The authors thank Manon Ranger and Alexandra Schulz for their assistance in preparing this protocol.

This study was approved by the Institutional Animal Care and Use Committees at Columbia University and the New York State Psychiatric Institute.
1. Tissue Preparation
<ol>
	<li>Order timed pregnant rats from 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 h 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 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 S1). 
	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 1A) and localize the bregma, marking it with a pen as the bone plates are removed (Figure 1B).</li>
	<li>Rapidly place the whole brain in a polymethyl methacrylate brain mold at room temperature for coronal slicing at room temperature (Figure 1C).</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 orientation of sections (Figure 2).</li>
	<li>Quickly add artificial cerebrospinal fluid (ACSF; 1.0 mM KH2PO4, 26 mM NaHCO3, 118.6 mM NaCl, 3.0 mM KCl, 203.3 mM 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 atlas33 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 2). 
	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 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 (see step 2.3).</li>
</ol>
2. Protein Extraction
<ol>
	<li>Prepare the protein extraction solution using the protein lysis kit (Table of Materials) on the day before expected pup delivery.</li>
	<li>Thaw (on ice) the frozen (-20 &#176;C) aqueous solution of the protease and phosphatase inhibitors of the lysis buffer kit and place the lysis buffer and DMSO solution of the proteases/phosphatases inhibitors on ice.</li>
	<li>Add 1.85 mL of lysis buffer into a clean, ice-cold 15 mL tube. Then, add 0.1 mL of aqueous solution of inhibitors and 0.05 mL of DMSO-dissolved inhibitors. Finally, cap and briefly vortex the tube and keep it at -20 &#176;C until use.</li>
	<li>Label 24 clean microcentrifuge tubes (0.5 mL each) for the lysis procedure. Designate 12 tubes per brain for the colostrum-unprimed group (U) [6 left (L) and 6 right (R) brain nuclei], and label according to nuclei acronym, side, and condition (e.g., NTS-L-U, NTS-R-U, etc.). Label the second group of 12 tubes for the colostrum-primed samples.</li>
	<li>Label two additional sets of tubes as done in step 2.4 for the stock protein extracts (using 1.5 mL Eppendorf-style tubes) and for the first set of sample preparation (using 0.5 mL tubes). Keep these tubes in two separate, labeled freezing boxes (each designed for 100 tubes). One box will be used for the unprimed samples and the other for the primed samples.</li>
	<li>Thaw the lysis solution on ice on the day that the pups are delivered, and while incubating brain slices in ACSF, aliquot 0.06 mL lysis solution into the lysis procedure tubes (from step 2.4) and add nuclei punches and incubate in ice for 60 min.</li>
	<li>Centrifuge the incubated lysed nuclei for 30 min in a cooled mini-centrifuge at 14000 rpm (10,000 x g) and carefully aspirate 0.055 mL of supernatant with a properly set pipette. Transfer the supernatant into the pre-cooled 1.5 mL stock tubes (from step 2.5) and put them on ice. Before freezing (at -20 &#176;C) the protein stock tubes, transfer 0.012 mL of supernatant into the 0.5 mL pre-cooled tubes for the first sample preparation (from step 2.5) and leave them on ice.</li>
</ol>
3. Sample Preparation for In-capillary Protein Measurement
<ol>
	<li>Use a kit and prepare the reagents for separation according to manufacturer&#8217;s directions. Add 0.003 mL of master-mix reagent to each of the 12 samples in the 0.5 mL labeled tubes (from step 2.5) on ice that contain 0.012 mL of the protein extracts.</li>
	<li>Turn on the heating block to 95 &#176;C and add 0.004 mL of the reagent prepared in step 3.1 to a biotinylated molecular-weight (MW) ladder in a tube with 0.016 mL of deionized water. To denature the ladder and samples, place the ladder tube and the 12 samples of unprimed protein extracts in the heat block at 95 &#176;C for 5 min and store them at 4 &#176;C until use.</li>
	<li>Repeat steps 2.0 to 3.2, from protein extraction to sample preparation, for the nuclei punched from colostrum-primed rats.</li>
</ol>
4. Electrophoresis Preparation
<ol>
	<li>Thaw the biotin labeling reagent (stored in the deep freezer at -80 oC) on the bench and prepare the protein detection kit on ice at 4 &#176;C following the manufacturer&#8217;s directions.</li>
	<li>Mix 0.15 mL of luminol with 0.15 mL of peroxide and load 0.01 mL into 25 wells (row E, wells 1-25) of the plate for the automated Western machine. Load 0.008 mL of Streptavidin-HRP from the kit into wells D1 to D25</li>
	<li>Load 0.01 mL of the antibody diluent solution into wells C1 to C25 and B1.</li>
	<li>Spin the 24 protein samples and ladder tubes briefly (2-3 seconds) in a minicentrifuge to pool down evaporated water from the tube caps.</li>
	<li>Load 0.003 mL of 12 unprimed samples into wells A2 to A13, 0.003 mL of 12 colostrum-primed samples into wells A14 to A25, and 0.005 mL of the biotinylated ladder into well A1.</li>
	<li>Leave row F empty and load 0.45 mL of wash buffer into each of the 5 compartments in each of the 3 rows below row F. Cover the plate with its plastic lid to avoid evaporation during the remaining procedures.</li>
