Name: a large lateral craniotomy procedure for mesoscale wide-field optical imaging of brain activity
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इस काम के द्वारा एक प्राकृतिक विज्ञान और कनाडा के इंजीनियरिंग अनुसंधान परिषद (NSERC) डिस्कवरी अनुदान # 40,352 समर्थित किया गया, अभिनव कार्यक्रम चेयर, एम एच एम के अलबर्टा अल्जाइमर रिसर्च प्रोग्राम के लिए कैम्पस अल्बर्टा, और NSERC BIF डॉक्टरल फेलोशिप और एम के लिए AIHS स्नातकोत्तर फैलोशिप में बनाएँ। हम इस प्रोटोकॉल के विकास के लिए और शल्य चिकित्सा प्रशिक्षण के लिए पु मिन वांग धन्यवाद, और बेह्रू मिर्ज़ा आगा और डि शाओ-पालन के लिए।

<html> निम्नलिखित प्रोटोकॉल Lethbridge पशु की देखभाल समिति के विश्वविद्यालय (एसीसी) दिशानिर्देशों का पालन करता है, और पशु की देखभाल पर कनाडा परिषद (CCAC) के मानकों के अनुसार किया जाता है। 1. तैयारी <ol><li> लंबे...

<ol>
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H., Kianianmomeni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics:+Methods+and+Protocols.">Optogenetics: Methods and Protocols.</a> Methods in Molecular Biology. 1408, 251-265 (2016).</li><li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging in neuroscience and development : a laboratory manual. , (2005).</li><li>Vanni, M. P., Murphy, T. 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H., 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=In+vivo+Large-Scale+Cortical+Mapping+Using+Channelrhodopsin-2+Stimulation+in+Transgenic+Mice+Reveals+Asymmetric+and+Reciprocal+Relationships+between+Cortical+Areas.">In vivo Large-Scale Cortical Mapping Using Channelrhodopsin-2 Stimulation in Transgenic Mice Reveals Asymmetric and Reciprocal Relationships between Cortical Areas.</a> Front Neural Circuits. 6, (2012).</li><li>Ferezou, I., 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=Spatiotemporal+dynamics+of+cortical+sensorimotor+integration+in+behaving+mice.">Spatiotemporal dynamics of cortical sensorimotor integration in behaving mice.</a> Neuron. 56 (5), 907-923 (2007).</li><li>Mohajerani, M. H., Aminoltejari, K., Murphy, T. 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एक बड़ी कपाल खिड़की के लिए यह नवीन प्रोटोकॉल सेरेब्रल कॉर्टेक्स के अस्थायी और पार्श्विका क्षेत्रों में एक साथ इमेजिंग सक्षम बनाता है। ऑप्टिकल इमेजिंग के साथ संयुक्त, यह सहज और प्रोत्साहन प्रेरित गत?...

