Name: surgery and behavioral testing in the tibial neuroma transposition model in rats
Uploaded: 2023-01-06T13:40:00
Description: Watch this Scientific Journal Video about Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats at JoVE.com

We would like to thank Sabine Versteeg for assisting during microsurgery and Anja van der Sar and Trudy Oosterveld-Romijn from the Common Animal Laboratory (Gemeenschappelijk Dieren Laboratorium) for their help in preparing the microscope and surgical room and taking care of the animals.
This research was funded by Axogen.

This research was performed in accordance with the IVD (Instantie voor Dierenwelzijn Utrecht) and the guidelines for animal research, project number AVD1150020198824.
1. Von Frey baseline measurements
<ol>
	<li>Prior to the surgery, perform baseline measurements according to the Von Frey testing procedure, described below in section 5 and section 6.</li>
</ol>
2. Anesthesia and preparation
NOTE: This study wa...

<ol>
	<li>Stokvis, A., vander Avoort, D. J., van Neck, J. W., Hovius, S. E., Coert, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+management+of+neuroma+pain:+a+prospective+follow-up+study.">Surgical management of neuroma pain: a prospective follow-up study.</a> Pain. 151 (3), 862-869 (2010).</li><li>Domeshek, 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=Surgical+treatment+of+neuromas+improves+patient-reported+pain,+depression,+and+quality+of+life.">Surgical treatment of neuromas improves patient-reported pain, depression, and quality of life.</a> Plastic and Reconstructive Surgery. 139 (2), 407-418 (2017).</li><li>Lame, I. E., Peters, M. L., Vlaeyen, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroma+formation+following+digital+amputations.">Neuroma formation following digital amputations.</a> Journal of Trauma. 23 (2), 136-142 (1983).</li><li>Bowen, J. B., Ruter, D., Wee, C., West, J., Valerio, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+muscle+reinnervation+technique+in+below-knee+amputation.">Targeted muscle reinnervation technique in below-knee amputation.</a> Plastic and Reconstructive Surgery. 143 (1), 309-312 (2019).</li><li>Jensen, T. 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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=Diagnostic+criteria+for+symptomatic+neuroma.">Diagnostic criteria for symptomatic neuroma.</a> Annals of Plastic Surgery. 82 (4), 420-427 (2019).</li><li>Liedgens, H., Obradovic, M., De Courcy, J., Holbrook, T., Jakubanis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+burden+of+illness+study+for+neuropathic+pain+in+Europe.">A burden of illness study for neuropathic pain in Europe.</a> Clinicoeconomics and Outcomes Research. 8, 113-126 (2016).</li><li>Langley, P. C., Van Litsenburg, C., Cappelleri, J. C., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+burden+associated+with+neuropathic+pain+in+Western+Europe.">The burden associated with neuropathic pain in Western Europe.</a> Journal of Medical Economics. 16 (1), 85-95 (2013).</li><li>Dworkin, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+the+clinical+importance+of+group+differences+in+chronic+pain+clinical+trials:+IMMPACT+recommendations.">Interpreting the clinical importance of group differences in chronic pain clinical trials: IMMPACT recommendations.</a> Pain. 146 (3), 238-244 (2009).</li><li>Mackinnon, S. E., Dellon, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+of+treatment+of+recurrent+dorsoradial+wrist+neuromas.">Results of treatment of recurrent dorsoradial wrist neuromas.</a> Annals of Plastic Surgery. 19 (1), 54-61 (1987).</li><li>Harden, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neuropathic+pain.+Mechanisms,+diagnosis,+and+treatment.">Chronic neuropathic pain. Mechanisms, diagnosis, and treatment.</a> Neurologist. 11 (2), 111-122 (2005).</li><li>Poppler, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+interventions+for+the+treatment+of+painful+neuroma:+a+comparative+meta-analysis.">Surgical interventions for the treatment of painful neuroma: a comparative meta-analysis.</a> Pain. 159 (2), 214-223 (2018).</li><li>Eberlin, K. R., Ducic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+algorithm+for+neuroma+management:+a+changing+treatment+paradigm.">Surgical algorithm for neuroma management: a changing treatment paradigm.</a> Plastic and Reconstructive Surgery Global Open. 6 (10), 1952 (2018).</li><li>Dorsi, M. 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=The+tibial+neuroma+transposition+(TNT)+model+of+neuroma+pain+and+hyperalgesia.">The tibial neuroma transposition (TNT) model of neuroma pain and hyperalgesia.</a> Pain. 134 (3), 320-334 (2008).</li><li>Tork, S., 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=Application+of+a+porcine+small+intestine+submucosa+nerve+cap+for+prevention+of+neuromas+and+associated+pain.">Application of a porcine small intestine submucosa nerve cap for prevention of neuromas and associated pain.</a> Tissue Engineering Part A. 26 (9-10), 503-511 (2020).</li><li>Miyazaki, R., Yamamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+morphine,+pregabalin,+gabapentin,+and+duloxetine+on+mechanical+allodynia+is+different+from+that+on+neuroma+pain+in+the+rat+neuropathic+pain+model.">The efficacy of morphine, pregabalin, gabapentin, and duloxetine on mechanical allodynia is different from that on neuroma pain in the rat neuropathic pain model.</a> Anesthesia and Analgesia. 115 (1), 182-188 (2012).</li><li>Tian, 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=Swimming+training+reduces+neuroma+pain+by+regulating+neurotrophins.">Swimming training reduces neuroma pain by regulating neurotrophins.</a> Medicine and Science in Sports Exercise. 50 (1), 54-61 (2018).</li><li>Decosterd, I., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spared+nerve+injury:+an+animal+model+of+persistent+peripheral+neuropathic+pain.">Spared nerve injury: an animal model of persistent peripheral neuropathic pain.</a> Pain. 87 (2), 149-158 (2000).</li><li>Poublon, A. R., 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=The+anatomical+relationship+of+the+superficial+radial+nerve+and+the+lateral+antebrachial+cutaneous+nerve:+A+possible+factor+in+persistent+neuropathic+pain.">The anatomical relationship of the superficial radial nerve and the lateral antebrachial cutaneous nerve: A possible factor in persistent neuropathic pain.</a> Journal of Plastic, Reconstructive and Aesthetic Surgery. 68 (2), 237-242 (2015).</li><li>Dixon, 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=Efficient+analysis+of+experimental+observations.">Efficient analysis of experimental observations.</a> Annual Review of Pharmacology and Toxicology. 20 (1), 441-462 (1980).</li><li>Austin, P. J., Wu, A., Moalem-Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+constriction+of+the+sciatic+nerve+and+pain+hypersensitivity+testing+in+rats.">Chronic constriction of the sciatic nerve and pain hypersensitivity testing in rats.</a> Journal of Visualized Experiments. (61), e3393 (2012).</li></ol>