	<li>Briefly vortex the thawed biotin labeling reagent and add 0.15 mL of the reagent to its designated tube; then add to it 0.15 mL of the total protein reconstitution agent and mix them to homogeneity.</li>
	<li>Remove the cover from the plate and load 0.01 mL of agents 1 and 2 into wells B2 to B25. Cover the plate and centrifuge it for 10 min at 1,000 x g to remove any air bubbles from the various solutions. Use an empty plate in the centrifuge for balance.</li>
</ol>
5. Electrophoresis
<ol>
	<li>While the plate is spinning, open a run file in the automated Western machine-attached computer by indicating in the dropdown page from &#8220;file&#8221; to run a total protein assay and clicking the respective spot.</li>
	<li>Annotate the samples by well in the computer. Then, remove the plate from the centrifuge remove the cover and carefully peel off the aluminum cover from the separation solution compartments.</li>
	<li>Place the plate in the automated Western instrument, peel off the cover from the capillary cartridge box, insert the cartridge in its designated place, and close the door.</li>
	<li>Click the &#8220;RUN&#8221; button, and when prompted with the type of the assay (&#8220;total protein&#8221;), type in the name of the samples (for example, &#8220;unprimed 2-13 and colostrum-primed 14-25&#8221;). Click &#8220;OK&#8221;, and when prompted by the activated run date and ID number of the run file, make a note of the time when the run ends.</li>
	<li>At the end of the run (170 min after the start), open the instrument door, remove the capillary cartridge, and discard it into the sharps disposal. Discard the plate in the biological matter disposal.</li>
	<li>Click the separation curves icon in the analysis page of the run file, and check that all the samples have run properly and are showing multiple protein curves in all capillaries.</li>
</ol>
6. Analysis of Signaling Proteins
<ol>
	<li>In a labeled 1.5 mL tube, add 0.003 mL of rabbit anti phospho-eIF2a (p-eIF2a) antibody and suspend it in 0.3 mL (1:100 dilution) in antibody diluent from the suitable detection kit. Then, keep it on ice.</li>
	<li>Label a luminol tube and add 0.15 mL of luminol and 0.15 mL of peroxide from this detection module kit and dispense 0.01 mL into wells E1 to E25 of a fresh, automated Western plate.</li>
	<li>Dispense 0.01 mL of the secondary anti rabbit antibody into wells D2 to D25, and add 0.01 mL of streptavidin-HRP from the kit to well D1.</li>
	<li>Dispense 0.01 mL of the primary antibody (from step 6.1) into wells C2 to C25, and add 0.01 mL of antibody diluent 2 solution to wells C1 and B1 to B25.</li>
	<li>Leave the row F wells empty and fill 0.45 mL of wash buffer into the 5 compartments of 3 rows below the F row.</li>
	<li>Briefly spin the refrigerated samples for 3 s, and add 0.003 mL of each sample to row A in the same order they were added for the total protein assay, starting from A2 to A25. Add 0.005 mL of the biotinylated MW ladder to well A1 and cover the plate.</li>
	<li>Centrifuge the plate as done in step 4.8. While the plate is spinning, open (in the automated Western-associated software and computer) a new run file and indicate in the dropdown page from &#8220;File&#8221; to run a molecular size assay by clicking the respective spot.</li>
	<li>In the assay page, type the sample names in each capillary, then type the name of the primary antibody in the allocated spot and the secondary anti-rabbit antibody below it.</li>
	<li>At the end of centrifugation, repeat steps 5.3&#8211;5.5, except the name given to the run file this time should be &#8220;p-eIF2a on unprimed 2&#8211;13 and colostrum-primed 14&#8211;25&#8221;.</li>
	<li>After the electrophoresis separation, check the run file for immune-reactivity peaks of antigens at sizes of 40&#8211;43 kDa. Where MW of peaks this size are missing, right-click below the curve and indicate inside the dropdown list to add MW to the peak, which ensures that the size and arbitrary quantity below the curve are recorded.</li>
</ol>
7. Processing the Results
<ol>
	<li>Open a spreadsheet file for total protein run in the automated Western run file and provide an ID number.</li>
	<li>Open the run file of the total protein assay at the analysis page at the curve mode and mark all the peaks in individual capillaries. Then, copy and paste them into the spreadsheet and sum the areas under the curve of all peaks recorded in the entire capillary.</li>
	<li>In a separate column, arrange the total amount of protein for each column with capillary numbers, names of the respective brain nuclei, and the ID numbers of the run files.</li>
	<li>Open a spreadsheet for the p-eIF2a antigen, and in a single column, record the area under the curve from each capillary side-by-side with its respective capillary number, name of brain nucleus, and ID number of the run file.</li>
	<li>Copy the total protein column (parallel to the p-eIF2a curve quantities) into a third spreadsheet and compute p-eIF2a:total protein ratios in a third column.</li>
	<li>Collect results from steps 4 to 6 for each brain nucleus, arrange them in groups of nuclei, and generate a bar graph.</li>
</ol>

