S.B.P is a recipient of a Loma Linda University Summer Undergraduate Research Fellowship.

The following protocol has been accepted and approved by the Institutional Animal Care and Use Committee (IACUC) of Loma Linda University. NIH guidelines for the ethical treatment of animals are followed in all animal experiments performed in the laboratory. All ethical standards were upheld by individuals performing this protocol.
1. Solutions
<ol>
	<li>Prepare artificial cerebral spinal fluid (aCSF).
	<ol>
		<li>Prepare fresh aCSF the evening before an experiment in 1 L ba...

<ol>
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Adapting the protocol presented here into an en bloc or slice workflow is advantageous for laboratories and studies that would like to utilize either en bloc brainstem-spinal cord and/or thin slice preparations for electrophysiology recordings. The dissection and slice method presented, combined with methods previously reported by others17,18,19, will allow reproducible preparation of robust and viable tissue that is widely adap...

The respiratory neural network of the brainstem provides a fertile domain for understanding the general characteristics of rhythmic neural networks. In particular, the interest is in the development of neonatal rodent breathing and understanding how the breathing rhythm develops. This may be done using a multi-level approach, including in vivo whole animal plethysmography, in vitro en bloc nerve recordings, and in vitro slice recordings that contain the breathing rhythm generator. Reductionist in vitro en bloc and slice recordings are an advantageous method to use when interrogating the mechanisms behind respiratory rhythmogenesis and neural circuitry in the brainstem-spinal cord region of developing rodents. The developing respiratory system includes approximately 40 cell types, characterized by firing pattern, including those of the central respiratory1,2. The central respiratory network includes a group of rhythmically active neurons located in the rostral ventrolateral medulla1,3. Mammalian respiratory rhythmogenesis is generated from an autorhythmic interneuron network dubbed the preB&#246;tzinger complex (pBC), which has been localized experimentally via both slice and en bloc preparations of neonatal mammalian brainstem-spinal cords3,4,5,6,7,8. This region serves a similar function to the sinoatrial node (SA) in the heart and generates an inspiratory timing system to drive respiration. From the pBC, the inspiratory rhythm is carried to other regions of the brainstem (including the hypoglossal motor nucleus) and spinal motor pools (such as the phrenic motor neurons that drive the diaphragm)9.
Rhythmic activity may be obtained using brainstem spinal cord en bloc preparations or slices from a variety of cell populations, including C3-C5 nerve rootlets, XII nerve rootlets, hypoglossal motor nucleus (XII MN), hypoglossal premotor neurons (XII pMN), and the pBC3,10,11,12. While these methods of data collection have been successful across a handful of laboratories, many of the protocols are not presented in a way that is fully reproducible for new researchers entering the field. Obtaining viable and rhythmically active en bloc and slice preparations requires an acute attention to detail through all steps of the dissection and slice cutting protocol. Previous protocols extensively describe the various recording procedures and electrophysiology, yet lack detail in the most critical part of obtaining a viable tissue preparation: performing the brainstem-spinal cord dissection and slice procedure.
Efficiently obtaining a rhythmically-active and viable en bloc or slice preparation brainstem-spinal cord electrophysiology recordings requires that all steps be performed correctly, carefully, and swiftly (typically, the whole procedure related here can be performed in approximately 30 min). Critical points of the brainstem-spinal cord electrophysiology protocol that have not been previously well described include the dissection of nerve rootlets and the slicing procedure on the vibratome. This protocol is the first to stepwise visually communicate the brainstem-spinal cord dissection for both new researchers and experts in the field. This protocol also thoroughly explains surgical techniques, landmarks, and other procedures to assist future researchers in standardizing slices and en bloc preparations to contain the exact circuitry desired in each experiment. The procedures presented here can be used in both rat and mouse neonatal pups.