Critical steps in the protocol 
The TNT model involves cutting the tibial nerve and transposing it laterally and subcutaneously to a pretibial location to enable sensitivity testing of the neuroma, in addition to plantar hyperalgesia over the sural nerve. In the TNT model, it is key that the place of the neuroma is visible to the researchers. Therefore, an albino rat strain is preferred because subcutaneous sutures are easily visible through the skin and the color of the suture should preferably be ...

Every wound, varying from simple lacerations to whole limb amputation, is accompanied by varying degrees of peripheral nerve injury. Such nerve injury can result in the formation of a neuroma, a disorganized entanglement of sprouting nerve fibers. Neuromas become painful in 8%-30% of patients, severely impacting their quality of life1,2,3,4,5. After limb amputation, neuroma pain develops in 50% of patients6,7,8. Reported symptoms include tenderness, spontaneous pain, allodynia, hyperalgesia, and mechanical or thermal hypersensitivity in the innervated area9. When not treated adequately within 1 year, neuroma pain can advance to a chronic pain state, resulting in high societal burden and associated medical costs10,11,12,13,14. Due to the poor efficacy of current pharmacological interventions, neuroma pain is preferably treated by surgical removal of the painful neuroma, and the nerve treated by various surgical techniques, as described in the literature15. It is important to note that complete pain relief is rare, pain often worsens over time, and 40% of patients do not benefit from the surgery, indicating that new treatments are needed1,16.
A standardized rat model of neuroma pain aids in understanding the mechanisms that drive neuroma pain, and may help identify new treatments or evaluate existing ones used in the clinic. The tibial neuroma transposition (TNT) model was first described by Dorsi et al. in 200817 and has been used by different research groups18,19,20. The overall goal of this method is to be able to test different treatment techniques for neuroma pain. The advantage of the model over, for example, the spared nerve injury (SNI) model21, is that it allows to test allodynia at the neuroma site. This is because the model involves transposing the proximal nerve ending of the tibial nerve to a subcutaneous pretibial position, where it can be probed with von Frey monofilaments. Moreover, allodynia develops at the plantar surface of the hind paw innervated by the intact sural nerve, which can be assessed independently from the neuroma pain in the same animal. This is similar to symptoms of neuroma pain in patients, where persistent neuropathic pain after removal of a painful neuroma is sometimes caused by the neighboring nerves22. Moreover, allodynia over a severed nerve with a neuroma is a different pain modality than allodynia over the intact neighboring nerve. Thus, this model facilitates assessment of the effect of new therapies on both allodynia present at the neuroma site and more widespread neuropathic pain tested in the plantar surface of the hind paw. As the surgery performed to create the TNT model can be challenging, this paper elaborates on the procedure to support researchers implementing the model in their facility.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aesthesio</td>
			<td>Linton Instrumentation</td>
			<td>514007 until 514015</td>
			<td>0.6 g until 15 g monofilaments</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Local Veterinary Pharmacy</td>
			<td>n/a</td>
			<td>The local veterinary pharmacy makes caprofen dilution</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Nobamed</td>
			<td>974255</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrocautery</td>
			<td>Fine Science Tools</td>
			<td>18010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Interchema BV</td>
			<td>400406</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethilon 4.0</td>
			<td>Johnson &#38; Johnson</td>
			<td>1854G</td>
			<td>IMPORTANT: the color should be blue or black</td>
		</tr>
		<tr>
			<td>Ethilon 8.0</td>
			<td>Johnson &#38; Johnson</td>
			<td>BV130-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflo, isoflurane Zoetis</td>
			<td>Dechra Veterinary Products</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh bottom cages</td>
			<td>StoeltingCo</td>
			<td>57816 and 57824</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder&#160;</td>
			<td>Fine Science Tools</td>
			<td>12076-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>Fine Science Tools</td>
			<td>15019-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tweezers</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Trademed</td>
			<td>H7 1000-FRE</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools</td>
			<td>12004-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment&#160;</td>
			<td>Local Veterinary Pharmacy</td>
			<td>n/a</td>
			<td>The local veterinary pharmacy makes the ophthalmic ointment</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo surgical microscope</td>
			<td>Leica</td>
			<td>A60 F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile sheet with hole</td>
			<td>Evercare OneMed</td>
			<td>1555-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade nr.15</td>
			<td>Fine Science Tools</td>
			<td>10015-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Fine Science Tools</td>
			<td>11617-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgery and behavioral testing in the tibial neuroma transposition model in rats

