Name: quantifying acute changes in renal sympathetic nerve activity in response to central nervous system manipulations in anesthetized rats
Uploaded: 2018-09-11T14:00:00
Description: Watch this Scientific Journal Video about Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats at JoVE.com

This study was supported by the National Institute for Nursing Research (K99/R00NR014369).

All methods described were approved by the Institutional Animal Care Committee at the University of Illinois at Chicago.
1. Create Bipolar RSNA Electrodes
<ol>
	<li>To create the electrode, cut two pieces of stainless steel wire each approximately 18 mm long. Cut one piece of polyethylene (PE-50) tubing approximately 15 mm long. Feed both pieces of wire into the tubing, leaving the wire protruding from both ends.</li>
	<li>Remove the insulation from the ends of the wires; trim the wires leav...

<ol>
	<li>Mancia, G., Grassi, 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+autonomic+nervous+system+and+hypertension.">The autonomic nervous system and hypertension.</a> Circulation Research. 114 (11), 1804-1814 (2014).</li><li>Kannan, A., Medina, R. I., Nagajothi, N., Balamuthusamy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Renal+sympathetic+nervous+system+and+the+effects+of+denervation+on+renal+arteries.">Renal sympathetic nervous system and the effects of denervation on renal arteries.</a> World Journal of Cardiology. 6 (8), 814-823 (2014).</li><li>Johns, E. J., Kopp, U. C., DiBona, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+control+of+renal+function.">Neural control of renal function.</a> Comprehensive Physiology. 1 (2), 767 (2011).</li><li>Fink, A. M., Dean, C., Piano, M. R., Carley, D. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Afferent+renal+denervation+impairs+baroreflex+control+of+efferent+renal+sympathetic+nerve+activity.">Afferent renal denervation impairs baroreflex control of efferent renal sympathetic nerve activity.</a> American Journal of Physiology, Regulatory, Integrative and Comparative Physiology. 295 (6), R1882-R1890 (2008).</li><li>Guild, S. 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=Quantifying+sympathetic+nerve+activity:+problems,+pitfalls+and+the+need+for+standardization.">Quantifying sympathetic nerve activity: problems, pitfalls and the need for standardization.</a> Experimental Physiology. 95 (1), 41-50 (2010).</li><li>Stocker, S. D., Hunwick, K. J., Toney, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+paraventricular+nucleus+differentially+supports+lumbar+and+renal+sympathetic+outflow+in+water-deprived+rats.">Hypothalamic paraventricular nucleus differentially supports lumbar and renal sympathetic outflow in water-deprived rats.</a> Journal of Physiology. 15 (563 Pt 1), 249-263 (2005).</li><li>Stocker, S. D., Gordon, K. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptors+in+the+hypothalamic+paraventricular+nucleus+contribute+to+insulin-induced+sympathoexcitation.">Glutamate receptors in the hypothalamic paraventricular nucleus contribute to insulin-induced sympathoexcitation.</a> Neurophysiology. 113 (5), 1302-1309 (2015).</li><li>Saponjic, J., Radulovacki, M., Carley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+glutamate+into+the+pedunculopontine+tegmental+nuclei+of+anesthetized+rat+causes+respiratory+dysrhythmia+and+alters+EEG+and+EMG+power.">Injection of glutamate into the pedunculopontine tegmental nuclei of anesthetized rat causes respiratory dysrhythmia and alters EEG and EMG power.</a> Sleep and Breathing. 9 (2), 82-91 (2005).</li><li>DiBona, G. F., Jones, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+renal+nerve+activity+responses+to+baroreceptor+denervation+in+hypertensive+rats.">Dynamic analysis of renal nerve activity responses to baroreceptor denervation in hypertensive rats.</a> Hypertension. 37 (4), 1153-1163 (2001).</li><li>Kunitake, T., Kannan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discharge+pattern+of+renal+sympathetic+nerve+activity+in+the+conscious+rat:+spectral+analysis+of+integrated+activity.">Discharge pattern of renal sympathetic nerve activity in the conscious rat: spectral analysis of integrated activity.</a> Journal of Neurophysiology. 84 (6), 2859-2867 (2000).</li><li>Machado, B. H., Bonagamba, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+L-glutamate+into+the+nucleus+tractus+solitarii+increases+arterial+pressure+in+conscious+rats.">Microinjection of L-glutamate into the nucleus tractus solitarii increases arterial pressure in conscious rats.</a> Brain Research. 576 (1), 131-138 (1992).</li><li>Murakami, 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=Inhalation+anesthesia+is+preferable+for+recording+rat+cardiac+function+using+an+electrocardiogram.">Inhalation anesthesia is preferable for recording rat cardiac function using an electrocardiogram.</a> Biological and Pharmaceutical Bulletin. 37 (5), 834-839 (2014).</li><li>Nakamura, T., Tanida, M., Niijima, A., Hibino, H., Shen, J., Nagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+stimulation+affects+renal+sympathetic+nerve+activity+and+blood+pressure+in+rats.">Auditory stimulation affects renal sympathetic nerve activity and blood pressure in rats.</a> Neuroscience Letters. 416 (2), 107-112 (2007).</li><li>Bardgett, M. E., McCarthy, J. J., Stocker, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+receptor+activation+in+the+rostral+ventrolateral+medulla+mediates+the+sympathoexcitatory+response+to+hyperinsulinemia.">Glutamatergic receptor activation in the rostral ventrolateral medulla mediates the sympathoexcitatory response to hyperinsulinemia.</a> Hypertension. 55 (2), 284-290 (2010).</li><li>Brozoski, D. T., Dean, C., Hopp, F. A., Seagard, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+blockade+of+endocannabinoids+in+the+NTS+modulates+baroreflex-evoked+sympathoinhibition.">Uptake blockade of endocannabinoids in the NTS modulates baroreflex-evoked sympathoinhibition.</a> Brain Research. 1059 (2), 197-202 (2005).</li><li>Barman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+can+we+learn+about+neural+control+of+the+cardiovascular+system+by+studying+rhythms+in+sympathetic+nerve+activity?.">What can we learn about neural control of the cardiovascular system by studying rhythms in sympathetic nerve activity?.</a> International Journal of Psychophysiology. 103, 69-78 (2016).</li><li>Charkoudian, N., Wallin, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+neural+activity+to+the+cardiovascular+system:+integrator+of+systemic+physiology+and+interindividual+characteristics.">Sympathetic neural activity to the cardiovascular system: integrator of systemic physiology and interindividual characteristics.</a> Comprehensive Physiology. 4 (2), 825-850 (2014).</li><li>Malpas, S. 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Critical steps for measuring RSNA include: (1) avoiding stretching of the renal artery and nerves when separating the kidney from the paraspinal muscle and when placing the nerve segment on the recording electrodes, (2) carefully dissecting the renal nerve fibers from the surrounding tissue/vessel, (3) ensuring that the electrode wires are free of tissue, blood, or lymph fluid, and (4) preventing the nerve from drying out by applying mineral oil to the renal nerve and silica gel to the nerve-electrode unit. For troublesh...

