Name: Autor em destaque: Revelando a codificação neural e os mecanismos de processamento visual no colículo superior
Uploaded: 2023-04-21T15:00:00
Description: Watch this Scientific Journal Video about Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus at JoVE.com

Este trabalho é apoiado pela Fundação Nacional de Ciências Naturais da China (32271060). Y.-t.L. desenhou a pesquisa, realizou o experimento, analisou os dados e escreveu o manuscrito. Z.L. e R.W. realizaram o experimento.

Todos os procedimentos experimentais foram realizados de acordo com as diretrizes de bem-estar animal e aprovados pela IACUC no Instituto Chinês de Pesquisa do Cérebro, Pequim.
OBS: O cronograma para este protocolo é o seguinte: 1) fazer a ventosa; 2) injetar o vírus; 3) implantar a placa cefálica; 4) após 3 semanas, implantar o plug; 5) após uma recuperação de ~3 dias e habituação na esteira, realizar imagens de dois fótons/campo largo.
1. Preparo...

Imagem de cálcio de dois fótons e campo amplo no colículo superior

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Etapas críticas do protocolo A etapa mais crítica é a craniotomia nas etapas 5.2 e 5.3. Primeiro, o osso 0,5 mm posterior ao lambda é espesso e tem vasos sanguíneos dentro, o que pode causar sangramento durante o processo de perfuração. Espuma de gel adequada deve ser preparada para parar o sangramento. Em segundo lugar, há uma boa chance de angiorrhexis ao remover o osso logo acima do seio transverso. Para solução de problemas, uma abordagem alternativa é afinar o osso dentro do oval e rem...

O colículo superior (CS) é um importante centro visual em todos os vertebrados. Em mamíferos, recebe entradas diretas da retina e do córtex visual1. Embora o registro óptico tenha sido amplamente aplicado no córtex 2,3,4,5, sua aplicação no CT é dificultada por acessos ópticos precários 6,7,8,9,10,11,12,13,14,15,16,17 ,18,19. O objetivo deste protocolo é fornecer detalhes sobre dois métodos complementares para o registro óptico da atividade neural no CT.
A CT está localizada abaixo do córtex e do seio transverso, o que limita o acesso óptico aos neurônios coliculares. Uma abordagem para superar essa limitação é aspirar a cortical sobreposta e expor o SC ântero-lateral7,9,10,13,14,19. No entanto, como o CT recebe entradas corticais, tal operação pode afetar a forma como os neurônios do CT respondem aos estímulos visuais. Para superar essa limitação, detalhamos aqui um protocolo alternativo para obter imagens da camada superficial do SC póstero-medial com um tampão de silicone, deixando o córtexintacto8,11. Especificamente, para alcançar a resolução de célula única, aplicamos microscopia de dois fótons para obter imagens de respostas de cálcio na CT póstero-medial de camundongos selvagens. Além disso, para obter ampla cobertura, aplicamos microscopia de campo largo para obter imagens de toda a CT de um camundongo mutante cujo córtex posterior não se desenvolveu20.
Os dois métodos descritos neste protocolo são complementares entre si. A imagem de cálcio de dois fótons sem ablação do córtex é apropriada para registrar a atividade neural em resolução de célula única com entradas corticais intactas. A imagem de cálcio de campo largo é apropriada para registrar a atividade neural em toda a CT enquanto sacrifica a resolução espacial.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50-mm lens</td>
			<td>Computar</td>
			<td>M5018-MP2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-mm coverslip</td>
			<td>Warner instruments</td>
			<td>CS-5R</td>
			<td></td>
		</tr>
		<tr>
			<td>bandpass filter</td>
			<td>Chroma Technology</td>
			<td>HQ575/250 m-2p</td>
			<td></td>
		</tr>
		<tr>
			<td>butyl cyanoacrylate</td>
			<td>Vetbond, World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera for monitoring pupil</td>
			<td>FLIR</td>
			<td>BFS-U3-04S2M-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>camera for widefield imaging</td>
			<td>Basler</td>
			<td>acA2000-165&#181;m</td>
			<td></td>
		</tr>
		<tr>
			<td>corona treater</td>
			<td>Electro-Technic Products</td>
			<td>BD-20AC</td>
			<td></td>
		</tr>
		<tr>
			<td>dichroic</td>
			<td>Chroma Technology</td>
			<td>T600/200dcrb&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>galvanometers</td>
			<td>Cambridge Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass bead sterilizer</td>
			<td>RWD</td>
			<td>RS1502</td>
			<td></td>
		</tr>
		<tr>
			<td>microdrill</td>
			<td>RWD</td>
			<td>78001</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>QUAD</td>
			<td></td>
		</tr>
		<tr>
			<td>photomultiplier tube</td>
			<td>Hamamatsu</td>
			<td>R3896</td>
			<td></td>
		</tr>
		<tr>
			<td>rotory encoder</td>
			<td>USdigital</td>
			<td>MA3-A10-125-N</td>
			<td></td>
		</tr>
		<tr>
			<td>self-curing dental adhesive resin cement&#160;</td>
			<td>SuperBond C&#38;B, Sun Medical Co, Ltd. Moriyama, Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>thermostatic heating pad&#160;</td>
			<td>RWD</td>
			<td>69020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti:Sapphire laser</td>
			<td>Spectra-Physics</td>
			<td>Mai Tai HP DeepSee</td>
			<td></td>
		</tr>
		<tr>
			<td>translucent silicone adhesive&#160;</td>
			<td>Kwik-Sil, World Precision Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>treadmill</td>
			<td>Xinglin Biology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Virus Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rAAV2/9-hsyn-Gcamp6m</td>
			<td>Vector Core at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J wild type</td>
			<td>Laboratory Animal Resource Center at Chinese Institute for Brain Research, Beijing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Emx1-Cre</td>
			<td>The Jackson Laboratory&#160;</td>
			<td>5628</td>
			<td></td>
		</tr>
		<tr>
			<td>Pals1flox/wt</td>
			<td>Christopher A. Walsh Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH Image</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labview</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

