Name: isolation of oxytocin receptor-rich nuclei from a neonatal rat brain
Uploaded: 2025-03-31T15:00:47.000+00:00
Duration: 1 min 25 s
Description: Source: Klein, B. Y. et al., Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After ...

isolation of oxytocin receptor-rich nuclei from a neonatal rat brain

Isolation of Oxytocin Receptor-Rich Nuclei from a Neonatal Rat Brain

After harvesting the brain, rapidly place the whole organ in a polymethylmethacrylate brain mold at room temperature, and immediately use a new razor blade to make 500-micrometer-thick slices of the tissue. Laying the slices rostral to caudal in a Petri dish as they are obtained, to maintain the correct orientation of the sections. When the entire brain has been sectioned, quickly add artificial cerebrospinal fluid without glucose to the dish and incubate the slices for 60 minutes at 28 to 30 degrees Celsius, with constant stirring on an orbital shaker.
Use a brain atlas and anatomic landmarks on each tissue to identify the brain nuclei to be punched, and place the slice with the nuclei of interest in a new Petri dish under a dissecting microscope. Once visualized, use a coring tool to quickly punch out four to six different nuclei, and rapidly immerse each punched nucleus in 0.06 milliliters of ice-cold protein extraction buffer in the appropriately labeled microcentrifuge tube, containing protease inhibitors and phosphatase inhibitors for 60 minutes.

isolation-of-oxytocin-receptor-rich-nuclei-from-a-neonatal-rat-brain

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue Preparation
<ol>
	<li>Order timed pregnant rats from the vendor.</li>
	<li>Follow timed pregnant rats by observing their growing abdomens in the weeks after their arrival and subsequently looking for pups on the expected delivery date by inspecting the cage every 2 hours until delivery begins.</li>
	<li>Remove pups with a gloved hand by their tail before their first feed for unprimed pups (no white milk belly is apparent when viewing the abdomen) or after the first feed for primed pups (at which point a white stomach will be visible on their abdomen) as described in the timeline (Figure 1). 
	NOTE: The first colostrum feed is termed as priming the pup; thus, a pup is unprimed until the first feed, after which they are colostrum-primed.</li>
	<li>Quickly decapitate the unanesthetized pup using sharp, clean surgical scissors.</li>
	<li>Remove the brain by cutting the skin down the midline and top surface of the skull to the nose. Then, using forceps, gently pry away the bone to expose the brain (Figure 2A) and localize the bregma, marking it with a pen as the bone plates are removed (Figure 2B).</li>
	<li>Rapidly place the whole brain in a polymethyl methacrylate brain mold at room temperature for coronal slicing at room temperature (Figure 2C).</li>
	<li>Without delay, make 500 &#956;m-thick slices using a fresh razor blade. Lay the slices rostral to caudal in a Petri dish to maintain the orientation of sections (Figure 3).</li>
	<li>Quickly add artificial cerebrospinal fluid (ACSF; 1.0 mM potassium dihydrogen phosphate, (KH2PO4), 26 mM sodium bicarbonate (NaHCO3), 118.6 mM Sodium chloride (NaCl), 3.0 mM potassium chloride (KCl), 203.3 mM magnesium chloride (MgCl2-6H2O)) without glucose and incubate the slices for 60 min at 28-30 &#176;C, constantly stirring on an orbital shaker to metabolically and differentially challenge the unprimed versus colostrum-primed tissues.</li>
	<li>Identify the brain nuclei that are required to punch using a brain atlas and anatomic landmarks on the tissue section. Place this slice with the nuclei of interest in a Petri dish and move it to the dissecting microscope.</li>
	<li>Once visualized, quickly punch out 4 of 6 different nuclei using a coring tool, selecting the size to best punch the nucleus in question and consistently between samples (Figure 3). 
	NOTE: The remaining brain slice will now have a hole where brain tissue was removed. In this study, we excised the following nuclei using the below coordinates. All anterior/posterior (A/P) coordinates are from Bregma (except nucleus of the solitary tract (NTS), which is with reference to the Calamus Scriptorius). All dorsal/ventral (D/V) coordinates are from the surface of the cortex (except NTS, which is from the surface of the medulla). The following coordinates include A/P, medial/lateral (M/L), and D/V in mm: 1) solitary tract nucleus (NTS, A/P, 0.4 to 0.8; L, &#177; 0.2; D/V, 0.3 (from the surface of the medulla), 2) paraventricular nucleus (PVN, -0.8; &#177; 0.2; 0), 3) supra-optic nucleus (SON, -1.1; &#177; 1.4; 4.3), 4) cortex (CX, partial cortex area 1, -2.8; &#177; 1.5; 0.6), 5) striatum nuclei (STR, -0.0; &#177; 1.6; 1.8), and 6) medial preoptic nucleus (MPO, -0.6; &#177; 0.2; 4.2).</li>
	<li>Rapidly immerse the punched nuclei in 0.06 mL of ice-cold protein extraction buffer containing protease inhibitors and phosphatase inhibitors for 60 minutes.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Pregnant Sprague-Dawley rats</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Punch device</td>
			<td>WellTech Rapid Core or Harris Uni-Core</td>
			<td>0.35, 0.50, 0.75, 1.0, 1.20, 1.50</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Klein, B. Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58341">Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding.</a> J. Vis. Exp. (2018)
This video demonstrates the isolation of oxytocin receptor-rich nuclei from the neonatal rat brain. Coronal slicing, followed by identification and precise punching of oxytocin receptor-rich brain nuclei, enables the effective extraction of oxytocin receptor-rich nuclei for further analysis.