<ol>
	<li>Hietaniemi, M., 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=Gene+expression+profiles+in+fetal+and+neonatal+rat+offspring+of+energy-restricted+dams.">Gene expression profiles in fetal and neonatal rat offspring of energy-restricted dams.</a> Journal of Nutrigenetics and Nutrigenomics. 2 (4-5), 173-183 (2009).</li><li>Okabe, 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=Homogenous+glycine+receptor+expression+in+cortical+plate+neurons+and+Cajal-Retzius+cells+of+neonatal+rat+cerebral+cortex.">Homogenous glycine receptor expression in cortical plate neurons and Cajal-Retzius cells of neonatal rat cerebral cortex.</a> Neuroscience. 123 (3), 715-724 (2004).</li><li>Mailleux, P., Takazawa, K., Erneux, C., Vanderhaeghen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+the+neurons+containing+inositol+1,4,5-trisphosphate+3-kinase+and+its+messenger+RNA+in+the+developing+rat+brain.">Distribution of the neurons containing inositol 1,4,5-trisphosphate 3-kinase and its messenger RNA in the developing rat brain.</a> Journal of Comparative Neurology. 327 (4), 618-629 (1993).</li><li>Carter, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+and+Human+Evolution.">Oxytocin and Human Evolution.</a> Current Topics in Behavioral Neuroscience. , (2017).</li><li>Sippel, L. M., 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=Oxytocin+and+Stress-related+Disorders:+Neurobiological+Mechanisms+and+Treatment+Opportunities.">Oxytocin and Stress-related Disorders: Neurobiological Mechanisms and Treatment Opportunities.</a> Chronic Stress (Thousand Oaks). 1, (2017).</li><li>Agnati, L. F., 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=Aspects+on+the+integrative+actions+of+the+brain+from+neural+networks+to+"brain-body+medicine".">Aspects on the integrative actions of the brain from neural networks to "brain-body medicine".</a> Journal of Receptors and Signal Transduction Research. 32 (4), 163-180 (2012).</li><li>Welch, M. G., Margolis, K. G., Li, Z., Gershon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+regulates+gastrointestinal+motility,+inflammation,+macromolecular+permeability,+and+mucosal+maintenance+in+mice.">Oxytocin regulates gastrointestinal motility, inflammation, macromolecular permeability, and mucosal maintenance in mice.</a> American Journal Physiology: Gastrointestinal and Liver Physiology. 307 (8), G848-G862 (2014).</li><li>Welch, M. G., 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=Expression+and+developmental+regulation+of+oxytocin+(OT)+and+oxytocin+receptors+(OTR)+in+the+enteric+nervous+system+(ENS)+and+intestinal+epithelium.">Expression and developmental regulation of oxytocin (OT) and oxytocin receptors (OTR) in the enteric nervous system (ENS) and intestinal epithelium.</a> Journal of Comparative Neurology. 512 (2), 256-270 (2009).</li><li>Prakash, B. S., Paul, V., Kliem, H., Kulozik, U., Meyer, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+oxytocin+in+milk+of+cows+administered+oxytocin.">Determination of oxytocin in milk of cows administered oxytocin.</a> Analytica Chimica Acta. 636 (1), 111-115 (2009).</li><li>Solangi, A. R., Memon, S. Q., Mallah, A., Khuhawar, M. Y., Bhanger, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+separation+of+oxytocin,+norfloxacin+and+diclofenac+sodium+in+milk+samples+using+capillary+electrophoresis.">Quantitative separation of oxytocin, norfloxacin and diclofenac sodium in milk samples using capillary electrophoresis.</a> Biomedical Chromatography. 23 (9), 1007-1013 (2009).</li><li>Klein, B. Y., 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=Oxytocin+modulates+markers+of+the+unfolded+protein+response+in+Caco2BB+gut+cells.">Oxytocin modulates markers of the unfolded protein response in Caco2BB gut cells.</a> Cell Stress and Chaperones. 19 (4), 465-477 (2014).</li><li>Klein, B. Y., Tamir, H., Hirschberg, D. L., Glickstein, S. B., Welch, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+modulates+mTORC1+pathway+in+the+gut.">Oxytocin modulates mTORC1 pathway in the gut.</a> Biochemical and Biophysical Research Communications. 432 (3), 466-471 (2013).</li><li>Klein, B. Y., Tamir, H., Ludwig, R. J., Glickstein, S. B., Welch, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colostrum+oxytocin+modulates+cellular+stress+response,+inflammation,+and+autophagy+markers+in+newborn+rat+gut+villi.">Colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi.</a> Biochemical an Biophysical Research Communications. 487 (1), 47-53 (2017).</li><li>Donnet-Hughes, 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=Potential+role+of+the+intestinal+microbiota+of+the+mother+in+neonatal+immune+education.">Potential role of the intestinal microbiota of the mother in neonatal immune education.</a> Proceedings of the Nutritional Society. 69 (3), 407-415 (2010).</li><li>Perez, P. F., 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=Bacterial+imprinting+of+the+neonatal+immune+system:+lessons+from+maternal+cells?.">Bacterial imprinting of the neonatal immune system: lessons from maternal cells?.</a> Pediatrics. 119 (3), e724-e732 (2007).</li><li>Takeda, S., Kuwabara, Y., Mizuno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentrations+and+origin+of+oxytocin+in+breast+milk.">Concentrations and origin of oxytocin in breast milk.</a> Endocrinolcia Japonica. 33 (6), 821-826 (1986).</li><li>Quintana, D. S., Outhred, T., Westlye, L. T., Malhi, G. S., Andreassen, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+oxytocin+administration+on+brain+activity:+a+systematic+review+and+meta-analysis+protocol.">The impact of oxytocin administration on brain activity: a systematic review and meta-analysis protocol.</a> Systematic Reviews. 5 (1), 205 (2016).</li><li>Dobbing, J., Sands, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+aspects+of+the+brain+growth+spurt.">Comparative aspects of the brain growth spurt.</a> Early Human Development. 3 (1), 79-83 (1979).</li><li>Orr, M. E., Garbarino, V. R., Salinas, A., Buffenstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+Postnatal+Brain+Development+in+the+Longest-Lived+Rodent:+Prolonged+Maintenance+of+Neotenous+Traits+in+the+Naked+Mole-Rat+Brain.">Extended Postnatal Brain Development in the Longest-Lived Rodent: Prolonged Maintenance of Neotenous Traits in the Naked Mole-Rat Brain.