Thank you to Mauricio R. Delgado, Stephanie R. Potts, and William T. McCuddy. Acquisition of the representative data was supported by funding from the National Institute on Drug Abuse to MRD (R01DA027764)

1. CPAW Creation
Note: the CPAW is versatile in its components and construction. While this protocol represents the approach taken by the author, alternative approaches are possible.
<ol>
	<li>Affix two to three pieces of Velcro (each about 4 in&#160;x 1 in) to each of the two gelpacs (6 in&#160;x 12&#160;in size) using rubber cement, equally spaced. Allow 24 hr to dry.</li>
	<li>Obtain a piece of thick fabric approximately 12 in&#160;x 16 in&#160;in size, fold in half lengthwise. The example 6 in&#160;x 12 in&#160;gelpacs will run lengthwise along each inner half of the folded fabric.</li>
	<li>Affix the complementary side of each Velcro piece (from step 1.1) to the inner sides of the fabric, spaced so that they will match placement of the Velcro on the gelpacs. Rubber cement or needle and thread may be used. This will allow gelpacs to be easily removed for cleaning and reattached for storage.</li>
	<li>Obtain two larger pieces of Velcro (approximately 2 in&#160;x 10 in). Separate. Cut one side to about a quarter of its original size (and discard the remainder) while keeping the other at the original 2 in&#160;x 10 in&#160;size.</li>
	<li>Affix the small piece of Velcro to the front and back of the outer portion of the fabric on the same side. Finally, affix the longer part of the Velcro to the analogous locations on the outer portion of the opposite side of the fabric. After this is completed, it should be possible to wrap the fabric around one&#39;s forearm and hand and fasten it in place using the Velcro straps.</li>
</ol>
2. CPAW Usage and Safety Precautions
<ol>
	<li>Inform participants in studies using CPAW multiple times, during consent and prior to CPAW administration, that they are free to withdraw and end the procedure at any time. Three discrete withdrawal statements are recommended. Additionally, be prepared to end the procedure if the participant becomes distressed.</li>
	<li>Example Instructional Text: &#34;In this study, we may ask you to participate in a stress procedure that involves placing an arm wrap made of cold gelpacs to your arm for a period of time. The wrap is designed to be stressful, and will thus be highly uncomfortable. It is important that you understand that it is your right to end the procedure and experiment at any time for any reason, whether the arm wrap is currently on your arm or not. To do this once in the MRI you may tell me verbally, you may kick your foot vigorously, or if necessary you may use the emergency squeeze-ball we will give you. Do you understand? Are you willing to continue?&#34;. Thereafter, it is advised that the participant be reminded twice more before MRI scanning with the statement, &#34;Remember, we will remove the arm wrap and end the experiment at your request at any time&#34;.</li>
	<li>Store CPAW in a freezer with a carefully monitored temperature. If the freezer cannot beset to a specific temperature (e.g.&#160;between 0-5 &#176;C) and is below 0 &#176;C, allow time for CPAW to warm to a safe temperature (no less than 0 &#176;C) after removing it from storage. Otherwise, the potential exists for a participant to experience a cold burn.</li>
	<li>Prior to employing CPAW, cover the gelpacs with a layer of plastic sheeting to avoid direct contact between gelpacs and the participant&#39;s skin. Measure the surface temperature of each gelpac (covered by the plastic sheeting) using an infrared thermometer in two-to-three locations along its length to ensure a safe temperature. If the temperature is too low, allow additional time to pass and measure again.</li>
	<li>When the desired temperature is reached, wrap CPAW around the participants forearm and hand affixing it in place with the aforementioned Velcro straps. Leave CPAW in place for the desired period of time (2 min is recommended). Remove it immediately upon the participant&#39;s request or after that time has elapsed.</li>
</ol>