calcium imaging in mouse superior colliculus

O colículo superior (CS), uma estrutura mesencefálica conservada evolutivamente em todos os vertebrados, é o centro visual mais sofisticado antes do surgimento do córtex cerebral. Ele recebe entradas diretas de ~30 tipos de células ganglionares da retina (RGCs), com cada uma codificando uma característica visual específica. Permanece indefinido se o CT simplesmente herda características retinianas ou se ocorre processamento adicional e potencialmente de novo no CS. Para revelar a codificação neural da informação visual no CS, fornecemos aqui um protocolo detalhado para registrar opticamente as respostas visuais com dois métodos complementares em camundongos acordados. Um método usa microscopia de dois fótons para obter imagens da atividade do cálcio em resolução de célula única sem ablar o córtex sobreposto, enquanto o outro usa microscopia de campo largo para obter imagens de toda a CT de um camundongo mutante cujo córtex é em grande parte subdesenvolvido. Este protocolo detalha esses dois métodos, incluindo preparação animal, injeção viral, implante de placa cefálica, implante de plug, aquisição de dados e análise de dados. Os resultados representativos mostram que a imagem de cálcio de dois fótons revela respostas neuronais visualmente evocadas em resolução de célula única, e a imagem de cálcio de campo largo revela atividade neural em toda a CT. Combinando esses dois métodos, pode-se revelar a codificação neural no CT em diferentes escalas, e tal combinação também pode ser aplicada a outras regiões cerebrais.

The superior colliculus (SC) is an important visual center in all vertebrates. In mammals, it receives direct inputs from the retina and the visual cortex1. While optical recording has been widely applied to the cortex2,3,4,5, its application in the SC is hindered by poor optical accesss6,7,8,9,10,11,12,13,14,15,16,17,18,19. The goal of this protocol is to provide details about two complementary methods for optical recording of the neural activity in the SC.
The SC is located beneath the cortex and transverse sinus, which limits optical access to the collicular neurons. One approach to overcome this limitation is to aspirate the overlaying cortex and expose the anterior-lateral SC7,9,10,13,14,19. However, because the SC receives cortical inputs, such an operation might affect how the SC neurons respond to visual stimuli. To overcome this limitation, we detail here an alternative protocol to image the superficial layer of the posterior-medial SC with a silicon plug, while leaving the cortex intact8,11. Specifically, to achieve single-cell resolution, we applied two-photon microscopy to image calcium responses in the posterior-medial SC of wild-type mice. In addition, to achieve broad coverage, we applied wide-field microscopy to image the entire SC of a mutant mouse whose posterior cortex has not developed20.
The two methods described in this protocol are complementary to each other. The two-photon calcium imaging without ablating the cortex is appropriate for recording neural activity at&#160;single-cell resolution with intact cortical inputs. The wide-field calcium imaging is appropriate for recording neural activity in the entire SC while sacrificing spatial resolution.