</a> Frontiers in Neuroscience. 10, 504 (2016).</li><li>Hansson, 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=Time-resolved+quantitative+proteome+analysis+of+in+vivo+intestinal+development.">Time-resolved quantitative proteome analysis of in vivo intestinal development.</a> Molecular and Cellular Proteomics. 10 (3), (2011).</li><li>Mochizuki, K., Yorita, S., Goda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+changes+in+the+jejunum+of+rats+during+the+transient+suckling-weaning+period.">Gene expression changes in the jejunum of rats during the transient suckling-weaning period.</a> Journal of Nutritional Science and Vitaminology (Tokyo). 55 (2), 139-148 (2009).</li><li>Rinaman, L., Banihashemi, L., Koehnle, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+life+experience+shapes+the+functional+organization+of+stress-responsive+visceral+circuits.">Early life experience shapes the functional organization of stress-responsive visceral circuits.</a> Physiology and Behavior. 104 (4), 632-640 (2011).</li><li>Johannes, G., Sarnow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-independent+polysomal+association+of+natural+mRNAs+encoding+c-myc,+BiP,+and+eIF4G+conferred+by+internal+ribosome+entry+sites.">Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.</a> RNA. 4 (12), 1500-1513 (1998).</li><li>Rinaman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindbrain+noradrenergic+A2+neurons:+diverse+roles+in+autonomic,+endocrine,+cognitive,+and+behavioral+functions.">Hindbrain noradrenergic A2 neurons: diverse roles in autonomic, endocrine, cognitive, and behavioral functions.</a> American Journal of Physiology: Regulatory, Integrative and Comparative Physiology. 300 (2), R222-R235 (2011).</li><li>Walker, C. D., Toufexis, D. J., Burlet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+and+limbic+expression+of+CRF+and+vasopressin+during+lactation:+implications+for+the+control+of+ACTH+secretion+and+stress+hyporesponsiveness.">Hypothalamic and limbic expression of CRF and vasopressin during lactation: implications for the control of ACTH secretion and stress hyporesponsiveness.</a> Progress in Brain Research. 133, 99-110 (2001).</li><li>Montiel-Castro, A. J., Gonzalez-Cervantes, R. M., Bravo-Ruiseco, G., Pacheco-Lopez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbiota-gut-brain+axis:+neurobehavioral+correlates,+health+and+sociality.">The microbiota-gut-brain axis: neurobehavioral correlates, health and sociality.</a> Frontiers in Integrative Neuroscience. 7, 70 (2013).</li><li>Blevins, J. E., Ho, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+oxytocin+signaling+in+the+regulation+of+body+weight.">Role of oxytocin signaling in the regulation of body weight.</a> Reviews in Endocrine and Metabolic Disorders. 14 (4), 311-329 (2013).</li><li>Welch, M. G., 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=Combined+administration+of+secretin+and+oxytocin+inhibits+chronic+colitis+and+associated+activation+of+forebrain+neurons.">Combined administration of secretin and oxytocin inhibits chronic colitis and associated activation of forebrain neurons.</a> Neurogastroenterology Motility. 22 (6), 654 (2010).</li><li>Berthoud, H. R., Neuhuber, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+chemical+anatomy+of+the+afferent+vagal+system.">Functional and chemical anatomy of the afferent vagal system.</a> Autonomic Neuroscience: Basic and Clinical. 85 (1-3), 1-17 (2000).</li><li>Taniuchi, S., Miyake, M., Tsugawa, K., Oyadomari, M., Oyadomari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+stress+response+of+vertebrates+is+regulated+by+four+eIF2alpha+kinases.">Integrated stress response of vertebrates is regulated by four eIF2alpha kinases.</a> Scientific Reports. 6, 32886 (2016).</li><li>Altschuler, S. M., Bao, X. M., Bieger, D., Hopkins, D. A., Miselis, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscerotopic+representation+of+the+upper+alimentary+tract+in+the+rat:+sensory+ganglia+and+nuclei+of+the+solitary+and+spinal+trigeminal+tracts.">Viscerotopic representation of the upper alimentary tract in the rat: sensory ganglia and nuclei of the solitary and spinal trigeminal tracts.</a> Journal of Comparative Neurology. 283 (2), 248-268 (1989).</li><li>Shapiro, R. E., Miselis, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+central+neural+connections+of+the+area+postrema+of+the+rat.">The central neural connections of the area postrema of the rat.</a> Journal of Comparative Neurology. 234 (3), 344-364 (1985).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1997).</li><li>Nayak, R., Pintel, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+viruses+can+induce+phosphorylation+of+eIF2alpha+via+PKR+activation,+which+can+be+overcome+by+helper+adenovirus+type+5+virus-associated+RNA.">Adeno-associated viruses can induce phosphorylation of eIF2alpha via PKR activation, which can be overcome by helper adenovirus type 5 virus-associated RNA.</a> Journal of Virology. 81 (21), 11908-11916 (2007).</li><li>Zaborske, J. M., 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=Genome-wide+analysis+of+tRNA+charging+and+activation+of+the+eIF2+kinase+Gcn2p.">Genome-wide analysis of tRNA charging and activation of the eIF2 kinase Gcn2p.</a> Journal of Biological Chemistry. 284 (37), 25254-25267 (2009).</li><li>Hollis, J. H., Lightman, S. L., Lowry, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+systemic+and+visceral+sensory+information+by+medullary+catecholaminergic+systems+during+peripheral+inflammation.">Integration of systemic and visceral sensory information by medullary catecholaminergic systems during peripheral inflammation.</a> Annals of the New York Academy of Sciences. 1018, 71-75 (2004).</li><li>Klein, B. Y., 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=Oxytocin+opposes+effects+of+bacterial+endotoxin+on+ER-stress+signaling+in+Caco2BB+gut+cells.">Oxytocin opposes effects of bacterial endotoxin on ER-stress signaling in Caco2BB gut cells.</a> Biochimica et Biophysica Acta. 1860 (2), 402-411 (2016).</li><li>Kaltschmidt, B., Kaltschmidt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+in+the+nervous+system.">NF-kappaB in the nervous system.</a> Cold Spring Harbor Perspectives in Biology. 1 (3), a001271 (2009).</li></ol>