craniotomy एक मानक neuroscientists द्वारा इस्तेमाल होने वाली मस्तिष्क के एक हिस्से को प्रकट करने के लिए प्रक्रिया है। इलेक्ट्रोफिजियोलॉजी की सुबह के बाद से, craniotomy तंत्रिका विज्ञान के क्षेत्र में अभूतपूर्व सफलताओं अनुमति दी गई है। इलेक्ट्रोड के साथ सेरेब्रल कॉर्टेक्स के घने मानचित्रण प्रयोगों परीक्षण परिकल्पना और सिद्धांतों इन नक्शों के आधार पर करने के लिए प्रेरित किया है। हमने हाल ही में एक नए युग जहां craniotomy cortical रक्त के प्रवाह को 1, 2, 3 और न्यूरोवैस्कुलर वास्तुकला 4 में vivo इमेजिंग के लिए किया जा रहा है, उजागर क्षेत्रों 5, 6, 7 के भीतर cortical गतिविधि के वास्तविक समय दृश्य को सक्षम करने में प्रवेश किया है। हालांकि कई अध्ययनों संरचना और cortical न्यूरॉन्स, glia के समारोह, और कोर अध्ययन करने के लिए इन विवो ऑप्टिकल इमेजिंग तकनीक के साथ संयुक्त craniotomies का उपयोग8 , 9 , टेलीक vasculature, आगे की जांच उजागर प्रांतस्था के छोटे क्षेत्रों (लेकिन देखें 10 ) द्वारा सीमित हैं। इस प्रोटोकॉल का उद्देश्य एक बड़े पार्श्व क्रैनीओटमी बनाने के लिए एक विधि प्रदान करना है, जो मस्तिष्क संबंधी कोर्टेक्स को स्क्वैमासेल हड्डी से मध्यवर्ती से उजागर करता है, और ब्रॉम्मा और लैम्ब्डा से परे का विस्तार करता है। यह बड़ी क्रानोयोटीमी एसोसिएशन cortices (रेट्रोप्लेनियल, सिंगुलेट और पिरियैटल), प्राथमिक और माध्यमिक मोटर, somatosensory, दृश्य और श्रवण प्रांतस्था के एक साथ देखने के लिए सक्षम बनाता है। इस पद्धति को पहले वोल्टेज संवेदनशील डाई इमेजिंग (वीएसडीआईआई) के साथ युग्मित किया गया है कि यह जांच करने के लिए कि सहज और उत्तेजना से प्रेरित cortical गतिविधि 5 , 11 , 12 के दौरान कितने कॉर्टिकल क्षेत्र एक दूसरे के साथ सहभागिता करते हैं। इस प्रक्रिया के सबसे चुनौतीपूर्ण पहलुओं में शामिल हैं सिर की स्थितिजानवर की, सिर प्लेट फिक्सिंग, और नकसीर से परहेज पार्श्विका हड्डी से अस्थायी मांसपेशी विभाजित किया। देखभाल भी एक परोक्ष कोण पर खोपड़ी घटता के रूप में ड्रिलिंग और खोपड़ी को हटाने की प्रक्रिया के दौरान लिया जाना चाहिए।

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		<tr>
			<td>Heating Pad&#160;</td>
			<td>FHC</td>
			<td>40-90-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps&#160;</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td>2 or more pairs are recommended</td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00, 15000-10</td>
			<td>1 pair should be designated for dura removal&#160;</td>
		</tr>
		<tr>
			<td>Jet tooth shade powder</td>
			<td>LANG Dental</td>
			<td>Jet Tooth Shade Powder</td>
			<td>to be mixed with the Jet Liquid</td>
		</tr>
		<tr>
			<td>Jet tooth shade liquid</td>
			<td>LANG Dental</td>
			<td>Jet Tooth Shade Liquid</td>
			<td>to be mixed wihth the Jet Powder&#160;</td>
		</tr>
		<tr>
			<td>Drill Heads - Carbide Burs FG 1/4 389</td>
			<td>Midwest Dental</td>
			<td>385201</td>
			<td></td>
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			<td>Agarose Powder</td>
			<td>Sigma-Aldrich</td>
			<td>A9793</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Sinclair Dental Canada</td>
			<td>Pfizer Gelfoam</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>124125</td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine 2% Epinephrine</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>125299</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethazone 5 mg/mL</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>125231</td>
			<td></td>
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		<tr>
			<td>Butyl cyanoacrylate glue (VetBond)</td>
			<td>Western Drug Distribution Centre Ltd</td>
			<td>12612</td>
			<td></td>
		</tr>
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a large lateral craniotomy procedure for mesoscale wide-field optical imaging of brain activity