<ol>
	<li>Sinha, R., Garcia, M., Paliwal, P., Kreek, M. J., Rounsaville, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-Induced+Cocaine+Craving+and+Hypothalamic-Pituitary-Adrenal+Responses+Are+Predictive+of+Cocaine+Relapse+Outcomes.">Stress-Induced Cocaine Craving and Hypothalamic-Pituitary-Adrenal Responses Are Predictive of Cocaine Relapse Outcomes.</a> Arch. Gen. Psychiatry. 63 (3), 324-331 (2006).</li><li>Dias-Ferreira, E., 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=Chronic+stress+causes+frontostriatal+reorganization+and+affects+decision-making.">Chronic stress causes frontostriatal reorganization and affects decision-making.</a> Science. 325, 621-625 (2009).</li><li>Schwabe, L., Wolf, O. 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Mem. 91, 377-381 (2009).</li><li>Cahill, L., Gorski, L., Le, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+human+memory+consolidation+with+post-learning+stress:+interaction+with+the+degree+of+arousal+at+encoding.">Enhanced human memory consolidation with post-learning stress: interaction with the degree of arousal at encoding.</a> Learn. Mem. 10, 270-274 (2003).</li><li>Duncko, R., Cornwell, B., Cui, L., Merikangas, K. R., Grillon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+exposure+to+stress+improves+performance+in+trace+eyeblink+conditioning+and+spatial+learning+tasks+in+healthy+men.">Acute exposure to stress improves performance in trace eyeblink conditioning and spatial learning tasks in healthy men.</a> Learn. Mem. 14, 329-335 (2007).</li><li>Lighthall, N. 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=Gender+differences+in+reward-related+decision+processing+under+stress.">Gender differences in reward-related decision processing under stress.</a> Soc. Cogn. Affect Neurosci. 7, 476-484 (2011).</li><li>Schwabe, L., Wolf, O. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Socially+evaluated+cold+pressor+stress+after+instrumental+learning+favors+habits+over+goal-directed+action.">Socially evaluated cold pressor stress after instrumental learning favors habits over goal-directed action.</a> Psychoneuroendocrinology. 35, 977-986 (2010).</li><li>Porcelli, A. J., Delgado, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stress+modulates+risk+taking+in+financial+decision+making.">Acute stress modulates risk taking in financial decision making.</a> Psychol. Sci. 20, 278-283 (2009).</li><li>al'Absi, M., Petersen, K. L., Wittmers, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+and+hemodynamic+predictors+of+pain+perception+in+men+and.">Adrenocortical and hemodynamic predictors of pain perception in men and.</a> 96, 197-204 (2002).</li><li>Smeets, T., Otgaar, H., Candel, I., Wolf, O. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=True+or+false?+Memory+is+differentially+affected+by+stress-induced+cortisol+elevations+and+sympathetic+activity+at+consolidation+and+retrieval.">True or false? Memory is differentially affected by stress-induced cortisol elevations and sympathetic activity at consolidation and retrieval.</a> Psychoneuroendocrinology. 33, 1378-1386 (2008).</li><li>McRae, A. L., 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=Stress+reactivity:+biological+and+subjective+responses+to+the+cold+pressor+and+Trier+Social+stressors.">Stress reactivity: biological and subjective responses to the cold pressor and Trier Social stressors.</a> Hum. Psychopharmacol. 21, 377-385 (2006).</li><li>Kirschbaum, C., Pirke, K. M., Hellhammer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'Trier+Social+Stress+Test'--a+tool+for+investigating+psychobiological+stress+responses+in+a+laboratory+setting.">The 'Trier Social Stress Test'--a tool for investigating psychobiological stress responses in a laboratory setting.</a> Neuropsychobiology. 28, 76-81 (1993).</li><li>Kudielka, B. M., Hellhammer, D. H., Kirschbaum, 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> Social neuroscience: Integrating biological and psychological explanations of social behavior. , 56-83 (2007).</li><li>Dickerson, S. S., Kemeny, M. 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Bull. 130, 355-391 (2004).</li><li>Schwabe, L., Haddad, L., Schachinger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPA+axis+activation+by+a+socially+evaluated+cold-pressor+test.">HPA axis activation by a socially evaluated cold-pressor test.</a> Psychoneuroendocrinology. 33, 890-895 (2008).</li><li>Smeets, T., 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=Introducing+the+Maastricht+Acute+Stress+Test+(MAST):+a+quick+and+noninvasive+approach+to+elicit+robust+autonomic+and+glucocorticoid+stress+responses.">Introducing the Maastricht Acute Stress Test (MAST): a quick and noninvasive approach to elicit robust autonomic and glucocorticoid stress responses.</a> Psychoneuroendocrinology. 37, 1998-2008 (2012).</li><li>Huettel, S. A., Song, A. W., McCarthy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Functional Magnetic Resonance Imaging.. , (2008).</li><li>Porcelli, A. J., Lewis, A. H., Delgado, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stress+influences+neural+circuits+of+reward+processing.">Acute stress influences neural circuits of reward processing.</a> Front. Neurosci. 6, 157 (2012).</li><li>Pruessner, J. C., Kirschbaum, C., Meinlschmid, G., Hellhammer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+formulas+for+computation+of+the+area+under+the+curve+represent+measures+of+total+hormone+concentration+versus+time-dependent+change.">Two formulas for computation of the area under the curve represent measures of total hormone concentration versus time-dependent change.</a> Psychoneuroendocrinology. 28, 916-931 (2003).</li><li>Porcelli, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+stress+influences+financial+decision+making.+In+M.+Mather+(Chair).">Acute stress influences financial decision making. In M. Mather (Chair).</a> How stress alters decision making. Symposium conducted at the meeting of the Assoc. Psychol. Sci. , (2010).</li><li>Levey, A. 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The authors declare no conflict of interest.