The authors have nothing to disclose.

A technique for microdissection of discrete, OTR-rich brain nuclei in the neonatal rat brain is presented in this paper. It is well recognized that neurons are highly specialized, even within well-characterized nuclei in the brain. This highly reproducible approach to isolate specific OTR-rich nuclei enables robust hypothesis testing. Using automated Western blotting, the consistency and reproducibility of the results were further improved. While a limitation of this technique remains modest brain punch variability; this...

In contrast to our understanding of early brain development occurring over the course of days-to-weeks postpartum, relatively little is known about the myriad of dynamic changes occurring in the first hours of life in rats. A key challenge has been the small size of the neonatal rat brain and a requirement for high-tech tools to isolate discrete brain regions or single cells. Studies often assess gene transcription and not translation1,2, which does not give a firm understanding of functional levels of activated signaling molecules. Others examine expression using immunohistochemistry to reference brain regions, which does not allow for the quantification of expression levels3. No study to date has examined the activation of signaling pathways associated with rats&#39; first colostrum feed in discrete brain regions, which requires rapid isolation and sacrifice and measurement of protein expression and protein phosphorylation using Western blotting. While brain microdissection is performed on older and larger brains, we have not identified a reference performing a non-single-cell brain punch in a P0 brain. This paper presents a protocol for isolating restricted regions of the neonatal brain using a relatively low-tech punch technique and a Western blotting procedure to measure protein expression in relatively small samples. This protocol may be suitable for research questions that require the assessment of protein expression and post-translational modifications (e.g., phosphorylation) in relatively restricted regions of small brains of any species, provided that the user can visually identify the brain region of interest with an atlas and identifiable landmarks.
This technique was developed to understand changes occurring in the brain as a result of the neonatal rats&#39; first colostrum feed, which is rich in oxytocin (OT). OT has been long known for its ability to stimulate milk let-down and uterine contraction. However, OT is now known to play a wide range of roles in the regulation of many bodily functions and behaviors4. For example, OT opposes stress and inflammation in conjunction with adaptive affiliative behaviors5, delays gastric emptying, and slows intestinal transit. OT receptors (OTR) have been identified in enteric neurons and intestinal epithelium6,7,8. The gastrointestinal effects of OT are particularly important to the infant during the early postnatal period. For instance, breastfeeding is associated with the delivery of significant quantities of OT to the neonatal gut9,10, and data show that the OTR is heavily overexpressed in duodenal villi during the milk suckling period8.
In vitro experiments using a gut cell line have demonstrated at the cellular level that oxytocin modulates important molecules in the stress signaling pathway11,12 and plays a regulatory role in translation of proteins12. These studies suggest that components of milk, including exogenous oxytocin from the mother, are important in the unfolded protein response in neonates to reduce cellular stress13.
In vivo and ex vivo studies have shown that colostrum OT modulates the cellular stress response, inflammation, and autophagy markers in newborn rat gut villi. Newborn enterocytes suffer substantial cellular stress on their luminal side when the gut is simultaneously exposed to microbiota from the mother in colostrum14,15 and numerous proteins, including hormones such as OT9,10,16.
The effects of OT on the brain have been studied17. However, the OT signaling mechanisms demonstrated in the gut during the early postnatal period have not been studied in the brain. In this paper, a method for isolating discrete brain nuclei in the neonatal rat brainstem and hypothalamus using electrophoresis is used to profile isolated brain regions. The overall goal of this method is to capture the state of cell signaling in brain areas as close as possible to birth, before and after the first milk suckling, in brain tissue with the lowest glial/neuronal index. The rationale for the development of this technique is that it allows for the rapid isolation of restricted, microscopic brain regions in neonatal pups with a more homogenous collection of neurons for ex vivo studies using an automated Western blotting methodology, offering highly consistent results on relatively small dissected samples. A shortcoming of prior work includes more gross dissection (brain slices or whole brain) and older animals18,19. The brains of young pups are incredibly dynamic, featuring waves of glial differentiation after birth. In order to study brain changes influenced by the pups&#39; first feeding, studying restricted neuronal nuclei with reproducible dissection is necessary.
Milk feed is usually analyzed for its immunological and nutritional impact on health or gene expression (for example, in enterocytes20,21), whereas its effect on brain areas during brain development is rarely studied. The effect of milk transit in the gut on brain function was analyzed in reference to gut cholecystokinin receptors vagal relay to brain stem nuclei, but not to intracellular signaling pathways22. There is a vast literature on vulnerability of the developing neonate brain to malnutrition of mothers during pregnancy23, but the stress and inflammation signals are not addressed. Importantly, the current method takes advantage of a phenomenon in day-zero rat newborns that isolates the blood-born colostrum stimuli from vagal relay of visceral stimuli. This is the so-called stress hypo-responsiveness period characterized by immature nucleus tractus solitarius (NTS)-hypothalamic circuit immediately after birth24,25 that restricts NTS, paraventricular nucleus (PVN), and supraoptic nucleus (SON) signals to blood-born stimuli.
This method is useful for analysis of multiple signaling pathways and relatively restricted to neuronal cells, provided that brain tissue is harvested at postnatal day-0 in rats, in addition to whether mothers have been challenged or not by any kind of treatment during pregnancy. Litters can be analyzed for the effects of colostrum feed versus pre-feeding signaling. When comparing signals between brain areas with poor versus rich protein yield, this method enables in-capillary determination of total protein of the polypeptide bands in capillaries run parallel to immune-quantitation of protein antigens. This method enables the quantitative comparison, using arbitrary units, of results obtained by the same antibody without standard quantitative curves and by reference to total protein per capillary. Comparing results obtained by different antibodies is possible only using quantitative standard curves.
This method allowed for the assessment of bidirectional signaling occurring between the gut and the brain and that can impact function in both organs26. The association between oxytocin and food intake, which has been extensively studied in recent years27, supports a link between increased oxytocin signaling and nutrient availability. These studies also support the converse concept that energy deficits are coupled with reductions in hypothalamic oxytocin signaling.
Earlier studies of the effect of OT on brain activity demonstrated that induced gut inflammation elicited cFos transcription in hypothalamic PVN, amygdala, and piriform cortex which was refractory to vagotomy28. However, systemic infusion of OT with secretin decreased the brain cFos response to the provoked inflammatory reaction in the gut28. This suggested that the effect of exogenous OT was carried out by routes other than vagal relays, possibly via blood-borne signaling molecules carried through the area postrema6,29.
In this study, the cellular stress signaling pathways that have previously observed in the gut were assessed in the brain. The hypothesis was that milk components may protect or defer the effect of inflammation on gut permeability to microbial and other metabolites, and in turn, the effects on brain function. The clear antagonistic differences in IkB versus BiP signaling found in villi, before and after priming by colostrum13, suggested that the brains of neonates, still in the process of developing, may sense these colostrum-induced gut signals.
Signaling protein markers used in previous gut experiments that are associated with endoplasmic reticulum stress were measured. They include the ER chaperone BiP, translation initiation factor eIF2a (which serves as a stress response integrator30), eIF2a kinase p-PKR, and two inflammation-signaling proteins (NF-kB and its inhibitor, IkB).
Six brain regions based on their ability in adults to secrete or respond to OT were chosen. The NTS, located at the upper medulla, is the first relay of the visceral input and receives direct signaling from vagal sensory neurons in the gut31 and possibly blood-born cytokines, toxins, and hormones via the adjacent area-postrema32. The PVN, supraoptic nucleus (SON), striatum nuclei (STR), cerebral cortex (CX), and medial preoptic nucleus (MPO) receive signaling from the gut via the NTS.
Results showed that the cellular stress response during the immediate postnatal period prior to colostrum priming and immediately after first feeding is different in NTS compared to PVN and SON. Signaling in CX, STR, and MPO differed from that of PVN and SON, as well. The distinct protective functions of OT shown previously to modulate cell stress and inflammation in the gut are likely sensed by some areas of the brain. Collectively, the data indicate that at the cellular level, during the first hours after birth, the brain responds to the metabolic stress associated with nutrient insufficiency. The data also show that the extent and direction of the modulating effects of the colostrum feed are region-dependent and that in some regions, they mirror OT effects shown previously in the gut.

