientras que está previsto procedimiento experimental. La duración del efecto de una dosis única de ketamina / xylazyne es de alrededor de 30 a 40 min, por lo tanto se recomienda aplicar adicional de ¼ a ½ dosis después de 25-30 min. NOTA: Se debe considerar también cuando la experimentación es la planificación que la frecuencia de las sesiones de formación de imágenes de un animal individual no debe exceder de 1 sesión por 2 días para evitar los efectos adversos de la reanestesia competitiva. </li></ol> 4. Laser lesión <ol><li> Después de la adquisición de la imagen de referencia de pila, utilice el protocolo de blanqueo para hacer una lesión micro. </li><li> Aumentar la potencia del láser al 100%. </li><li> Aumentar el tiempo de exposición a 100-1.000 ms por región de interés (100-1,000x más que en el caso de las imágenes estándar). </li><li> Esquema de la zona de la lesión. </li><li> Hacer la lesión por el cambio en el blanqueo. </li><li> Vuelva al modo de imagen normal y continuar con las grabaciones time-lapse. </li></ol> 5. Procesamiento y Análisis de Datos <ol><li> Proceda con desmezcla espectral usando desmezcla plugin en ImageJ 8 (Joachim Walter, <a href="http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html">http://rsbweb.nih.gov/ij/plugins/spectral-unmixing.html</a> ). </li><li> Abra la pila de imágenes y dividir los canales. </li><li> Encuentra el área libre de los nervios o de pelo para las mediciones de fondo. Ponga una región of interés en esa área. </li><li> Esquema cuidadosamente la parte del cabello donde se distingue claramente de los nervios. </li><li> Esquema del nervio en la parte de la imagen donde es separado del cabello. </li><li> Ahorra desmezcla matriz. </li><li> Aplicar matriz desmezcla medido para toda la pila. </li><li> Guarde los nuevos pilas de imágenes procesadas en formato * .tif. </li></ol>

<ol>
	<li>Lumpkin, E. A., Caterina, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+sensory+transduction+in+the+skin.">Mechanisms of sensory transduction in the skin.</a> Nature. 445, 858-865 (2007).</li><li>Kennedy, W. R., Wendelschafer-Crabb, G., Johnson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+epidermal+nerves+in+diabetic+neuropathy.">Quantitation of epidermal nerves in diabetic neuropathy.</a> Neurology. 47, 1042-1048 (1996).</li><li>Wu, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morton's+interdigital+neuroma:+a+clinical+review+of+its+etiology,+treatment,+and+results.">Morton's interdigital neuroma: a clinical review of its etiology, treatment, and results.</a> J. Foot Ankle Surg. 35, 112-119 (1996).</li><li>Lauria, G., Lombardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+biopsy:+a+new+tool+for+diagnosing+peripheral+neuropathy.">Skin biopsy: a new tool for diagnosing peripheral neuropathy.</a> BMJ. 334, 1159-1162 (2007).</li><li>Cheng, C., Guo, G. F., Martinez, J. A., Singh, V., Zochodne, D. 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Los autores declaran que no tienen intereses financieros en competencia.

En este protocolo de vídeo se demuestra el método para longitudinal dos fotones de imágenes no invasiva de las terminaciones nerviosas individuales. La dinámica de la inervación de la piel se ve afectada en enfermedades tales como la psoriasis y la neuropatía periférica 2, y en lesiones traumáticas 9. Dos fotones de imágenes permite el análisis detallado de las estructuras de fibras nerviosas en la matriz de colágeno. El uso de ratones transgénicos reportero...

A correction was made to <a href="/video/51045">In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin</a>.
Leonard Khiroug was removed as an author.

Erratum: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Terminaciones nerviosas cutáneas experimentan cambios dinámicos en diferentes estados fisiopatológicos. Las fibras nerviosas pueden pasar por el proceso de degeneración y regeneración o reestructuración en el curso de estas enfermedades como la neuropatía periférica 2 o Morton neuroma 3. Después de una lesión traumática, una parte importante de la dinámica de terminaciones nerviosas en la piel es la reinervación de la zona dañada. Sin embargo, el enfoque común para la investigación de las terminaciones nerviosas es ex vivo seccionamiento histológico que carece de información en tiempo real sobre los procesos en curso 4. El uso de marcadores fluorescentes codificadas genéticamente, es posible realizar un seguimiento de las terminaciones nerviosas en la piel de animales vivos, obteniendo así información rica y significativamente más pertinente sobre los cambios estructurales. La investigación de las terminaciones nerviosas cutáneas es posible utilizando microscopía de fluorescencia convencional, sin embargo, la luz fuerte dispersión de la tejido de la piel socava fuertemente en la calidadde los datos adquiridos 5. Microscopía multifotónica permite la adquisición de imágenes de alta resolución en los tejidos debido a la fuerte dispersión de la suma no lineal de la energía de los fotones de luz de excitación resultantes en la emisión de fluorescencia sólo desde el punto focal del objetivo. Este efecto conduce a un fuerte incremento en la profundidad de penetración y la mejora de la relación señal-ruido para la medición en los tejidos de la piel 6. Utilizando el mismo láser como para formación de imágenes, es posible producir la disección selectiva de las fibras nerviosas 7. En el siguiente protocolo se muestra el método de formación de imágenes longitudinal de las terminaciones nerviosas cutáneas in vivo en ratones transgénicos reportero combinado con lesión selectiva por láser utilizando el sistema de microscopio multifotónica disponible comercialmente.