In this article an alternative to the traditional cold pressor test was demonstrated - the cold pressor arm wrap. CPAW is a procedure that produces psychophysiological and neuroendocrine stress responses comparable to the CPT. Specifically, robust increases in SAM (i.e.&#160;sympathetic ANS) activation and mild-to-moderate increases in HPA (i.e.&#160;cortisol) activity were observed. CPAW can be created from a range of commonly found components, and serves as a viable alternative to the traditional (water-based) CPT. An additional advantage of CPAW is that it can be easily employed in research contexts where it could be impractical, even unfeasible, to use CPT (e.g.&#160;MRI).
As mentioned previously, CPAW requires additional safety precautions beyond those associated with CPT. Exclusionary criteria (largely shared by CPT) should include: cold urticaria, Reynaud&#39;s Disease, aneurysm, pregnancy, history of cardiovascular illness, diabetes, chronic rheumatologic disease, and untreated blood pressure abnormalities. Given that CPAW can potentially be cooled to unsafe temperatures below that of liquid water, strict temperature measurement prior to application is critical to avoid risk of participant injury. As the apparatus is physically fastened to the participant&#39;s arm it is vital that it be made clear at several points that they can end the procedure at any time at their request. Further, the experimenter must be prepared to terminate the procedure if the participant becomes distressed. Thus, the procedure requires that the researcher be vigilant on multiple fronts.
Several limitations exist in use of CPAW. For example, in the context of MRI studies a short-bore MRI may be necessary as fastening CPAW to a participants arm might not be possible in a long-bore MRI. That said, adapting CPAW for use on a foot or leg is feasible and would be compatible with a long-bore setup. Titration of CPAW temperature to a highly precise temperature is also difficult. Research indicates that deviations of even 2 &#176;C are associated with variations in tolerance time and pain ratings31. To avoid this, use of a freezer that can be cooled to a specific temperature is advised. Additionally, it is advised to have a second CPAW available if the device will be employed multiple times in close succession (to avoid it being warmed beyond the desired temperature). Though in the representative results CPAW use was not associated with increases in head motion, researchers should pay special attention to head movement in the fMRI setting - especially if CPAW is being applied during (rather than prior to) fMRI scanning. While subjective stress ratings, HPA, and SAM activation suggest that CPAW is an effective stressor comparable to the traditional CPT, future research should focus on careful comparison of the extent of HPA and SAM activity with that evoked by the traditional CPT as well as other physiological indices (e.g.&#160;heart rate and blood pressure).