Pre-clinical discoveries about autonomic control of the cardiovascular system inform strategies for managing disorders such as hypertension, heart failure, and chronic kidney disease. Over-activity of the sympathetic nervous system and reduced vagal cardiac tone contribute to elevated blood pressure (BP)1. Chronically elevated renal sympathetic outflow enhances catecholamine secretion and decreases renal blood flow, with deleterious consequences to the cardiovascular/renal systems2,3. To define the neurobiological pathways leading to autonomic dysfunction, studies in rodents are important for determining how central nervous system (CNS) neurons regulate sympathetic parameters. The purpose of this protocol is to provide technical information about measuring renal sympathetic nerve activity (RSNA) and BP and to outline the techniques for quantifying acute sympathetic changes in response to CNS manipulations in anesthetized rats.
Acute (non-survival) RSNA measurements (lasting min to h) are useful when scientists will probe the CNS pharmacologically, electrically, or optogenetically in anesthetized rats to determine the functions of specific nuclei. Using these methods, structures such as the solitary nucleus, periaqueductal gray, pedunculopontine tegmentum, and rostral ventrolateral medulla have been investigated to define neurobiological pathways regulating sympathetic parameters4,5,6,7. This approach is important for identifying CNS targets to be investigated further in chronic models of autonomic dysfunction8,9. To complete these experiments, the laboratory requires a soldering iron, surgical microscope, stereotaxic frame, microelectrode amplifier, and audio monitor. Depending on the factors present in the laboratory that contribute to electrical noise, the surgical/recording area may require a Faraday cage/grounding strap to reduce electrical noise in the RSNA recording. If brain analyses will require tissue fixation, a perfusion pump and fume hood are required. Data can be digitized and recorded using multiple physiologic software/data acquisition (analogue-digital converter) units4,5, with different analysis options and compatibilities for incorporating telemetric signals.