<table border="0" cellpadding="0" cellspacing="0" width="1231">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round cover glass&#160;</td>
			<td style="width:209px;">Electron Microscopy Science</td>
			<td style="width:119px;">72296-05</td>
			<td style="width:687px;">5 mm diameter #1.5 thickness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eye drops Viscotears</td>
			<td style="width:209px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>2mg/g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superglue Loctite 401</td>
			<td style="width:209px;">Henkel</td>
			<td>135429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketaminol</td>
			<td style="width:209px;">Intervet</td>
			<td style="width:119px;"></td>
			<td>Ketamine 50 mg/ml, working solution 10 mg/ml (use it 80 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rompun</td>
			<td style="width:209px;">Bayer Healthcare</td>
			<td style="width:119px;"></td>
			<td>Xylazine 20 mg/ml, working solution 1.25 mg/ml (use it 10 &#181;g per gramm of animal weight)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td style="width:687px;">Working solution is a mixture of Ketamine 10 mg/ml and Xylazine 1.25 mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol 70%</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Distilled water or Milli-Q water</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes 1ml&#160;</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30G 1/2&#34; needles</td>
			<td style="width:209px;">BD</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plastic packaging material</td>
			<td style="width:209px;"></td>
			<td style="width:119px;"></td>
			<td>Could be purchaized from general hardware store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FV-1000MPE microscope</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">FV-1000MPE</td>
			<td>Microscope with motorized stage and rigid metal bar for fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25X XLPlan objective</td>
			<td style="width:209px;">Olympus</td>
			<td style="width:119px;">XLPLN 25XWMP</td>
			<td>Water imersion objective optimized for multiphoton imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mai-Tai DeepSee laser (2W)</td>
			<td style="width:209px;">SpectraPhysics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heating pad</td>
			<td style="width:209px;">Supertech</td>
			<td style="width:119px;">TMP-5b</td>
			<td>Heating pad with a temperature controller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal ring fixator</td>
			<td style="width:209px;">Neurotar Ltd.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:209px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Open source software for image processing and analysis, http://rsbweb.nih.gov/ij/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thy1-YFPH mice strain</td>
			<td style="width:209px;">JaxLab</td>
			<td style="width:119px;">003782</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-photon microscopy of single nerve endings in skin

Las terminaciones nerviosas en la piel están implicadas en procesos fisiológicos tales como la detección de 1, así como en procesos patológicos tales como dolor neuropático 2. Su posicionamiento de cerca a la superficie facilita imágenes microscópicas de las terminaciones nerviosas de la piel en la que viven los animales intactos. Usando microscopía multifotónica, es posible obtener imágenes finas superar el problema de la fuerte dispersión de la luz del tejido de la piel. Reportero ratones transgénicos que expresan EYFP bajo el control del promotor Thy-1 en las neuronas (incluyendo neuronas sensoriales periferia) son muy adecuados para los estudios longitudinales de las terminaciones nerviosas individuales durante períodos prolongados de tiempo hasta varios meses o incluso largo de la vida. Además, usando el mismo láser de femtosegundo como para la formación de imágenes, es posible producir lesiones altamente selectivos de fibras nerviosas para los estudios de la reestructuración de la fibra nerviosa. A continuación, presentamos un protocolo sencillo y fiable para multifotón longitudinal de imágenes in vivo ymicrocirugía láser basado en las terminaciones nerviosas de la piel de ratones.

Usando la técnica descrita es posible un seguimiento de la misma fibra después de la lesión y para estudiar la degradación de las terminaciones nerviosas dañadas (Figura 1). Adquisición de la pila con el espesor de 120-150 micras es generalmente adecuado para la formación de imágenes repetitivo durante varios días con el fin de mantener a toda la fibra en el campo de visión. La lesión típicamente se puede producir de forma concisa cuando el material de los envase...

Watch this Scientific Journal Video about In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin at JoVE.com

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Se presenta el protocolo para la formación de imágenes longitudinal dinámico y lesión selectiva por láser de las terminaciones nerviosas en ratones transgénicos reportero.

En Vivo De dos fotones microscopía de terminaciones nerviosas individuales en la piel

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

in-vivo-two-photon-microscopy-of-single-nerve-endings-in-skin

Research

JoVE Journal

Neuroscience

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

10.7K vistas.  University of Helsinki.  Se presenta el protocolo para la formación de imágenes longitudinal dinámico y lesión selectiva por láser de las terminaciones nerviosas en ratones transgénicos reportero. 

Video: In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Imágenes in vivo de la médula espinal de ratón Utilizando microscopía de dos fotones

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Investigación

Neurociencias

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimagen Funcional Utilizando ultrasonidos hematoencefálica perturbación de la barrera y la RM-manganeso mayor

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

In vivo de dos fotones de imágenes de dependientes Experience-Los cambios moleculares en las neuronas corticales

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultánea de dos fotones<em&gt; En Vivo</em&gt; Imágenes de entradas sinápticas y Objetivos postsinápticos en la corteza del ratón retroesplenial

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Cuantificación de Cerebral Vascular Arquitectura utilizando microscopía de dos fotones en un modelo de ratón de la neuroinflamación inducida por el VIH

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

Seguimiento óptico del nervio terminal de estar etiquetado en folículo piloso lanceoladas Los finales de la<em&gt; Ex Vivo</em&gt; Piel de la oreja de ratón

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Proyección de imagen de calcio de dos fotones en las dendritas neuronales en rebanadas de cerebro

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

En Vivo Proyección de imagen de dos fotones de neuronas corticales en ratones neonatales

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

En Vivo Microscopía bicolor tridimensional para estudiar las respuestas vasculares realizadas por la eyección ATP local utilizando una micro-pipette de vidrio

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

Visualización de la proteína quinasa Una actividad en ratones de trabajo fijos en la cabeza utilizando la microscopía de imágenes de por vida con fluorescencia de dos fotones in Vivo

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...