Stress exposure is associated with activation of two stress-responsive biological systems: the hypothalamic-pituitary-adrenal (HPA) and sympatho-adrenomedullary (SAM) axes5. When stress is of sufficient intensity to disrupt an organism&#39;s homeostatic balance, SAM quickly triggers increased activity in the sympathetic branch of the autonomic nervous system (ANS). This is associated with release of catecholamines (e.g.&#160;adrenaline and noradrenaline; CA) from the adrenal medulla and sympathetic nerve terminals. Increased levels of these excitatory chemicals are associated with elevations in heart rate and blood pressure that characterize the classic &#34;fight-or-flight&#34; response6. Although subsequent activation of the parasympathetic ANS will eventually inhibit this excitation, ascending CA neurons interfacing with brainstem structures contribute to slower HPA activation via the hypothalamus and eventual glucocorticoid (i.e.&#160;cortisol in humans) release from the adrenal cortex7. Understanding the relationship between stress modality and the specific dynamics of the stress response itself will be critical as research on the relationship between stress and the neural correlates of various psychological processes (e.g.&#160;memory, learning, and decision-making) advances.
Multiple stress induction tasks have been developed for use in laboratory research. Generally, these can be classified as systemic (i.e.&#160;representing a reflexive response to a direct threat to homeostatic balance) or processive/psychogenic (i.e.&#160;requiring forebrain processing and appraisal prior to engagement of the stress response)8.&#160;Notably, these two distinct stress modalities Fare associated with differential patterns of SAM and HPA activation. The cold pressor test (CPT) is a common and well-validated means of inducing systemic stress involving immersion of an individual&#39;s dominant hand in ice water (typically between 0-5 &#176;C) for a period of time4,9,10. CPT has been used in a wide range of research examining the influence of stress on memory11-13, learning14, and decision-making15-17.&#160;Studies using CPT as a means of stress induction have, however, yielded mixed results in terms of cortisol reactivity. Overall mild-to-moderate cortisol increases have been observed post-CPT13,18,19, though SAM activation is robust and reliable4,20. In contrast, processive laboratory stressors such as the Trier Social Stress Task (TSST)21,22, during which participants give a 5 min speech followed by 5 min of mental arithmetic while being watched and/or videotaped, have been associated with two- to three-fold increases in cortisol23.
To maximize both SAM and HPA activation, some researchers have taken the approach of combining CPT with social evaluative components similar to those present in the TSST. For example, in the socially evaluated cold pressor test (SECPT) participants are asked to perform a traditional CPT while being video-taped and watched by an experimenter24. Similarly, the Maastricht Acute Stress Test integrates SECPT with public speaking and mental arithmetic components of the TSST25.&#160;While these approaches represent a significant step forward in stress research it remains important to examine the specific, and potentially dissociable, influence of SAM and HPA activation on the neural correlates of behavior, cognition, and emotion. Unfortunately CPT, involving water, is often impractical to perform in studies involving magnetic resonance imaging (MRI) unless completed prior to entry into the MRI itself. For example, spilled water can be a threat to sensitive and expensive MRI equipment (e.g.&#160;the head coil) and can interfere with MRI signal due to its high proton density26.&#160;Thus, as research involving CPT expands towards use in neuroimaging contexts modifications to the traditional CPT procedure are necessary. To that end, an alternative is proposed - the cold pressor arm wrap (CPAW).
Rather than hand immersion in ice-cold water, the CPAW involves an arm wrap composed of MRI-safe gelpacs cooled to a temperature similar to the traditional CPT. It can be attached to the forearm and hand of a participant, and is easily removed at their request. Further, CPAW is versatile in its composition from a simple collection of common components. It is important to note that additional safety concerns exist in CPAW use, as it can potentially be cooled to a temperature much lower than liquid water. That said, these can be accounted for with minimal effort on the part of the researcher. Proper storage and use of the CPAW will be demonstrated&#160;with special attention paid to safety protocols.