Craniotomy इन विवो प्रयोगों के लिए मस्तिष्क को बेनकाब करने के एक सामान्य प्रदर्शन किया प्रक्रिया है। माउस अनुसंधान में, सबसे प्रयोगशालाओं एक छोटे से craniotomy, आमतौर पर 3 मिमी x 3 मिमी का उपयोग करें। यह प्रोटोकॉल एक काफी बड़ा 7 मिमी x 6 मिमी कपाल माउस अस्थायी और पार्श्विका cortices पर एक मस्तिष्क गोलार्द्ध के सबसे उजागर खिड़की बनाने के लिए विधि का परिचय (- 4.5 मिमी, पार्श्व 0 - 6 मिमी जैसे, 2.5 bregma)। इस सर्जरी प्रदर्शन करने के लिए, सिर लगभग 30 डिग्री झुका किया जाना चाहिए और लौकिक पेशी के बहुत से मुकर जाना चाहिए। हड्डी को हटाने की बड़ी राशि के कारण, इस प्रक्रिया केवल पशु शल्य चिकित्सा और प्रयोग के दौरान संज्ञाहरण के साथ तीव्र प्रयोगों के लिए करना है। इस अभिनव बड़े पार्श्व कपाल खिड़की का मुख्य लाभ यह कॉर्टेक्स के दोनों औसत दर्जे का और पार्श्व क्षेत्रों के लिए एक साथ अभिगम प्रदान करना है। इस बड़े एकतरफा कपाल खिड़की कोशिकाओं के बीच तंत्रिका गतिकी का अध्ययन करने के लिए इस्तेमाल किया जा सकता है,साथ ही बहु इलेक्ट्रोड electrophysiological रिकॉर्डिंग, न्यूरोनल गतिविधि (जैसे, आंतरिक या बाह्य इमेजिंग), और optogenetic उत्तेजना की इमेजिंग के संयोजन के द्वारा विभिन्न cortical क्षेत्रों के बीच के रूप में। साथ ही, इस बड़े craniotomy भी cortical रक्त वाहिकाओं के एक बड़े क्षेत्र को उजागर करता है, पार्श्व cortical वाहिका संरचना के सीधे प्रभाव के लिए अनुमति देता है।

<html> 6 मिमी पार्श्व - एक गोलार्द्ध के भीतर cortical क्षेत्रों के बीच संबंधों का अध्ययन करने के लिए, हम एक बड़े सैजिटल साइनस और 5 फैली craniotomy इस्तेमाल किया। यह कपाल खिड़की शामिल प्राथमिक (मोटर, somatosensory, दृश्?...

Watch this Scientific Journal Video about A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity at JoVE.com

A Large Lateral Craniotomy Procedure for Mesoscale Wide-field Optical Imaging of Brain Activity

यह प्रोटोकॉल माउस सेरेब्रल कॉर्टेक्स के अस्थायी और पार्श्विका क्षेत्रों पर एक बड़ी एकतरफा craniotomy बनाने के लिए विधि प्रस्तुत करता है। यह एक cortical गोलार्द्ध के एक विशाल क्षेत्र में वास्तविक समय इमेजिंग के लिए विशेष रूप से उपयोगी है।

मस्तिष्क गतिविधि के मेसोस्केल वाइड-फील्ड ऑप्टिकल इमेजिंग के लिए एक बड़े पार्श्व क्रैनीओटमी प्रक्रिया

The craniotomy is a commonly performed procedure to expose the brain for in vivo experiments. In mouse research, most labs utilize a small craniotomy, ...

