Name: 저자 스포트라이트: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus
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

이 연구는 중국 국립 자연 과학 재단 (32271060)의 지원을 받고 있습니다. Y.-t.L.은 연구를 설계하고, 실험을 수행하고, 데이터를 분석하고, 원고를 썼습니다. Z.L.과 R.W.가 실험을 수행하였다.

모든 실험 절차는 동물 복지 지침에 따라 수행되었으며 베이징에 있는 중국 뇌 연구소의 IACUC의 승인을 받았습니다.
참고: 이 프로토콜의 타임라인은 다음과 같습니다: 1) 흡입 컵을 만드십시오. 2) 바이러스를 주입합니다. 3) 헤드플레이트를 이식합니다. 4) 3 주 후, 플러그를 이식하십시오. 5) 트레드밀에서 ~3일의 회복 및 습관화 후 이광자/광시야 이미징을 수행합니다.
<p clas...

Superior Colliculus의 Two-photon 및 Wide-field Calcium 이미징

<ol>
	<li>May, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+superior+colliculus:+laminar+structure+and+connections.">The mammalian superior colliculus: laminar structure and connections.</a> Progress in Brain Research. 151, 321-378 (2006).</li><li>Denk, W., Strickler, J. H., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>Ohki, K., Chung, S., Ch'ng, Y. H., Kara, P., Reid, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+with+cellular+resolution+reveals+precise+micro-architecture+in+visual+cortex.">Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex.</a> Nature. 433 (7026), 597-603 (2005).</li><li>Ratzlaff, E. H., Grinvald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tandem-lens+epifluorescence+macroscope:+Hundred-fold+brightness+advantage+for+wide-field+imaging.">A tandem-lens epifluorescence macroscope: Hundred-fold brightness advantage for wide-field imaging.</a> Journal of Neuroscience Methods. 36 (2-3), 127-137 (1991).</li><li>de Vries, S. E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+standardized+physiological+survey+reveals+functional+organization+of+the+mouse+visual+cortex.">A large-scale standardized physiological survey reveals functional organization of the mouse visual cortex.</a> Nature Neuroscience. 23 (1), 138-151 (2020).</li><li>Mrsic-Flogel, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+map+of+visual+space+in+the+superior+colliculus+of+mice+lacking+early+retinal+waves.">Altered map of visual space in the superior colliculus of mice lacking early retinal waves.</a> The Journal of Neuroscience. 25 (29), 6921-6928 (2005).</li><li>Cang, J., Wang, L., Stryker, M. P., Feldheim, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+ephrin-as+and+structured+activity+in+the+development+of+functional+maps+in+the+superior+colliculus.">Roles of ephrin-as and structured activity in the development of functional maps in the superior colliculus.</a> The Journal of Neuroscience. 28 (43), 11015-11023 (2008).</li><li>Feinberg, E. H., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orientation+columns+in+the+mouse+superior+colliculus.">Orientation columns in the mouse superior colliculus.</a> Nature. 519 (7542), 229-232 (2015).</li><li>Ahmadlou, M., Heimel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+for+concentric+orientations+in+the+mouse+superior+colliculus.">Preference for concentric orientations in the mouse superior colliculus.</a> Nature Communications. 6, 6773 (2015).</li><li>de Malmazet, D., K&#252;hn, N. K., Farrow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinotopic+separation+of+nasal+and+temporal+motion+selectivity+in+the+mouse+superior+colliculus.">Retinotopic separation of nasal and temporal motion selectivity in the mouse superior colliculus.</a> Current Biology. 28 (18), 2961-2969 (2018).</li><li>Li, Y. T., Turan, Z., Meister, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+motion+direction+in+the+mouse+superior+colliculus.">Functional architecture of motion direction in the mouse superior colliculus.</a> Current Biology. 30 (17), 3304-3315 (2020).</li><li>Gribizis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+cortex+gains+independence+from+peripheral+drive+before+eye+opening.">Visual cortex gains independence from peripheral drive before eye opening.</a> Neuron. 104 (4), 711-723 (2019).</li><li>Inayat, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+in+the+most+superficial+lamina+of+the+mouse+superior+colliculus+are+highly+selective+for+stimulus+direction.">Neurons in the most superficial lamina of the mouse superior colliculus are highly selective for stimulus direction.