Este trabajo fue apoyado por subvenciones de Samsung Science and Technology Foundation (Número de proyecto SSTF-BA1801-10).

<ol>
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Las técnicas de cirugía hábiles son esenciales para lograr imágenes ópticas de calcio in vivo exitosas con microscopía en miniatura de montaje en la cabeza en regiones cerebrales más profundas como la amígdala como la describimos aquí. Por lo tanto, aunque este protocolo proporciona una guía para procesos quirúrgicos optimizados de fijación de placa base e implantación de lentes GRIN, es posible que sean necesarios procesos de optimización adicionales para pasos críticos. Como se menciona en la sección de...

El calcio es un segundo mensajero omnipresente, jugando un papel crucial en casi todas las funciones celulares8. En las neuronas, el disparo potencial de acción y la entrada sináptica causan un cambio rápido de libre intracelular [Ca2+]9,10. Por lo tanto, el seguimiento de los transitorios de calcio proporciona una oportunidad para monitorear la actividad neuronal. Los GECIs son potentes herramientas que permiten monitorear [Ca2+] en poblaciones celulares definidas y compartimentos intracelulares11,12. Entre muchos tipos diferentes de indicadores de calcio a base de proteínas, GCaMP, una sonda Ca2+ basada en una sola molécula GFP13,es la GECI más optimizada y por lo tanto ampliamente utilizada. A través de múltiples rondas de ingeniería, se han desarrollado una serie de variantes de GCaMP12,14,15,16. Utilizamos uno de los GCaMPs recientemente desarrollados, GCaMP7b, en este protocolo16. Los sensores GCaMP han contribuido en gran medida al estudio de las funciones de circuito neural en una serie de organismos modelo como la toma de imágenes de los transitorios Ca2+ durante el desarrollo17,la imagen in vivo en una capa cortical específica18,la medición de la dinámica de circuito en el aprendizaje de tareas motoras19 y la imagen de la actividad del conjunto celular relacionada con la memoria de miedo asociativa en el hipocampo y la amígdala20,21.
La imagen óptica de los GECIs tiene varias ventajas22. La codificación genética permite que los GECIs se expresen de forma estable durante un período de tiempo a largo plazo en un subconjunto específico de células definidas por perfil genético o patrones específicos de conectividad anatómica. Las imágenes ópticas permiten un monitoreo simultáneo crónico in vivo de cientos a miles de neuronas en animales vivos. Se han desarrollado algunos sistemas ópticos de imágenes para imágenes in vivo y análisis de GECIs dentro del cerebro de ratones que se comportan libremente con microscopios de fluorescencia miniaturizados de montaje en la cabeza21,23,24,25. A pesar de que la técnica de imagen óptica in vivo basada en GECIs, la lente GRIN y un microscopio en miniatura de montaje en la cabeza son una poderosa herramienta para estudiar el vínculo entre la actividad y el comportamiento de los circuitos neuronales, aplicar esta tecnología a la amígdala ha sido difícil debido a varios problemas técnicos relacionados con la orientación de la lente GRIN a las células que expresan GECIs en la amígdala sin causar artefactos de movimiento que reducen severamente la calidad de la adquisición de imágenes y la búsqueda de células que expresan GECIs. Este protocolo tiene como objetivo proporcionar una guía útil para los procedimientos quirúrgicos de fijación de placa base e implantación de lentes GRIN que son pasos críticos para obtener imágenes ópticas de calcio in vivo exitosas en la amígdala. Aunque este protocolo se dirige a la amígdala, la mayoría de los procedimientos descritos aquí son comúnmente aplicables a otras regiones cerebrales más profundas. Aunque este protocolo se basa en un sistema en particular (por ejemplo, Inscopix), el mismo propósito puede lograrse fácilmente con otros sistemas alternativos.