<table border="0" cellpadding="0" cellspacing="0" width="573">
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	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">2 Cryo-Max Cold Packs (6 in x 12 in)</td>
			<td style="width:71px;">Modular Thermal Technologies</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">This is an example gelpac &#8211; others of a similar size would serve as well.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Infrared Thermometer</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">02-401-1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Biopac MP150 Data Acquisition System</td>
			<td style="width:71px;">Biopac Systems, Inc.</td>
			<td style="width:119px;">MP150</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Biopac GSR EDA Galvanic Skin Response Amplifier</td>
			<td style="width:71px;">Biopac Systems, Inc.</td>
			<td style="width:119px;">GSR100C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">2 Biopac Unshielded Leads</td>
			<td style="width:71px;">Biopac Systems, Inc.</td>
			<td style="width:119px;">LEAD110A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">2 Biopac disposable Electrodermal Electrodes</td>
			<td style="width:71px;">Biopac Systems, Inc.</td>
			<td style="width:119px;">EL507</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Salivary Cortisol Enzyme Immunoassay Kit</td>
			<td style="width:71px;">Salimetrics, LLC.</td>
			<td style="width:119px;">I-3002</td>
			<td style="width:167px;">If assaying will not be conducted in-house, only collection supplies below are needed.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Salimetrics Oral Swab</td>
			<td style="width:71px;">Salimetrics, LLC.</td>
			<td style="width:119px;">5001.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Swab Storage Tube</td>
			<td style="width:71px;">Salimetrics, LLC.</td>
			<td style="width:119px;">5001.05</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

an alternative to the traditional cold pressor test: the cold pressor arm wrap

Recently research on the relationship between stress and cognition, emotion, and behavior has greatly increased. These advances have yielded insights into important questions ranging from the nature of stress&#39; influence on addiction1 to the role of stress in neural changes associated with alterations in decision-making2,3. As topics being examined by the field evolve, however, so too must the methodologies involved. In this article a practical and effective alternative to a classic stress induction technique, the cold pressor test (CPT), is presented: the cold pressor arm wrap (CPAW). CPT typically involves immersion of a participant&#39;s dominant hand in ice-cold water for a period of time4. The technique is associated with robust activation of the sympatho-adrenomedullary (SAM) axis (and release of catecholamines; e.g.&#160;adrenaline and noradrenaline) and mild-to-moderate activation of the hypothalamic-pituitary-adrenal (HPA) axis with associated glucocorticoid (e.g.&#160;cortisol) release. While CPT has been used in a wide range of studies, it can be impractical to apply in some research environments. For example use of water during, rather than prior to, magnetic resonance imaging (MRI) has the potential to damage sensitive and expensive equipment or interfere with acquisition of MRI signal. The CPAW is a practical and effective alternative to the traditional CPT. Composed of a versatile list of inexpensive and easily acquired components, CPAW makes use of MRI-safe gelpacs cooled to a temperature similar to CPT rather than actual water. Importantly CPAW is associated with levels of SAM and HPA activation comparable to CPT, and can easily be applied in a variety of research contexts. While it is important to maintain specific safety protocols when using the technique, these are easy to implement if planned for. Creation and use of the CPAW will be discussed.