<table>
	<tbody>
		<tr>
			<td>Stainless steel wire</td>
			<td>A-M Systems; Sequim, WA</td>
			<td>791000</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Polyethylene (PE-50) tubing</td>
			<td>VWR; Radnor, PA</td>
			<td>63019-048</td>
			<td>RSNA electrode; vessel cannulation</td>
		</tr>
		<tr>
			<td>Miniature pin connector</td>
			<td>A-M Systems; Sequim, WA</td>
			<td>520200</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Crimping tool</td>
			<td>Daniels Manufacturing Corp.; Orlando, FL</td>
			<td>M22520</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Connector strip</td>
			<td>Amphenol; Clinton Township, MI</td>
			<td>221-2653</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>J-B Kwik Epoxy</td>
			<td>J-B Weld, Sulphur Springs, TX</td>
			<td>8270</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Silicone</td>
			<td>Permatex; Hartford, CT</td>
			<td>2222</td>
			<td>RSNA electrode</td>
		</tr>
		<tr>
			<td>Heparin sodium; Injectable (10 mL vial, 1000 U/mL)</td>
			<td>KV Veterinary Supply; David City, NE</td>
			<td>P03466</td>
			<td>Venous line patency</td>
		</tr>
		<tr>
			<td>Phenylephrine HCl; Injectable (1 mL vial; 10 mg/mL)</td>
			<td>ACE Surgical Supply; Brockton, MA</td>
			<td>950-6312</td>
			<td>Testing renal sympathoinhibition</td>
		</tr>
		<tr>
			<td>Single-hook elastic surgical stays</td>
			<td>Harvard Apparatus; Holliston, MA</td>
			<td>72-2595</td>
			<td>Incision</td>
		</tr>
		<tr>
			<td>Silk surgical tape</td>
			<td>3M, Minneapolis, MN</td>
			<td>1538-0</td>
			<td>Secure surgical stays</td>
		</tr>
		<tr>
			<td>Needles, 20 G</td>
			<td>Sigma-Aldrich; St. Louis, MO</td>
			<td>Z192554-100EA</td>
			<td>Vessel cannulation</td>
		</tr>
		<tr>
			<td>Dumont #7 curved forceps</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>11274-20</td>
			<td>Vessel cannulation</td>
		</tr>
		<tr>
			<td>5-0 silk suture ties</td>
			<td>Braintree Scientific; Braintree, MA</td>
			<td>SUT-S 106</td>
			<td>Vessel cannulation</td>
		</tr>
		<tr>
			<td>Delicate hemostatic forceps</td>
			<td>Roboz Surgical Instrument Co.; Gaithersburg, MD</td>
			<td>RS-7117</td>
			<td>Vessel cannulation and RSNA surgery</td>
		</tr>
		<tr>
			<td>Crile Hemostatic forceps</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>13004-14</td>
			<td>Needle bending</td>
		</tr>
		<tr>
			<td>Telemetry transmitter</td>
			<td>Data Sciences International; Minneapolis, MN</td>
			<td>PA-10</td>
			<td>Mean arterial pressure monitoring (telemetry)</td>
		</tr>
		<tr>
			<td>Re-gel syringe</td>
			<td>Data Sciences International; Minneapolis, MN</td>
			<td>276-0038-001</td>
			<td>Transmitter reuse (telemetry)</td>
		</tr>
		<tr>
			<td>Disposable pressure transducer</td>
			<td>Transpac; San Clemente, CA</td>
			<td>MI-1224</td>
			<td>Mean arterial pressure monitoring</td>
		</tr>
		<tr>
			<td>Clear-Cuff pressure infuser</td>
			<td>MILA International Inc.; Florence, KY</td>
			<td>2281339</td>
			<td>Mean arterial pressure monitoring</td>
		</tr>
		<tr>
			<td>Vessel cannulation forceps</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>00574-11</td>
			<td>Catheter insertion</td>
		</tr>
		<tr>
			<td>Black monofilament nylon 4-0 suture on reverse cutting needle</td>
			<td>McKesson Medical-Surgical; San Francisco, CA</td>
			<td>S661GX</td>
			<td>Secure telemetry transmitter</td>
		</tr>
		<tr>
			<td>Telemetry receiver</td>
			<td>Data Sciences International; Minneapolis, MN</td>
			<td>RPC-1</td>
			<td>Mean arterial pressure monitoring (telemetry)</td>
		</tr>
		<tr>
			<td>LabChart Pro (software), PowerLab (acquisition hardware)</td>