<table border="0" cellpadding="0" cellspacing="0" style="width:666px;" width="665">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">26G needle</td>
			<td style="width:121px;">BD</td>
			<td align="right" style="width:161px;">302002</td>
			<td style="width:167px;">Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AAV1-Syn-GCaMP7b-WPRE</td>
			<td style="width:121px;">Addgene</td>
			<td style="width:161px;">104493-AAV1</td>
			<td>Surgery</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">AAV2/1-CaMKii&#945;-GFP</td>
			<td style="width:121px;">custom made</td>
			<td style="width:161px;"></td>
			<td>Surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acrylic-Dental cement (Ortho-jet Acrylic Pink)</td>
			<td style="width:121px;">Lang</td>
			<td style="width:161px;">1334-pink</td>
			<td>Surgery &#38; Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Air flow manipulator</td>
			<td style="width:121px;">Neurotar</td>
			<td style="width:161px;">NTR000253-04</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amoxicillin</td>
			<td style="width:121px;">SIGMA</td>
			<td style="width:161px;">A8523-5G</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Baseplate</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-002192</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Baseplate cover</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-002193</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Behavioral apparatus (chamber)</td>
			<td style="width:121px;">Coulbourn Instrument</td>
			<td style="width:161px;">Testcage</td>
			<td>Behavior test</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Behavioral apparatus (software)</td>
			<td style="width:121px;">Coulbourn Instrument</td>
			<td style="width:161px;">Freeze Frame</td>
			<td>Behavior test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carbon cage</td>
			<td style="width:121px;">Neurotar</td>
			<td style="width:161px;">180mm x 70mm</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carprofen</td>
			<td style="width:121px;">SIGMA</td>
			<td style="width:161px;">PHR1452-1G</td>
			<td>Surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Data processing software</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">INSCOPIX Data Processing Software</td>
			<td>Baseplate Attachment &#38; Behavior test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dexamethasone</td>
			<td style="width:121px;">SIGMA</td>
			<td style="width:161px;">D1756-500MG</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Drill</td>
			<td style="width:121px;">Seyang</td>
			<td style="width:161px;">marathon-4</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Drill bur</td>
			<td style="width:121px;">ELA</td>
			<td style="width:161px;">US1/2, Shank104</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass needle</td>
			<td style="width:121px;">WPI</td>
			<td style="width:161px;">PG10165-4</td>
			<td>Surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GRIN lens (INSCOPIX Proview Lens Probe)</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-002208</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hamilton Syringe</td>
			<td style="width:121px;">Hamilton</td>
			<td align="right" style="width:161px;">84875</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Head plate</td>
			<td style="width:121px;">Neurotar</td>
			<td style="width:161px;">Model 5</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hex-key</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-004195</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laptop computer</td>
			<td style="width:121px;">Samsung</td>
			<td style="width:161px;">NT950XBV</td>
			<td>Surgery &#38; Baseplate Attachment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lens holder, Stereotaxic rod (INSCOPIX proview implant kit)</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-004223</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope gripper</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">1050-002199</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope, DAQ software, hardware</td>
			<td style="width:121px;">INSCOPIX</td>
			<td style="width:161px;">nVista 3.0</td>
			<td>Baseplate Attachment &#38; Behavior test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mobile homecage</td>
			<td style="width:121px;">Neurotar</td>
			<td style="width:161px;">MHC V5</td>
			<td>Baseplate Attachment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moterized arm</td>
			<td style="width:121px;">Neurostar</td>
			<td style="width:161px;">Customized</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moterized arm software</td>
			<td style="width:121px;">Neurostar</td>
			<td style="width:161px;">Customized</td>
			<td>Surgery</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NI board</td>
			<td style="width:121px;">National instrument</td>
			<td style="width:161px;"></td>
			<td>Behavior test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Removable epoxy bond</td>
			<td style="width:121px;">WPI</td>
			<td style="width:161px;">Kwik-Cast</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Resin cement (Super-bond)</td>
			<td style="width:121px;">Sun medical</td>
			<td style="width:161px;">Super bond C&#38;B</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Skull screw</td>
			<td style="width:121px;">Stoelting</td>
			<td align="right" style="width:161px;">51457</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereotaxic electrode holder</td>
			<td style="width:121px;">ASI</td>
			<td style="width:161px;">EH-600</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereotaxic frame</td>
			<td style="width:121px;">Stoelting</td>
			<td align="right" style="width:161px;">51600</td>
			<td>Surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereotaxic manipulator</td>
			<td style="width:121px;">Stoelting</td>
			<td align="right" style="width:161px;">51600</td>
			<td>Baseplate Attachment</td>
		</tr>
	</tbody>
</table>

successful in vivo calcium imaging with a head-mount miniaturized microscope in the amygdala of freely behaving mouse