The tibial neuroma transposition (TNT) is a rat model in which allodynia at the neuroma site (tibial nerve) can be independently evaluated from allodynia at the plantar surface of the hind paw innervated by the intact sural nerve. This TNT model is suitable to test therapies for neuroma pain, such as the potential superiority of certain surgical therapies that are already used in the clinic, or to evaluate new drugs and their effect on both pain modalities in the same animal. In this model, a distal lesion (neurotmesis) is made in the tibial nerve, and the proximal nerve end is transposed and fixed subcutaneously and pretibially to enable assessments of the neuroma site with a 15 g Von Frey monofilament. To assess allodynia over the sural nerve, Von Frey monofilaments can be used via the up-down method on the plantar lateral region of the hind paw. After cutting the tibial nerve, mechanical hypersensitivity develops at the neuroma site within 1 week after surgery and persists at least until 12 weeks after surgery. Allodynia at the sural innervated plantar surface develops within 3 weeks after surgery compared to the contralateral limb. At 12 weeks, a neuroma forms on the proximal end of the severed tibial nerve, indicated by dispersion and swirling of axons. For the TNT model surgery, multiple critical (micro)surgical steps need to be followed, and some surgery practice under terminal anesthesia is advised. Compared to other neuropathic pain models, such as the spared nerve injury model, allodynia over the neuroma site can be independently tested from sural nerve hypersensitivity in the TNT model. However, the neuroma site can be tested only in rats, not in mice. The tips and directions provided in this protocol can help research groups working on pain successfully implement the TNT model in their facility.

Assessment at the neuroma site showed increased sensitivity to the application of the 15 g von Frey monofilament. At baseline, rats typically responded to 10%-15% (&#177; 13%) of the 25 applications of a 15 g monofilament. The response rate increased to 45%-50% (&#177; 24%) 1 week after TNT surgery. On the contralateral side, the number of responses after surgery was similar to those at baseline (Figure 2A). Around 20% of the rats did not develop a painful neuroma; the response rate did not ...

Watch this Scientific Journal Video about Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats at JoVE.com

Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats

This protocol describes the tibial neuroma transposition model, which entails a lesion of the tibial nerve with subsequent transposition of the proximal nerve end toward a subcutaneous pretibial or lateral position. Behavioral testing of neuroma pain and plantar hyperalgesia is quantified using Von Frey monofilaments.

The tibial neuroma transposition (TNT) is a rat model in which allodynia at the neuroma site (tibial nerve) can be independently evaluated from allodynia ...