CPAW has been used in a previously published study examining the influence of acute stress on neural reward processing while participants underwent functional MRI scanning27. Thirty-two participants (16 females, 16 males; mean age = 23.41 years, SD years = 4.07) were randomly assigned between-subjects to a CPAW or no stress control group (an arm wrap composed of towels at room temperature). A baseline salivary cortisol sample was acquired at the beginning of the protocol followed by CPAW (or no stress control) application for 2 min, after which a 10&#160;min behavioral task was performed. After the first behavioral task, this pattern repeated - a second saliva sample was acquired, followed by CPAW (or no stress control) for 2 min, and a second 10 min&#160;behavioral task. A third saliva sample was acquired after the second behavioral task. In a post-experimental questionnaire, participants were asked to report subjective ratings of perceived stress, including how the arm wrap made them feel (good to bad) and how stressful the experience was (high to low) on a 7-point Likert scale.
Compared to the no stress group, participants who experienced CPAW reported the arm wrap as feeling significantly more stressful, t(30) = 3.46, p&#60; 0.01,&#160;d = 1.22, and as feeling significantly worse, t(30) = 4.42, p&#60; 0.001, d = 1.56. Three participants were excluded from salivary cortisol analysis, one due to corrupted samples and two due to an inability to collect samples during the experiment. Thus, final cortisol analyses were conducted on 29 of the 32 participants (13 no stress control, 16 CPAW). Area under the curve with respect to increase (AUCI)was calculated using the trapezoidal method for both groups (see Table 1 for mean salivary cortisol of each sample by experimental group in nmol/L)28. A one-tailed independent t-test between the no stress control and CPAW group AUCI indicated a significant increase in salivary cortisol for the CPAW group, t(27) = 1.78, p&#60; 0.05, d = 0.69 (See Figure 1). Only two participants exhibited head motion in excess of one voxel (i.e.&#160;3 mm),but both were in the no stress group. No participants withdrew from the study due to CPAW exposure. Preliminary analysis of an additional dataset provides convergent support for the efficacy of CPAW in terms of SAM activation (i.e.&#160;skin conductance)29. Twenty-eight participants (14 females, 14 males; mean age = 21.18 years, SD years = 2.97) performed a no stress control arm wrap (as above) for 2 min followed by a 15 min&#160;behavioral task. After this they performed the CPAW for 2 min, followed by a second 15 min&#160;behavioral task (all within-subjects). Skin conductance levels (SCL) were measured during each of the arm wrap exposures (no stress control then CPAW) using a BIOPAC module and analyzed using AcqKnowledge software. The average SCL waveform of each 2 min arm wrap was computed (in microsiemens, &#181;s) and normalized using a square-root transform30. A paired t-test indicated significantly elevated SCL during CPAW exposure above the no stress control, t(27) = 5.49, p&#60; .001, d = .65 (see Figure 2).
<img alt="Table 1" fo:content-width="4in" fo:src="/files/ftp_upload/50849/50849table1highres.jpg" src="/files/ftp_upload/50849/50849table1.jpg" width="500px" /> 
Table 1.&#160;Mean salivary cortisol levels in nmol/L at baseline, after task run 1, and after task run 2 for CPAW and no stress control groups (Mean &#177; SEM). Adapted from Porcelli et al.27 with permission. <a href="https://www.jove.com/files/ftp_upload/50849/50849table1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50849/50849fig1highres.jpg" src="/files/ftp_upload/50849/50849fig1.jpg" width="500px" /> 
Figure 1. Salivary cortisol area under the curve with respect to increase (AUCI) for CPAW and no stress control groups. Negative AUCI&#160;values, which indicate a net decrease in salivary cortisol, were retained as an &#34;index of decrease&#34;28. Adapted from Porcelli et al.27 with permission. <a href="https://www.jove.com/files/ftp_upload/50849/50849fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50849/50849fig2highres.jpg" src="/files/ftp_upload/50849/50849fig2.jpg" width="500px" /> 
Figure 2. Square-root transformed mean skin conductance levels (in microsiemens; &#181;s) during CPAW or no stress control arm wrap. <a href="https://www.jove.com/files/ftp_upload/50849/50849fig2highres.jpg" target="_blank">Click here to view larger image</a>.

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An Alternative to the Traditional Cold Pressor Test: The Cold Pressor Arm Wrap

The cold pressor test is a classic stress induction task used in a wide range of research examining the relationship between stress and cognition, emotion, and behavior. In this article, a practical and versatile alternative to the traditional cold pressor test is presented: the cold pressor arm wrap.

Recently research on the relationship between stress and cognition, emotion, and behavior has greatly increased. These advances have yielded insights into ...

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14.4K Views.  Marquette University. The cold pressor test is a classic stress induction task used in a wide range of research examining the relationship between stress and cognition, emotion, and behavior. In this article, a practical and versatile alternative to the traditional cold pressor test is presented: the cold pressor arm wrap.