<table><tbody><tr><td>Bradford solution</td><td>Bio Rad</td><td></td><td></td></tr><tr><td>Protein lysis kit</td><td>Protein simple</td><td>CBS403</td><td>Bicine/CHAPS</td></tr><tr><td>WES kits</td><td>Protein simple</td><td>WES-Mouse 12-230 master kit (PS-MK15), WES-Rabbit 12-230 master kit (PS-MK14), WES 12-230 kDa total Protein master kit (PS-TP07)</td><td></td></tr><tr><td>anti-mouse IgG HRP conjugate</td><td>Protein simple</td><td></td><td></td></tr><tr><td>Rabbit anti-phospho-eIF2a</td><td>Cell Signaling technology</td><td>SER51, 9721</td><td></td></tr><tr><td>mouse mAb anti-PKR</td><td>Cell Signaling technology</td><td>2103</td><td></td></tr><tr><td>Rabbit anti-phospho-PKR</td><td>Millipore</td><td>Thr451, 07-886</td><td></td></tr><tr><td>Rabbit mAb anti-PKR</td><td>Cell Signaling technology</td><td>12297</td><td></td></tr><tr><td>rabbit mAb anti-GAPDH</td><td>Cell Signaling technology</td><td>2118</td><td></td></tr><tr><td>mouse mAb anti-phospho-IKB</td><td>Cell Signaling technology</td><td>9246</td><td></td></tr><tr><td>mouse mAb anti-IKB</td><td>Cell Signaling technology</td><td>4814</td><td></td></tr><tr><td>rabbit anti-BiP</td><td>Cell Signaling technology</td><td>3183</td><td></td></tr><tr><td>Rabbit anti GCN2</td><td>Cell Signaling technology</td><td>3302</td><td></td></tr><tr><td>Rabbit mAb anti-phospho-GCN2</td><td>BIORBYT</td><td>T899</td><td></td></tr><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>

assessing cellular stress and inflammation in discrete oxytocin-secreting brain nuclei in the neonatal rat before and after first colostrum feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