			<td>AD Instruments; Colorado Springs, CO</td>
			<td>ML846, MX2 matrix 2.0 (Compatible with Data Science International telemetry)</td>
			<td>3 options for software/acquisition hardware</td>
		</tr>
		<tr>
			<td>SciWorks (software), DataWave (acquisition hardware)</td>
			<td>DataWave Technologies, Loveland, CO</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Spike 2 (software), Micro1401-3</td>
			<td>Cambridge Electronic Design Ltd., London UK</td>
			<td>1401-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-drill</td>
			<td>Roboz Surgical Instrument Co.; Gaithersburg, MD</td>
			<td>RS-6300</td>
			<td>CNS surgery</td>
		</tr>
		<tr>
			<td>Stereotaxic surgery frame</td>
			<td>Stoelting; Wood Dale, IL</td>
			<td>51600</td>
			<td>CNS surgery</td>
		</tr>
		<tr>
			<td>Microelectrode amplifier with 10X pre-amplifier</td>
			<td>A-M Systems; Sequim, WA</td>
			<td>1800-2</td>
			<td>RSNA recording</td>
		</tr>
		<tr>
			<td>Retractors</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>17009-07</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Micro-dissecting tweezers</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>11251-10</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Micro-hook</td>
			<td>Fine Science Tools; Foster City, CA</td>
			<td>10064-14</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Fisher Scientific; Waltham, MA</td>
			<td>8042-47-5</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Audio monitor</td>
			<td>A-M Systems; Sequim, WA</td>
			<td>3300</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Wacker, Munchen; Germany</td>
			<td>RT601A-B</td>
			<td>RSNA surgery</td>
		</tr>
		<tr>
			<td>Electrical clips</td>
			<td>Tyco Electronics; Schaffhausen, Switzerland</td>
			<td>EB0283-000</td>
			<td>Grounding or securing perfusion needle</td>
		</tr>
		<tr>
			<td>Bonn scissors, straight/sharp points</td>
			<td>Roboz Surgical Instrument Co; Gaithersburg, MD</td>
			<td>RS-5840</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Gavage needle</td>
			<td>Harvard Apparatus; Holliston, MA</td>
			<td>75-0286</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Masterflex perfusion pump</td>
			<td>Cole-Parmer; Vernon Hills, IL</td>
			<td>7524-10</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Masterflex platinum-cured silicone tubing</td>
			<td>Cole-Parmer; Vernon Hills, IL</td>
			<td>96410-15</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Formalin (10% buffered solution; 4 L)</td>
			<td>Sigma-Aldrich; St. Louis, MO</td>
			<td>HT501128</td>
			<td>Perfusion</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich; St. Louis, MO</td>
			<td>S0389</td>
			<td>Cryoprotection</td>
		</tr>
	</tbody>
</table>

quantifying acute changes in renal sympathetic nerve activity in response to central nervous system manipulations in anesthetized rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Figure 1 illustrates a sample RSNA and BP recording from a Nembutal-anesthetized rat. An intravenous injection of phenylephrine was used to induce an increase in mean arterial pressure and to evoke the baroreflex and transient sympathoinhibition4,6. To quantify RSNA, the raw RSNA was rectified and averaged for non-overlapping 10 s segments; the noise estimate was subtracted from each segment.
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Watch this Scientific Journal Video about Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats at JoVE.com

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

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