El monitoreo in vivo en tiempo real de las actividades neuronales en animales en movimiento libre es uno de los enfoques clave para vincular la actividad neuronal con el comportamiento. Para ello, se ha desarrollado y aplicado con éxito a muchas estructuras cerebrales1,2,3,4,5,6una técnica de imágenes in vivo que detecta transitorios de calcio en las neuronas utilizando indicadores de calcio codificados genéticamente (GECIs), un microscopio de fluorescencia miniaturizado y una lente de índice refractivo degradado (GRIN). Esta técnica de imágenes es particularmente potente porque permite la toma de imágenes simultáneas crónicas de poblaciones celulares genéticamente definidas durante un período a largo plazo hasta varias semanas. Aunque útil, esta técnica de imagen no se ha aplicado fácilmente a las estructuras cerebrales que se encuentran en lo profundo del cerebro como la amígdala, una estructura cerebral esencial para el procesamiento emocional y la memoria del miedo asociativo7. Hay varios factores que dificultan la aplicación de la técnica de imagen a la amígdala. Por ejemplo, los artefactos de movimiento generalmente ocurren con más frecuencia durante las imágenes realizadas en las regiones cerebrales más profundas porque un microscopio de montaje en la cabeza implantado en las profundidades del cerebro es relativamente inestable. Otro problema es que el ventrículo lateral se coloca cerca de la lente GRIN implantada y su movimiento durante la respiración puede causar artefactos de movimiento altamente irregulares que no se pueden corregir fácilmente, lo que dificulta la formación de una vista de imagen estable. Además, debido a que las células de la amígdala suelen estar silenciosas en un estado de reposo o anestesiado, es difícil encontrar y enfocar las células objetivo que expresan GECI en la amígdala durante el procedimiento de placa base para obtener imágenes posteriores. Este protocolo proporciona una guía útil sobre cómo dirigirse eficientemente a las células que expresan GECI en la amígdala con microscopio miniaturizado de montaje en la cabeza para obtener imágenes de calcio in vivo exitosas en una región cerebral más profunda. Se observa que este protocolo se basa en un sistema particular (por ejemplo, Inscopix) pero no se limita a él.

Validación de la implantación de lentes GRIN Antes de conectar crónicamente la placa base al cerebro mediante la cementación, la implantación de la lente GRIN debe ser validada. En animales con implantación exitosa de lentes, tanto las células que expresan GCaMP como los vasos sanguíneos se observaron claramente dentro de un rango de plano focal determinado por la distancia entre la lente objetiva del microscopio y la lente GRIN implantada(Figura 2</stron...

Watch this Scientific Journal Video about Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse at JoVE.com

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

La imagen de calcio microendoscópica in vivo es una herramienta invaluable que permite el monitoreo en tiempo real de las actividades neuronales en animales que se comportan libremente. Sin embargo, aplicar esta técnica a la amígdala ha sido difícil. Este protocolo tiene como objetivo proporcionar una guía útil para atacar con éxito las células de amígdala con un microscopio miniaturizado en ratones.

Imágenes de calcio in vivo exitosas con un microscopio miniaturizado head-mount en la amígdala del ratón que se comporta libremente

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this ...

successful-vivo-calcium-imaging-with-head-mount-miniaturized

Research

JoVE Journal

Neuroscience

12.0K vistas.  Korea Advanced Institute of Science and Technology (KAIST). La imagen de calcio microendoscópica in vivo es una herramienta invaluable que permite el monitoreo en tiempo real de las actividades neuronales en animales que se comportan libremente. Sin embargo, aplicar esta técnica a la amígdala ha sido difícil. Este protocolo tiene como objetivo proporcionar una guía útil para atacar con éxito las células de amígdala con un microscopio miniaturizado en ratones.

Video: Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

Formación de imágenes in vivo de calcio neuronal en C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Investigación

Neurociencias

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

En Vivo Cerebral Funcional Imaging Enfoque Basado en calcio bioluminiscente Indicador GFP-aequorina

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

Imagen radiométrica calcio de neuronas individuales en comportamiento Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

A largo plazo conflicto sensorial en comportarse libremente ratones

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imágenes transpupilares de dos fotones in vivo de la retina del ratón

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.