The representative bands of immunoreactivity relative to total protein show that there are brain nuclei with very low harvested protein. This requires the use of the automated Western blot technique, which is highly sensitive compared to the canonical Western blot. This approach can be run with fortyfold less protein per capillary compared to the per-lane in Western blots.
Differential effects of colostrum priming on BiP levels in ...

Watch this Scientific Journal Video about Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding at JoVE.co

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

assessing-cellular-stress-inflammation-discrete-oxytocin-secreting

Research

JoVE Journal

Immunology and Infection

7.9K Views.  Columbia University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de postnatale ontwikkeling veld over de manier waarop melk zogen beïnvloedt stress signalering in de hersenen. Deze techniek kan worden gebruikt om specifieke hersengebieden vast te leggen tijdens de eerste natuurlijke hongerperiode en om ze te vergelijken met de respectieve gebieden kort na de eerste natuurlijke biest feed. Begrijpen hoe zogen de fysiologie van gedrag en emotie beïnvloedt, maakt het gebruik van een anta...

Video: Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

Behavior

Neurodevelopmental Reflex Testing in Neonatal Rat Pups

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are also referred to as primitive reflexes. They are sensitive and consistent with later outcomes. Abnormal reflexes are described as an absence, persistence, reappearance, or latency of reflexes, which are predictive indices of infants that are at high risk for neurodevelopmental disorders. Animal models of neurodevelopmental disabilities, such as cerebral palsy, often display aberrant developmental reflexes, as would be observed in human infants. The techniques described assess a variety of neurodevelopmental reflexes in neonatal rats. Neurodevelopmental reflex testing offers the investigator a testing method that is not otherwise available in such young animals. The methodology presented here aims to assist investigators in examining developmental milestones in neonatal rats as a method of detecting early-onset brain injury and/or determining the effectiveness of therapeutic interventions. The methodology presented here aims to provide a general guideline for investigators.

Behavioral testing is the gold standard for determining outcomes following brain injury, and can identify the presence of developmental disabilities in infants and children. Neurodevelopmental reflexes are an early indicator of these abnormalities. A host of easily accomplished developmental reflex tests in the neonatal rodent were developed and described here.

Neurodevelopmental reflex testing is commonly used in clinical practice to assess the maturation of the nervous system. Neurodevelopmental reflexes are ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Neuroscience

Assessment of Stress Effects on Cognitive Flexibility using an Operant Strategy Shifting Paradigm

Stress affects cognitive function. Whether stress enhances or impairs cognitive function depends on several factors, including the 1) type, intensity, and duration of the stressor; 2) type of cognitive function under study; and 3) timing of the stressor in relation to learning or executing the cognitive task. Furthermore, sex differences among the effects of stress on cognitive function have been widely documented. Described here is an adaptation of an automated operant strategy shifting paradigm to assess how variations in stress affect cognitive flexibility in male and female Sprague Dawley rats. Specifically, restraint stress is used before or after training in this operant-based task to examine how stress affects cognitive performance in both sexes. Particular brain areas associated with each task in this automated paradigm have been well-established (i.e., the medial prefrontal cortex and orbitofrontal cortex). This allows for targeted manipulations during the experiment or the assessment of particular genes and proteins in these regions upon completion of the paradigm. This paradigm also allows for the detection of different types of performance errors that occur after stress, each of which has defined neural substrates. Also identified are distinct sex differences in perseverative errors after a repeated restraint stress paradigm. The use of these techniques in a preclinical model may reveal how stress affects the brain and impairs cognition in psychiatric disorders, such as post-traumatic stress disorder (PTSD) and major depressive disorder (MDD), which display marked sex differences in prevalence.

Stressful life events impair cognitive function, increasing the risk of psychiatric disorders. This protocol illustrates how stress affects cognitive flexibility using an automated operant strategy shifting paradigm in male and female Sprague Dawley rats. Specific brain areas underlying particular behaviors are discussed, and translational relevance of results are explored.

Stress affects cognitive function. Whether stress enhances or impairs cognitive function depends on several factors, including the 1) type, intensity, and ...