<img alt="Figure 1" src="/files/ftp_upload/23205/23205_Figure1.jpg" /> 
Figure 1: Schematic showing the protocol timeline.
<img alt="Figure 2" src="/files/ftp_upload/23205/23205_Figure2.jpg" /> 
Figure 2: Dissected neonatal rat brain. (A) The dissected brain is shown with bones removed from rostral to bregma. (B) The brain is shown after a line has been drawn at bregma, after which the remaining bone plates were removed. (C) The brain is shown in a polymethyl methacrylate brain mold just before being sliced with a razor blade.
<img alt="Figure 3" src="/files/ftp_upload/23205/23205_Figure3.jpg" /> 
Figure 3: Brain slices arranged from rostral to caudal with punches (red circles). Abbreviations: CX: cortex, parietal lobe; MPO: medial preoptic area; NTS: nucleus tractus solitaries; PVN: paraventricular nucleus; SON: supraoptic nucleus; and STR: striatum.

Source: Klein, B. Y. et al., Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After ...

The brain&#39;s oxytocin receptor-rich nuclei are clusters of neurons that express oxytocin receptors, which interact with oxytocin, a neuropeptide.
To isolate oxytocin receptor-rich nuclei, begin with a neonatal rat brain.
Place the brain in a mold with a slicing guide. Slice the entire brain to expose different regions with nuclei.
Transfer these slices to a dish and immobilize them.
Add artificial cerebrospinal fluid, or aCSF, to maintain neuronal viability and incubate under gentle rotation.
Using a published brain atlas as a reference, identify anatomical landmarks to locate oxytocin-receptor-rich nuclei within the slices.
Transfer an identified brain slice of interest to a new plate.
Under a microscope, locate the nuclei within the slice and punch it.
Repeat the process for other identified sections to isolate the oxytocin-rich nuclei.
Transfer the isolated nuclei to a tube containing ice-cold protein extraction buffer supplemented with protease and phosphatase inhibitors to preserve the oxytocin receptors.

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Isolation of Oxytocin Receptor-Rich Nuclei from a Neonatal Rat Brain

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Isolation of Oxytocin Receptor-Rich Nuclei from a Neonatal Rat Brain