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			<td height="21" style="height:21px;width:359px;">NaCl</td>
			<td style="width:288px;">Fisher Scientific</td>
			<td style="width:320px;">S271-500</td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma Aldrich</td>
			<td>P5405-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>BP328-1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4 &#8226;H2O</td>
			<td>Sigma Aldrch</td>
			<td>S9638-25G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">CaCl2&#8226;2H2O</td>
			<td>Sigma Aldrich</td>
			<td>C7902-500G</td>
			<td></td>
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			<td height="21" style="height:21px;">MgSO4&#8226;7H2O</td>
			<td>Sigma Aldrich</td>
			<td>M7774-500G</td>
			<td></td>
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			<td height="21" style="height:21px;">D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cold-Light source Halogen lamp 150W</td>
			<td>AmScope</td>
			<td>H2L50-AY</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dissection Microscope</td>
			<td>Leica</td>
			<td>M-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome 1000 Plus</td>
			<td>Vibratome</td>
			<td>W3 69-0353</td>
			<td></td>
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			<td height="21" style="height:21px;">Magnetic Base</td>
			<td>Kanetic</td>
			<td>MB-B-DG6C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane, USP</td>
			<td>Patterson Veterinary</td>
			<td>NDC 14043-704-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sword Classic Double Edge Blades</td>
			<td>Wilkinson</td>
			<td>97573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histoclear</td>
			<td>Sigma-Aldrich</td>
			<td>H2779</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5/45 Forcep</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
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			<td height="21" style="height:21px;">Scalpel Blades #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handel #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring Scissors Straight&#160;</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narrow Pattern Forcep Serrated/straight</td>
			<td>Fine Science Tools</td>
			<td>11002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Castroviejo Micro Dissecting Spring Scissors; Straight</td>
			<td>Roboz</td>
			<td>RS-5650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vannas Scissors 3&#34; Curved</td>
			<td>Roboz</td>
			<td>RS-5621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 0</td>
			<td>Fine Science Tools/8840604</td>
			<td>26000-35&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 0, SS</td>
			<td>Fine Science Tools</td>
			<td>26001-35</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 00</td>
			<td>Fine Science Tools</td>
			<td>26000-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 00, SS</td>
			<td>Fine Science Tools</td>
			<td>26001-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 000</td>
			<td>Fine Science Tools</td>
			<td>26000-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins, 000, SS</td>
			<td>Fine Science Tools</td>
			<td>26001-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minutien pins, 0.10 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minutien pins, 0.15 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minutien pins, 0.2 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher Tissue prep Parafin&#160;</td>
			<td>fisher</td>
			<td>T56-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graphite&#160;</td>
			<td>fisher&#160;</td>
			<td>G67-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Delrin Plastic&#160;</td>
			<td>Grainger</td>
			<td>3HMT2</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">18 Gauge Hypodermic Needle</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
	</tbody>
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preparation of rhythmically-active in vitro neonatal rodent brainstem-spinal cord and thin slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

The method presented here allows a researcher interested in obtaining rhythmically active slices of brainstem to reproducibly and reliably cut a viable, robust slice that will allow recording of fictive motor output for many hours. All of the minimally necessary neural circuit elements for generating and transmitting inspiratory rhythm can be captured in a thin slice using this method. These elements include: the preB&#246;tzinger Complex, premotor neurons projecting to the hypoglossal mo...

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Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