Autor em destaque: Revelando a codificação neural e os mecanismos de processamento visual no colículo superior 

Imagem de cálcio no colículo superior de camundongos

My research focuses on neural coding and mechanisms for visual processing. We are trying to answer how various information is encoded in the brain, especially in the superior colliculus under the underlying neuro-mechanisms. To understand visual processing, scientists combine various approaches, including neuronal morphology, antegrade and retrograde tracing, IL sequencing, neuro modeling, machine learning, is understood well.The challenge is to remove the skull over the SSA, cutting and left in the bone in one larger piece, is likely to fail because the bone is attached to the dura. By combining two photo on a wide field calcium image, our recent work revealed the function architecture of motion direction in the mouse SC at both single cell resolution and a global scale. We found that neurons with similar preference form patches up to 500 micrometer under global to the upward on the nasal motion.With our protocol, we are addressing two research gaps. First, researchers can perform long-term cast imaging in mouse SC at a single cell resolution without breaking the cortex. Secondly, researchers can record the neuroactivity across the entire SC using widefield microscoping.Our protocol provides a measure to image the posterior medial SC at single cell resolution with a intact cortex in mice. Also, we use a biocompatible plug to expose the SC, which reduces infection for chronic imaging. Our results provide a way to study the neural coding of visual information across a large visual field.Combined with optogenetics, one can study half inputs from different brain regions modulating the neuroactivity in SC.

As Figuras 1A,B mostram como fazer a ventosa e os plugs, respectivamente. A Figura 2 mostra como implantar o plugue com sucesso. Após o implante do plug, o SC póstero-medial é exposto, como mostra a Figura 2D. A Figura 3 mostra as respostas de cálcio dos neurônios SC de um exemplo de camundongo selvagem fotografado usando microscopia de dois fótons. O prisma triangular, que é fac...

Watch this Scientific Journal Video about Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus at JoVE.com

Este protocolo detalha o procedimento para obter imagens de respostas de cálcio no colículo superior (CS) de camundongos acordados, incluindo a obtenção de imagens da atividade de um único neurônio com microscopia de dois fótons, deixando o córtex intacto em camundongos selvagens, e a obtenção de imagens de todo o SC com microscopia de campo largo em camundongos mutantes do córtex parcial.

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

Minha pesquisa se concentra em codificação neural e mecanismos de processamento visual. Estamos tentando responder como várias informações são codificadas no cérebro, especialmente no colículo superior sob os neuro-mecanismos subjacentes. Para entender o processamento visual, os cientistas combinam várias abordagens, incluindo morfologia neuronal, traçado anterógrado e retrógrado, sequenciamento de IL, modelagem neuro, aprendizado de máquina, é bem compreendido.O desafio é remover o crânio sobre o SSA, cortando e deixado no osso em um pedaço maior, é provável que falhe porque o osso está ligado à dura-máter. Combinando duas fotos em uma imagem de cálcio de campo amplo, nosso trabalho recente revelou a arquitetura de função da direção do movimento no SC do camundongo em resolução de célula única e escala global. Descobrimos que neurônios com preferência semelhante formam manchas de até 500 micrômetros sob o movimento global para cima no movimento nasal.Com nosso protocolo, estamos abordando duas lacunas de pesquisa. Primeiro, os pesquisadores podem realizar imagens de gesso de longo prazo em SC de camundongos em uma única resolução de célula sem quebrar o córtex. Em segundo lugar, os pesquisadores podem registrar a neuroatividade em toda a CS usando microscópio de campo largo.Nosso protocolo fornece uma medida para obter imagens das CT póstero-mediais em resolução de célula única com um córtex intacto em camundongos. Além disso, usamos um plugue biocompatível para expor o SC, o que reduz a infecção por exames crônicos. Nossos resultados fornecem uma maneira de estudar a codificação neural da informação visual através de um grande campo visual.Combinado com a optogenética, pode-se estudar meias entradas de diferentes regiões cerebrais modulando a neuroatividade em CT.

calcium-imaging-in-mouse-superior-colliculus

Research

JoVE Journal

Neuroscience

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.

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.

Neurociência

Preparation of a Suction Cup and Plugs for Calcium Imaging in Mouse

Begin the process of creating a suction cup by depositing a drop of PBS in an acrylic dish. Use a 21 gauge flat needle to fill the tip of the needle with PBS by capillarity. Cover the tip of the needle with a translucent silicone adhesive, and set it for approximately 10 minutes.Cut the silicone adhesive with scissors to a disc with a two millimeter diameter. To prepare the plugs, take a 0.75 millimeter thick plastic shim stock and cut out a one by one centimeter square in the center. Clean the shim stock and two acrylic blocks with alcohol, and wipe them with lens paper.Place the shim stock on one acrylic block, and deposit silicone adhesive into the center to fill 90%of the opening. Add another acrylic block. Apply approximately one kilogram of force, and wait 20 minutes.Under a surgical microscope, cut the silicone adhesive with a scalpel into one by 1.5 millimeter triangular prisms. Remove any dust from the triangular prisms with sticky plastic tape, and place them on a glass cover slip. Place the cover slip with the triangular prisms under a corona treater and turn it on.Bring the corona treater closer to the cover slip until lightning appears, and hold it in that position for a few minutes until the prisms and cover slip are attached. After placing the plugs in a Petri dish, incubate at 70 degrees Celsius overnight.