This method helps finding better treatments for neuroma pain. The advantage of this technique is that you can measure both neuroma pain and generalized pain independently in the same rep model. The TNT model is particularly fitted to the testing of neuroma pain, as the neuroma itself can be mechanically stimulated with the Von Frey monofilaments.Begin by placing the anesthetized rat on its back with its head either to the left or right. Place the leg to be operated on near the surgeon. Exorotate the lower hind limb so the medial malleolus faces upward.Before placing the rat under a stereo surgical microscope with 6x magnification, disinfect the shaved area with at least three alternating rounds of iodine-based scrub and alcohol. Locate the knee and gently make a longitudinal incision of two to three centimeters over the medial side of the hind paw from midcalf to ankle using a scalpel. If needed, open the skin and subcutis further with microscissors until the muscle layers are visible.Identify the superficial neurovascular bundle as two to three white, purple, or red lines that can move freely over the muscle layers. Keep the neurovascular bundle intact. Dissect the posterior from this bundle.Next, dissect to open the fascia in between the gastrocnemius muscles. Between the fascia of the muscles, the deeper neurovascular bundle with the tibial nerve can be found, which is about three times the size of the superficial neurovascular bundle. Use the tibial bone as an additional landmark, as the tibial nerve flies just posterior to the tibial bone.To dissect the tibial nerve free from the surrounding vascular bundles, bluntly move the tibial nerve and cut the exposed tissue that shows some stretch while moving the tibial nerve. Once the whole tibial nerve is exposed, place the muscle layers back to avoid dehydration of the nerve until the dissection of the pretibial skin from the subcutaneous muscle. To dissect the pretibial skin from the subcutaneous muscle layer and make a subcutaneous tunnel, hold the skin up and push the blunt tip into the tissue parallel to the skin using a blunt microsurgery tool.Ensure that the end of the tunnel is located pretibially or laterally for easy accessibility of the area for testing of the neuroma. After cutting the tibial nerve at the distal end near the ankle, change the microscope magnification to 10x or 16x and identify the epineurium of the tibial nerve proximal to the cut made near the ankle. Or, in the case of a more proximal bifurcation of the tibial nerve, identify the epineurium of both the medial and lateral planter branches proximal to the cut made near the ankle.Carefully place an 8-0 nylon suture through the epineurium of the proximal nerve end by holding the epineurium with tweezers and putting the needle between the nerve and epineurium with a bite of approximately 0.5 millimeters. Take a bite with the needle subcutaneously at the end of the subcutaneous tunnel and pull the suture to transpose the nerve laterally into the subcutaneous tunnel. Fix the suture by making a knot.Place a thicker suture of 4-0 with a dark color flushed to the fixated nerve end without penetrating the skin. Ensure that the suture is visible outside the skin. Check if the nerve stays in place after moving the paw and muscles.Cut off the suture ends with a slightly longer suture end on the 4-0, then on the 8-0 suture. Change the magnification of the microscope back to 6x and close the skin with intraepidermal sutures using the 8-0 suture. Gently clean the skin with 0.9%sodium chloride using a cotton swab.If the room is cold, place a heating pad under a part of a clean cage and place the rat in the cage under a paper towel in a comfortable position. Ensure easy access to food and water. To stimulate the sterile nerve for hypersensitivity, apply the monofilament on the lateral side close to the hair border without touching the foot pads.To stimulate the tibial nerve for hyposensitivity, apply the monofilament in the middle of the planter surface of the hind paw. For neuroma site testing, hold the rats with their nose pointed toward the elbow fold. If the rat is held in the right hand, his left hind paw should hang freely between the right thumb and index finger.After verifying that the rat is calm and comfortable while holding, at baseline, place the 15-gram monofilament gently on the pretibial surface of the exposed hind paw. After the surgery, place the 15-gram monofilament on the visible suture and apply sufficient force to the monofilament to bend the hair and hold for one second. Von Frey of the neuroma site showed that at baseline, rats responded to 10%to 15%of the applications of a 15-gram monofilament.One week after the tibial neuroma transposition surgery, the response rate increased from 45%to 50%On the contralateral side, the responses after surgery were similar to the baseline. Around 20%of the rats did not develop a painful neuroma and the response rate did not increase compared to the baseline. At the end of the transected and transposed tibial nerve stump 12 weeks after surgery, all rats developed a neuroma.Transection of the tibial nerve reduced mechanical sensitivity at the middle of the planter side of the hind paw innervated by the tibial nerve. Hyposensitivity present at one week after surgery was significantly different from the contralateral side and baseline from three weeks after surgery and remained until 12 weeks after surgery. At the lateral part of the planter side of the hind paw innervated by the intact sural nerve, the rats developed significantly different mechanical hypersensitivity from the contralateral side and baseline from one week after surgery.It persisted for 12 weeks after surgery. At the contralateral paw, mechanical sensitivity was not affected compared to baseline in the areas innervated either by the sural or tibial nerve. Remember to create an off-length of the tibial nerve for range of motion for lateral transposition.Also remember to mark this neuroma site with a dark suture.

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Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...