This method facilitates the reproducible dissection and slicing of rodent brainstems for the captured analysis of the Mirian Medullary Respiratory Network. This technique offers great experimental flexibility, making it possible to prepare either an in-vitro unblock or a slice preparation for recording. This method may be used for electrophysiology experiments to explore the neural control circuit that generates breathing rhythms and to understand the pathology and disregulation of breathing in genetically modified rodents.After dissection of the neuraxis, quickly transfer the isolated trunk of the animal to an aerated dissection chamber under a dissection microscope and place the tissue dorsal-side up, with the rostral end facing the front of the chamber. Pin the tissue at the shoulders and the most caudal end of the spinal cord and make a midsagittal incision through the skull following the parietal suture to avoid damaging the cortex and the brainstem underlying the skull. Beginning at the sagittal suture and working laterally, snip the occipital sutures of the skull to create flaps of bone that may be reflected and pinned to anchor and stabilize the rostral part of the skull.After reflecting both skull flaps, excise the remainder of the cerebral cortex, leaving the caudal portion of the cerebellum relatively intact to perform a dorsal laminectomy. Use micro spring scissors and forceps to remove the musculature surrounding the skull and vertebral column. Remove the tissue along the dorsal side of the ribcage, leaving the ribs intact and carefully snip away the lateral processes of the vertebral lamina.After cutting away any tissue overlying the pons and medulla, the vermis, cerebellum, pons and beginning of the spinal cord, will be clearly visible. To perform a ventral laminectomy, place the tissue dorsal-side down and pin at the ribcage and the most caudal end of the spine. Use the skull flaps to pin down the rostral-side of the tissue and remove the ventral half of the ribcage, including the sternum and all of the abdominal organs.Dissect away the soft tissue attached to the ribcage to expose the ribs and spinal cord and remove the tongue, esophagus, trachea, larynx and all of the other soft tissue and musculature overlying the base of the skull and the spinal column. In order to identify the hard pallet, dissect the tissue overlying this rectangular plate of bone at the base of the skull, that includes a v-shape indentation. Then cut along the midline of the pallet, carefully lifting the tissue upwards and perform a transverse cut to remove it.To begin a ventral laminectomy, remove the lamina, exposing the ventral surface of the brainstem and the spinal cord from the first cervical vertebra to approximately the thoracic vertebra seven and snip five to 10 millimeters along both sides of the spinal column at the lamina. When the spinal cord has been exposed, snip the rootlets approximately 20 to 25 millimeters bilaterally, along the spinal column to approximately T7 and carefully lift the rostral edge of C1, C2 and C3 with hooked or bent forceps to allow snipping beneath the bone for removal of the three vertebra. When the desired length of spinal cord has been isolated from the vertebral column, make a transverse cut to remove the spinal cord tissue.After removing the dura, place the brainstem in the center of the paraffin platform on the plastic cutting block and use fine insect pins, trimmed to no more than one centimeter in length to pin the caudal end of the brainstem through the distal spinal cord. Then align the paraffin covered cutting block with the pinned brainstem in the Vibratone block holder, so that the blade will cut perpendicular to the rostral face of the brainstem. Next, make an initial slice to remove 200 to 300 micrometers of uneven extraneous tissue on the rostral-most end of the tissue, making small adjustments as necessary to ensure PARP linearity and small cuts to remove any uneven tissue.The glossopharyngeal rootlets will become visible at the lateral edge of the brainstem, cut a 300 to 500 micrometer slice of brainstem from these rostral neuroanatomical markers to capture the pre-Botzinger complex and the associated transmission circuitry. All of the slicing procedure steps are critical for creating a reproducible, viable slice, with all of the necessary neuronal circuitry in the correct orientation to produce robust rhythm. All of the minimally necessary neural circuit elements for generating and transmitting inspiratory rhythm can be captured in a thin slice using this method.Including the pre-Botzinger complex, pre-motor neurons projecting to the hypoglossal motor neurons and the hypoglossal nerve rootlets. The pre-Botzinger complex, the hypoglossal or 12th cranial nerve rootlets and C4 nerve rootlets can then be used for inspiratory recording. The key aspect of this procedure is the careful isolation of the rootlets and confirmation that the brainstem is not damaged before cutting the final slice.Following this procedure, electrophysiology recordings can be made using patch clamp methods, extra cellular recordings or suction electrodes to record the electrical activity produced by the network. Previously published protocols have provided little step-by-step detail, making it difficult for new researchers to use this method without traveling to another laboratory and spending time with a seasoned investigator.

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Research

JoVE Journal

Neuroscience

11.4K visualizações.  Loma Linda University. Este método facilita a dissecção reprodutível e o corte de troncos cerebrais de roedores para a análise capturada da Rede Respiratória Medullary Mirian. Esta técnica oferece grande flexibilidade experimental, possibilitando a preparação de um desbloqueio in vitro ou uma preparação de fatia para gravação. Este método pode ser usado para experimentos de eletrofisiologia para explorar o circuito de controle neural que gera ritmos respiratórios e para entender a patologia e a desre...