Here we present how to expose the geniculate ganglion of a live, anesthetized laboratory mouse and how to use calcium imaging to measure the responses of ensembles of these neurons to taste stimuli, allowing for multiple trials with different stimulants. This allows for in depth comparisons of which neurons respond to which tastants.

Imágenes de calcio in vivo de las respuestas de las neuronas ganglionares geniculadas de ratón a los estímulos del gusto

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imágenes de calcio en la aceituna inferior de ratón

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the other hand, two-photon imaging can resolve the activity of local neural circuits at the single-cell level. It is critical to make a large cranial window to perform multiple-scale analysis using both imaging techniques in the same mouse. To achieve this, one must remove a large section of the skull and cover the exposed cortical surface with transparent materials. Previously, glass skulls and polymer-based cranial windows have been developed for this purpose, but these materials are not easily fabricated. The present protocol describes a simple method for making a large cranial window consisting of commercially available polyvinylidene chloride (PVDC) wrapping film, a transparent silicone plug, and a cover glass. For imaging the dorsal surface of an entire hemisphere, the window size was approximately 6 x 3 mm2. Severe brain vibrations were not observed regardless of such a large window. Importantly, the condition of the brain surface did not deteriorate for more than one month. Wide-field imaging of a mouse expressing a genetically-encoded calcium indicator (GECI), GCaMP6f, specifically in astrocytes, revealed synchronized responses in a few millimeters. Two-photon imaging of the same mouse showed prominent calcium responses in individual astrocytes over several seconds. Furthermore, a thin layer of an adeno-associated virus was applied to the PVDC film and successfully expressed GECI in cortical neurons over the cranial window. This technique is reliable and cost-effective for making a large cranial window and facilitates the investigation of the neural and glial dynamics and their interactions during behavior at the macroscopic and microscopic levels.

In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

The present protocol describes making a large (6 x 3 mm2) cranial window using food wrap, transparent silicone, and cover glass. This cranial window allows in vivo wide-field and two-photon calcium imaging experiments in the same mouse.

In vivo Imágenes de calcio de campo amplio y dos fotones de un ratón usando una ventana craneal grande

Wide-field calcium imaging from the mouse&#39;s neocortex allows one to observe cortex-wide neural activity related to various brain functions. On the ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Imágenes subcelulares del manejo neuronal del calcio in vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

Imágenes de calcio de dos fotones y de campo amplio en el colículo superior

Calcium Imaging in Mouse Superior Colliculus

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the emergence of the cerebral cortex. It receives direct inputs from ~30 types of retinal ganglion cells (RGCs), with each encoding a specific visual feature. It remains elusive whether the SC simply inherits retinal features or if additional and potentially de novo processing occurs in the SC. To reveal the neural coding of visual information in the SC, we provide here a detailed protocol to optically record visual responses with two complementary methods in awake mice. One method uses two-photon microscopy to image calcium activity at single-cell resolution without ablating the overlaying cortex, while the other uses wide-field microscopy to image the whole SC of a mutant mouse whose cortex is largely undeveloped. This protocol details these two methods, including animal preparation, viral injection, headplate implantation, plug implantation, data acquisition, and data analysis. The representative results show that the two-photon calcium imaging reveals visually evoked neuronal responses at single-cell resolution, and the wide-field calcium imaging reveals&#160;neural activity across the entire SC. By combining these two methods, one can reveal the neural coding in the SC at different scales, and such combination can also be applied to other brain regions.

Autor destacado: Desvelando la codificación neuronal y los mecanismos de procesamiento visual en el colículo superior 

This protocol details the procedure for imaging calcium responses in the superior colliculus (SC) of awake mice, including imaging single-neuron activity with two-photon microscopy while leaving the cortex intact in wild-type mice, and imaging the entire SC with wide-field microscopy in partial-cortex mutant mice.

Imágenes de calcio en el colículo superior de ratón

The superior colliculus (SC), an evolutionarily conserved midbrain structure in all vertebrates, is the most sophisticated visual center before the ...