The overall goal of this large lateral craniotomy is to perform real time imaging over an expansive area of a cortical hemisphere. The main advantage of this technique is that large areas of the cortex are exposed for observation and can be used in conjunction with technologies such as optogenetics. This technique can provide insights into characterizing the neural dynamics between cells as well as that between different brain circuits at different temporal and spatial resolutions.Visual demonstration of this method is critical due to the increased risk of hemmorage and cortical damage since the dura is often attached to brain surface and blood vessels. This method can be applied to study the neurobiological mechanisms underlying brain circuit development and its changes throughout life in response to new experiences or pathologies such as Alzheimer's Disease or stroke. To begin this procedure, autoclave all the surgical supplies and ensure that sterility is maintained throughout the surgery.Ensure there is enough brain buffer for the surgery. Next, transfer an anesthetized mouse to the head holder setup and place it on a thermo-regulating heating pad set to 37 degrees Celsius. Secure the mouse's upper teeth in a teeth holder.Then rotate its head towards the left approximately 30 degrees to expose the right lateral side of the head. Subsequently, secure the mouse's head with the blunt end of the ear bars. Then apply a thalmic ointment to prevent corneal drying.To reduce cerebral edema, inject it with dexamethasone at four milligrams per kilogram intermuscularly. Wipe the skin over the surgical area with cotton swabs dipped in 4%chlorhexidine three times and then with 70%alcohol three times. Inject lidocaine subcutaneously over the craniotomy site.Under a dissecting microscope, lift up the skin at one millimeter left of the midline just behind the ear with forceps and make a small horizontal incision with surgical scissors. Make a five to six millimeter lateral cut towards the right ear and then cut towards the rostral end of the head. At the initial incision point, insert the scissors and cut 10 millimeters rostrally.After that, cut the skin around the right ear and near the right eye to expose the right side of the skull and temporal muscle. Ensure that the widest part of the exposed area is at least seven millimeters. Then trim the skin further if the surgical area needs to be extended.Using a cotton swab, rub the surface of the skull in a circular motion to remove the periosteum from the skull. Ensure that no periosteum remains and that the skull is completely dry, which is critical for successful attachment of the head plate. Next, using spring scissors and forceps, separate the temporal muscle from the skull.Cut and retract the muscle laterally until it reaches the squamosal bone. Take extreme care not to damage the superficial temporal vein that runs along the level of the squamosal bone near the eye, otherwise hemorrhage might occur. Control bleeding with gel foam pre-soaked in brain buffer.For serious hemorrhage, use a heat cauterizer. Drop butyl cyanoacrylate glue onto bleeding sites. To begin this procedure, first mark bregma to orient the underlying brain regions, apply ethyl cyanoacrylate glue around the bottom edge of the head plate and glue the head plate over the craniotomy area.After that, fill the opening space between the skull and the head plate with dental cement, leaving only the craniotomy area exposed. Then wait for the dental cement to dry and harden. Subsequently, outline the cranial window region by lightly scoring the surface of the skull with the dental drill.Gently trace the drill along the original scoring to deepen it, ensuring that the drill does not penetrate through the skull into the brain. In the meantime, switch between drilling and dabbing the skull's surface with moistened rolled tissues every few minutes. This very challenging start requires surgeons to drill through the aged skull to free it for removal without damaging the underlying cortex.Slow is loose, smooth is fast. During drilling, periodically check for buckling of the skull by gently pressing on it with forceps or the non-moving drill bit. When the bone begins the buckle, stop drilling and immerse the entire window in brain buffer.Wait for at least five minutes before skull removal to soften the bone and to reduce the chance of the dura sticking to the bone, making the skull removal process easier. Next, perform the skull removal process beginning at the anterior edge. Gently pry the loose skull from the dura using forceps.Once the bone is loose and floating on the dura, firmly grasp it with forceps and lift it away from the dura. Ensure the bone never penetrates into the brain. Use forceps and spring scissors to gently tear and cut away the dura.Care must be taken to ensure the brain parenchyma and blood vessels are not damaged. Continue until all dura is removed from the cranial window site. When performed correctly, the brain will appear to be very clean with distinct blood vessels and no blemishes.Spray the coverglass with 70%ethanol and use an air canister to gently dry. Ensure the glass is completely clean with no spots or dust present. Next, prepare 1.3%agarose by heating 200 milligrams of agarose powder dissolved in 15 milliliters of brain buffer.Place a thermometer in the hot agarose and cool the agarose down to just above solidifying temperature at about 40 degrees Celsius. Cooling can be expedited by running cold water over the tube, taking care not to allow any water to enter the agar. Gently stir with the thermometer while monitoring its temperature.Immediately before applying the agar, remove the brain buffer from the cranial well. Ensure no bubbles or particles are present as these will interfere with imaging. Now draw up the agarose with a transfer pipe head and drop the agarose directly on the brain.Then quickly place the coverslip over the surface and fasten the coverslip with the agarose drops on the corners. Shown here is a photo micrograph of the wide unilateral craniotomy with bregma indicated by a white circle in each image. Patterns of cortical activation are shown in a mouse anesthetized with 0.5%isoflurane after stimulation of the contralateral whisker, auditory stimulation, contralateral forelimb stimulation, contralateral hindlimb stimulation and visual stimulation of the contralateral eye with a light-emitting diode.There was midline activation from all forms of sensory stimulation at 10 to 25 milliseconds after primary sensory cortex activation. Once mastered, this technique can be completed in three to four hours. When attempting this procedure, it's important to remember to keep the blood loss to minimum and to ensure the cortex is not damaged.Using this procedure, other imaging techniques such as optical imaging can help to answer important questions such as how neural dynamics are influenced by spontaneous activity in the normal and diseased brain. Following its development, this technique helped to pave the way for researchers in the field of systems neuroscience to explore interactions and neural dynamics between widely spaced cortical regions in mice. This video shows you how to perform a large lateral craniotomy in the mouse with minimal blood loss and cortical damage, enabling you to collect the necessary data to answer your most pressing scientific questions.

Research

JoVE Journal

Neuroscience

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