</a> The Journal of Neuroscience. 35 (20), 7992-8003 (2015).</li><li>Barchini, J., Shi, X., Chen, H., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+encoding+of+motion+contrast+in+the+mouse+superior+colliculus.">Bidirectional encoding of motion contrast in the mouse superior colliculus.</a> eLife. 7, 35261 (2018).</li><li>Savier, E. L., Chen, H., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+locomotion+on+visual+responses+in+the+mouse+superior+colliculus.">Effects of locomotion on visual responses in the mouse superior colliculus.</a> The Journal of Neuroscience. 39 (47), 9360-9368 (2019).</li><li>Schr&#246;der, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arousal+modulates+retinal+output.">Arousal modulates retinal output.</a> Neuron. 107 (3), 487-495 (2020).</li><li>Ge, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+waves+prime+visual+motion+detection+by+simulating+future+optic+flow.">Retinal waves prime visual motion detection by simulating future optic flow.</a> Science. 373 (6553), (2021).</li><li>Chen, H., Savier, E. L., DePiero, V. J., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+evidence+for+stereotypical+direction+columns+in+the+mouse+superior+colliculus.">Lack of evidence for stereotypical direction columns in the mouse superior colliculus.</a> The Journal of Neuroscience. 41 (3), 461-473 (2021).</li><li>Kasai, M., Isa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+light+isoflurane+anesthesia+on+organization+of+direction+and+orientation+selectivity+in+the+superficial+layer+of+the+mouse+superior+colliculus.">Effects of light isoflurane anesthesia on organization of direction and orientation selectivity in the superficial layer of the mouse superior colliculus.</a> The Journal of Neuroscience. 42 (4), 619-630 (2022).</li><li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+apical+complex+couples+cell+fate+and+cell+survival+to+cerebral+cortical+development.">The apical complex couples cell fate and cell survival to cerebral cortical development.</a> Neuron. 66 (1), 69-84 (2010).</li><li>Kaifosh, P., Zaremba, J. D., Danielson, N. B., Losonczy, A. S. I. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Python+software+for+analysis+of+dynamic+fluorescence+imaging+data.">Python software for analysis of dynamic fluorescence imaging data.</a> Frontiers in Neuroinformatics. 8, 80 (2014).</li><li>Pnevmatikakis, E. A., Giovannucci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NoRMCorre:+An+online+algorithm+for+piecewise+rigid+motion+correction+of+calcium+imaging+data.">NoRMCorre: An online algorithm for piecewise rigid motion correction of calcium imaging data.</a> Journal of Neuroscience Methods. 291, 83-94 (2017).</li><li>Kerlin, A. M., Andermann, M. L., Berezovskii, V. K., Reid, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+tuned+response+properties+of+diverse+inhibitory+neuron+subtypes+in+mouse+visual+cortex.">Broadly tuned response properties of diverse inhibitory neuron subtypes in mouse visual cortex.</a> Neuron. 67 (5), 858-871 (2010).</li><li>G&#246;bel, W., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+calcium+imaging+of+neural+network+function.">In vivo calcium imaging of neural network function.</a> Physiology. 22 (6), 358-365 (2007).</li><li>Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L., Tank, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+large-scale+neural+activity+with+cellular+resolution+in+awake,+mobile+mice.">Imaging large-scale neural activity with cellular resolution in awake, mobile mice.</a> Neuron. 56 (1), 43-57 (2007).</li><li>Evans, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synaptic+threshold+mechanism+for+computing+escape+decisions.">A synaptic threshold mechanism for computing escape decisions.</a> Nature. 558 (7711), 590-594 (2018).</li></ol>

프로토콜의 중요한 단계 가장 중요한 단계는 5.2단계와 5.3단계의 개두술입니다. 첫째, 람다 뒤쪽 0.5mm의 뼈는 두껍고 내부에 혈관이 있어 시추 과정에서 출혈이 발생할 수 있습니다. 출혈을 멈추기 위해 적절한 젤 폼을 준비해야 합니다. 둘째, 횡동(transverse sinus) 바로 위의 뼈를 제거할 때 혈관 손상증이 발생할 가능성이 높습니다. 문제 해결을 위한 한 가지 다른 방법은 타원형 내부?...