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and offspring, including the development of neuropsychiatric disorders. Risk factors linked to the emergence of affective dysregulation in the maternal-infant dyad have been extensively studied. Exposure to psychosocial stress during pregnancy has consistently emerged as one of the strongest predictors. Several rodent models have been created to explore this association; however, these models rely on the use of physical stressors or a limited number of psychosocial stressors presented in a repetitive fashion, which do not accurately capture the type, intensity, and frequency of stressors experienced by women. To overcome these limitations, a chronic psychosocial stress (CGS) paradigm was generated that employs various psychosocial insults of different intensity presented in an unpredictable fashion. The manuscript describes this novel CGS paradigm where pregnant female mice, from gestational day 6.5 to 17.5, are exposed to various stressors during the day and overnight. Day stressors, two per day separated by a 2 h break, range from exposure to foreign objects or predator odor to frequent changes in bedding, removal of bedding, and cage tilting. Overnight stressors include continuous light exposure, changing cage mates, or wetting bedding. We have previously shown that exposure to CGS results in the development of maternal neuroendocrine and behavioral abnormalities, including increased stress reactivity, the emergence of fragmented maternal care patterns, anhedonia, and anxiety-related behaviors, core features of women suffering from perinatal mood and anxiety disorders. This CGS model, therefore, becomes a unique tool that can be used to elucidate molecular defects underlying maternal affective dysregulation, as well as trans-placental mechanisms that impact fetal neurodevelopment and result in negative long-term behavioral consequences in the offspring.

The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...

Mouse Maternal Autonomic Changes from Pregnancy to Pup-Rearing

Analysis of Electrocardiograms and Behavior in Mice from Pregnancy to Lactation Period

Changes in the mother-offspring relationship are presumably accompanied by dynamic changes in the autonomic nervous system. Although temporal measurements of autonomic activity have been performed in human mothers and infants, the analysis of long-term changes remains unexplored. Mouse mothers can form social bonds with their pups and have a short period of pregnancy and lactation, which makes them useful for the examination of physiological changes from pregnancy to pup-rearing. Therefore, a telemetry system was used for several weeks to measure the changes in the autonomic nervous system and the behavior of mouse mothers. The current results showed that an electrocardiogram (ECG) could be stably recorded regardless of the movements of mothers and parturition. ECG analysis showed that the heart rate gradually decreased from pregnancy to lactation, and sympathetic activity sharply increased as the pups developed. Furthermore, the simultaneous recording of behavior and ECG in the home cage enabled us to understand the behavior-dependent influences on the ECG, thereby revealing the characteristics of autonomic nervous activity during each behavior. Thus, the present experimental method helps to understand how the physiological characteristics of mothers change from pregnancy through pup rearing, supporting the healthy development of pups.

A simultaneous recording of autonomic activity and detailed maternal behavior of mother mice from pregnancy to lactation was achieved using a telemetry system. This method helps to understand the dynamics of the physiological and behavioral characteristics in mothers from pregnancy to weaning.

Changes in the mother-offspring relationship are presumably accompanied by dynamic changes in the autonomic nervous system. Although temporal measurements ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

Medicine

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Biology

Creating a Preterm Rat Model for Early Postnatal Pain Research

A Preterm Rat Model for Pain Studies

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain sensitivity. The primary objective of this protocol was to investigate the impact of neonatal pinprick stimuli on the pain threshold in the later stages of life using a preterm rat model. By establishing this model, we aim to advance the research on understanding and managing early postnatal pain associated with prematurity. The findings of this study indicate that while the baseline thresholds to mechanical stimuli remained unaffected, there was a notable increase in mechanical hypersensitivity following complete Freund's adjuvant (CFA) injection in adult rats. Interestingly, compared with male rats, female rats demonstrated heightened inflammatory hypersensitivity. Notably, maternal behavior, the weight of the litters, and the growth trajectory of the offspring remained unchanged by the stimulation. The manifestation of altered nociceptive responses in adulthood after neonatal painful stimuli could be indicative of changes in sensory processing and the functioning of glucocorticoid receptors. However, further research is needed to understand the underlying mechanisms involved and to develop interventions for the consequences of prematurity and neonatal pain in adults.

Here, we present a concise protocol for creating a preterm rat model, facilitating research on early postnatal pain management. The method involves performing a cesarean section three days before the expected birth, extracting preterm rat pups via hysterectomy, and integrating them with the biological offspring of a surrogate dam.

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain ...

Modeling Early-Life Adversity in Mice Using Limited Bedding and Nesting

Limited Bedding and Nesting as a Model for Early-Life Adversity in Mice

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing neuropsychiatric disorders such as depression. Animal models for ELA have been used to study the impact of chronic stress on brain development, and typically rely on manipulating the quality and/or quantity of maternal care, as this is the major source of early-life experiences in mammals, including humans. Here, a detailed protocol for employing the Limited Bedding and Nesting (LBN) model in mice is provided. This model mimics a low-resource environment, which provokes fragmented and unpredictable patterns of maternal care during a critical developmental window (postnatal days 2-9) by limiting the amount of nesting materials given to the dam to build a nest for her pups and separating the mice from the bedding via a mesh platform in the cage. Representative data are provided to illustrate the changes in maternal behavior, as well as the diminished pup weights and long-term changes in basal corticosterone levels, that result from the LBN model. As adults, offspring reared&#160;in the LBN environment have been shown to exhibit an aberrant stress response, cognitive deficits, and anhedonia-like behavior. Therefore, this model is an important tool to define how the maturation of stress-sensitive brain circuits is altered by ELA and results in long-term behavioral changes that confer vulnerability to mental disorders.

Author Spotlight: Exploring Microglial Interactions with Stress-Response Circuitry Using the Limited Bedding and Nesting Model

This protocol describes an animal model for studying how early-life adversity, provoked by an impoverished environment and unpredictable maternal care during the early postnatal period, affects brain development and the future risk of mental disorders.

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

Samenvatting

Meer video's verkennen