Preparo de ventosa e plugs para obtenção de imagens de cálcio em camundongos

Viral Construct (AAV) Injection in Mouse Superior Colliculus for Calcium Imaging

Begin by securing the anesthetized mouse onto a stereotaxic frame with ear bars. Apply ophthalmic ointment on the mouse's eyes to prevent drying, and maintain the body temperature of the mouse at 37 degrees Celsius with a thermostatic heating pad. After shaving the fur and sterilizing the surgical site, inject lidocaine, tilidine, and meloxicam.Once the skin over the SC is removed, clean the skull with a cotton swab. Adjust the stereotaxic frame until the skull surface is flat. Using a drill, create a whole 0.5 millimeter lateral, and 0.42 millimeter anterior to the lambda, until the dura is exposed.Inject an adeno-associated virus, or AAV, expressing GCaMP6m, at depths of one millimeter and 1.6 millimeters from the Lambda using a beveled glass micro-pipette. After the injection, slowly withdraw the injector and proceed with the implantation of the head plate.

Injeção de Constructo Viral (AAV) no Colículo Superior de Camundongos para Imagem com Cálcio

Implantation of the Headplate in Mouse for Calcium Imaging 

After injecting the viral construct into the mouse brain, clean the muscles and tissues over the skull, and scratch the skull with a blade. Prepare the dental adhesive resin cement by mixing 3/4 spoonful of polymer, three drops of monomer, and one drop of catalyst in a ceramic bowl on ice. Apply the mixture to the head plate and the skull.Attach them and wait for the adhesive to set for five minutes. Finally, inject antibiotics to prevent infections. After removing the mouse from the stereotaxic frame, place it on a heating pad for recovery, and return it to the home cage after recovering from anesthesia.

Implante da Headplate em Camundongos para Imagem por Cálcio 

Implanting the Plug in the Mouse for Calcium Imaging

Three weeks after the viral infection, anesthetize and fix the mouse with a head plate on a stereotaxic frame. Soak a gel foam in saline to stop potential bleeding. Use a micro-drill to create a three by two millimeter oval, centered at 0.5 millimeter posterior to the lambda.Thin the boundary of the oval until it cracks, then thin the skull before the oval recording window to close the cover slip to the SC.Using fine forceps, take off the bone flaps. Make a cut down the dura, posterior to the transverse sinus and remove the piece of dura. Dry the skull and the recording window with gel foam and cotton swabs.Then deposit a drop of silicone adhesive into the recording window. Use the suction cup to hold the plug with negative pressure, and use a motorized micro-manipulator to move the suction cup and lower the plug into the silicone adhesive until the cover slip touches the skull. Once the head plate is cleaned, apply butyl cyanoacrylate and resin cement around the boundary of the plug to fix it to the head plate and proceed with imaging.

Implantando o plugue no mouse para imagens de cálcio

Two-Photon Calcium Imaging of Mouse Superior Colliculus Neurons

After implanting the head plate and plug, secure the animal on a rotating treadmill with its head plate. Place the treadmill beneath the two-photon microscope and adjust the height to an appropriate position. Place a black aluminum cone between the objective and the head plate to prevent light contamination from the monitor for visual stimulation.To perform two-photon imaging, adjust the laser power at the sample plane between 20 and 80 milliwatts. Scan a 600-by-600 micrometer field of view at 4.8 hertz with a spatial resolution of 2.4 micrometers per pixel and image neural activity up to 350 micrometers in depth. Using a rotary encoder, record the mouse's locomotion on the treadmill.Use a camera with a 50 millimeter lens to record its pupil size and position. Lastly, synchronize these recordings, and image acquisitions to visual stimulation by recording triggers sent by the stimulation computer. The calcium responses of SC neurons from a wild type mouse and the standard deviation projection of the fluorescence across images are presented.

Imagem de cálcio de dois fótons de neurônios do colículo superior de camundongos

Wide-Field Calcium Imaging of Mouse Superior Colliculus Neurons

To begin, inject 100 nanoliters of AAV expressing GCaMP6m at a depth of 200 micrometers at the center of each side of the SC to the mutant mouse whose posterior cortex has failed to develop. After approximately three weeks, implant a five millimeter diameter cover slip over the SC.After fixing the cover slip, acquire images using a CMOA camera after passing an emission filter at 10 hertz. The calcium responses of SC neurons from a partial cortex mutant mouse were imaged using wide-field microscopy.The acquired image was down sampled to one quarter of the original size, and visually evoked calcium responses are shown.