상구(superior colliculus, SC)는 모든 척추동물에서 중요한 시각 중추입니다. 포유류에서는 망막과 시각 피질(visual cortex)1로부터 직접 입력을받는다. 광학 기록은 피질 2,3,4,5에 널리 적용되어 왔지만, SC에서의 적용은 열악한 광학 액세스 6,7,8,9,10,11,12,13,14,15,16,17에 의해 방해를 받는다 ,18,19. 이 프로토콜의 목표는 SC에서 신경 활동을 광학적으로 기록하기 위한 두 가지 보완 방법에 대한 세부 정보를 제공하는 것입니다.
SC는 피질과 횡동(transverse sinus) 아래에 위치하여 측부 뉴런에 대한 광학적 접근을 제한합니다. 이러한 한계를 극복하기 위한 한 가지 접근법은 중첩 피질을 흡인하고 전방 SC 7,9,10,13,14,19를 노출시키는 것입니다. 그러나 SC는 대뇌 피질 입력을 받기 때문에 이러한 작업은 SC 뉴런이 시각적 자극에 반응하는 방식에 영향을 미칠 수 있습니다. 이 한계를 극복하기 위하여는, 우리는 피질을 온전하게 남겨두면서 실리콘 마개를 가진 후방-내측 SC의 표층을, 8,11 남겨 두는 양자택일 의정서를 여기에서 상세히 기술한다. 특히, 단일 세포 분해능을 달성하기 위해 야생형 마우스의 후방-내측 SC에서 칼슘 반응을 이미지화하기 위해 이광자 현미경을 적용했습니다. 또한 넓은 범위를 달성하기 위해 광시야 현미경을 적용하여 후방 피질이 발달하지 않은 돌연변이 쥐의 전체 SC를 이미지화했습니다20.
이 프로토콜에 설명된 두 가지 방법은 서로 보완적입니다. 피질을 절제하지 않는 2광자 칼슘 이미징은 손상되지 않은 피질 입력으로 단일 세포 해상도로 신경 활동을 기록하는 데 적합합니다. 광시야 칼슘 이미징은 공간 해상도를 희생하면서 전체 SC의 신경 활동을 기록하는 데 적합합니다.

<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

모든 척추동물에서 진화적으로 보존된 중뇌 구조인 상구(superior colliculus, SC)는 대뇌피질이 출현하기 전에 가장 정교한 시각 중추입니다. ~30가지 유형의 망막 신경절 세포(RGC)로부터 직접 입력을 받으며, 각 세포는 특정 시각적 특징을 인코딩합니다. SC가 단순히 망막 특징을 물려받는지, 아니면 SC에서 추가적이고 잠재적으로 새로운 처리가 발생하는지는 여전히 파악하기 어렵습니다. SC에서 시각 정보의 신경 코딩을 밝히기 위해 깨어 있는 쥐에서 두 가지 보완적인 방법으로 시각적 반응을 광학적으로 기록하는 자세한 프로토콜을 제공합니다. 한 가지 방법은 이광자 현미경을 사용하여 중첩 피질을 절제하지 않고 단일 세포 해상도로 칼슘 활성을 이미지화하는 반면, 다른 방법은 광시야 현미경을 사용하여 피질이 거의 발달하지 않은 돌연변이 쥐의 전체 SC를 이미지화합니다. 이 프로토콜은 동물 준비, 바이러스 주입, 헤드플레이트 이식, 플러그 주입, 데이터 수집 및 데이터 분석을 포함한 이 두 가지 방법을 자세히 설명합니다. 대표적인 결과는 이광자 칼슘 이미징이 단일 세포 해상도에서 시각적으로 유발된 신경 반응을 나타내고 광시야 칼슘 이미징이 SC 전체에 걸쳐 신경 활동을 나타낸다는 것을 보여줍니다. 이 두 가지 방법을 결합하면 SC의 신경 코딩을 서로 다른 규모로 밝힐 수 있으며 이러한 조합은 다른 뇌 영역에도 적용될 수 있습니다.

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.

저자 스포트라이트: Unveiling Neural Coding and Mechanisms of Visual Processing in the Superior Colliculus 

Mouse Superior Colliculus의 칼슘 이미징

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.

그림 1A,B는 각각 흡입 컵과 플러그를 만드는 방법을 보여줍니다. 그림 2는 플러그를 성공적으로 이식하는 방법을 보여줍니다. 플러그를 이식한 후 그림 2D와 같이 후방-내측 SC가 노출됩니다. 그림 3은 이광자 현미경을 사용하여 이미지화한 야생형 쥐의 SC 뉴런의 칼슘 반응을 보여줍니다. 현미경?...

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

이 프로토콜은 야생형 마우스에서 피질을 그대로 유지하면서 이광자 현미경으로 단일 뉴런 활동을 이미징하고 부분 피질 돌연변이 마우스에서 광시야 현미경으로 전체 SC를 이미징하는 것을 포함하여 깨어 있는 마우스의 상구(SC)에서 칼슘 반응을 이미징하는 절차를 자세히 설명합니다.

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

제 연구는 신경 코딩과 시각 처리 메커니즘에 중점을 두고 있습니다. 우리는 다양한 정보가 뇌, 특히 근본적인 신경 메커니즘 아래의 상구에서 어떻게 암호화되는지에 대한 답을 찾으려고 노력하고 있습니다. 시각 처리를 이해하기 위해 과학자들은 신경 형태학, 전행 및 역행 추적, IL 시퀀싱, 신경 모델링, 기계 학습을 포함한 다양한 접근 방식을 결합합니다.문제는 SSA 위의 두개골을 제거하고 뼈를 잘라 하나의 큰 조각으로 남겨두는 것인데, 뼈가 경막에 부착되어 있기 때문에 실패할 가능성이 높습니다. 광시야 칼슘 이미지에 두 장의 사진을 결합함으로써 우리의 최근 연구는 단일 세포 분해능과 글로벌 규모 모두에서 마우스 SC의 운동 방향의 기능 구조를 밝혔습니다. 우리는 비슷한 선호도를 가진 뉴런이 코운동에서 위쪽으로 500마이크로미터 아래까지 패치를 형성한다는 것을 발견했습니다.우리의 프로토콜을 통해 우리는 두 가지 연구 격차를 해결하고 있습니다. 첫째, 연구원들은 피질을 손상시키지 않고 단일 세포 해상도에서 마우스 SC에서 장기 캐스트 이미징을 수행할 수 있습니다. 둘째, 연구자들은 광시야 현미경 검사를 사용하여 전체 SC의 신경 활성을 기록할 수 있습니다.당사의 프로토콜은 마우스의 온전한 피질을 사용하여 단일 세포 해상도에서 후방 내측 SC를 이미지화하는 측정값을 제공합니다. 또한 생체 적합성 플러그를 사용하여 SC를 노출시켜 만성 영상의 감염을 줄입니다. 우리의 결과는 넓은 시각 분야에서 시각 정보의 신경 코딩을 연구하는 방법을 제공합니다.광유전학과 결합하면 SC의 신경 활성을 조절하는 다른 뇌 영역의 절반 입력을 연구할 수 있습니다.

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.

연구

신경과학

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.

마우스의 칼슘 이미징을 위한 흡입 컵 및 플러그 준비

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.

칼슘 이미징을 위한 Mouse Superior Colliculus의 Viral Construct (AAV) 주입

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.

칼슘 이미징을 위해 마우스에 헤드플레이트 이식 

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.

칼슘 이미징을 위해 마우스에 플러그 이식

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.

Mouse Superior Colliculus Neuron의 Two-Photon Calcium 이미징

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.