Video: An Alternative to the Traditional Cold Pressor Test: The Cold Pressor Arm Wrap

Use of an Eight-arm Radial Water Maze to Assess Working and Reference Memory Following Neonatal Brain Injury

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food deprivation, and may introduce scent cue confounds. The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs. Specifically, subjects are required to avoid the arms previously used for escape during each testing day (working memory) as well as avoid the fixed arms, which never contain escape platforms (reference memory). Re-entries into arms that have already been used for escape during a testing session (and thus the escape platform has been removed) and re-entries into reference memory arms are indicative of working memory deficits. Alternatively, first entries into reference memory arms are indicative of reference memory deficits. We used this maze to compare performance of rats with neonatal brain injury and sham controls following induction of hypoxia-ischemia and show significant deficits in both working and reference memory after eleven days of testing. This protocol could be easily modified to examine many other models of learning impairment.

The eight-arm radial water maze is designed to evaluate reference and working memory performance simultaneously by requiring subjects to use extra-maze cues to locate escape platforms and remedies the limitations observed in land based radial arm maze designs.

Working and reference memory are commonly assessed using the land based radial arm maze.&#160;However, this paradigm requires pretraining, food ...

Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in na&#239;ve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.

A method to assess exercise endurance in laboratory mice without the use of a shock grid is demonstrated. This method is a humane refinement that can decrease the confounding effects of stress on experimental parameters.

Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most ...

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer's Disease

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of four mountains within a computer-generated landscape is tested using a delayed match-to-sample paradigm. Allocentric spatial memory is assessed by altering the viewpoint, colors and textures between the initially presented and target images.
Allocentric spatial memory is a key function of the hippocampus, one of the earliest brain regions to be affected in Alzheimer&#39;s disease (AD) and impairment of hippocampal function predates the onset of dementia. It was hypothesized that performance on the 4MT would aid the diagnosis of predementia AD, which manifests clinically as Mild Cognitive Impairment (MCI).
The 4MT was applied to patients with MCI, stratified further based on cerebrospinal fluid (CSF) AD biomarker status (10 MCI biomarker positive, 9 MCI biomarker negative), and with mild AD dementia, as well as healthy controls. Comparator tests included tests of episodic memory and attention widely accepted as sensitive measures of early AD. Behavioral data were correlated with quantitative MRI measures of the hippocampus, precuneus and posterior cingulate gyrus.
4MT scores were significantly different between the two MCI groups (p = 0.001), with a test score of &#8804;8/15 associated with 100% sensitivity and 78% specificity for the classification of MCI with positive AD biomarkers, i.e., predementia AD. 4MT test scores correlated with hippocampal volume (r = 0.42) and cortical thickness of the precuneus (r = 0.55).
In conclusion, the 4MT is effective in identifying the early stages of AD. The short duration, easy application and scoring, and favorable psychometric properties of the 4MT fulfil the need for a simple but accurate diagnostic test for predementia AD.

The 4 Mountains Test: A Short Test of Spatial Memory with High Sensitivity for the Diagnosis of Pre-dementia Alzheimer&#039;s Disease

This article describes the 4 Mountains Test (4MT), a hippocampus-dependent test of working allocentric spatial memory. The hippocampus is affected early in Alzheimer's disease (AD) and this article outlines the 4MT methodology and results of patient testing, which demonstrates the value of the 4MT in the diagnosis of pre-dementia AD.

This protocol describes the administration of the 4 Mountains Test (4MT), a short test of spatial memory, in which memory for the topographical layout of ...

The Other End of the Leash: An Experimental Test to Analyze How Owners Interact with Their Pet Dogs

It has been suggested that the way in which owners interact with their dogs can largely vary and influence the dog-owner bond, but very few objective studies, so far, have addressed how the owner interacts with the dog. The goal of the present study was to record dog owners&#39; interaction styles by means of objective observation and coding. The experiment included eight standardized situations in which owners of pet dogs were asked to perform specific tasks including both positive (i.e. playing, teaching a new task, showing a preference towards an object in a food searching task, greeting after separation) and potentially distressing tasks (i.e. physical restriction during DNA sampling, putting a T-shirt onto the dog, giving basic obedience commands while the dog was distracted). The video recordings were coded off-line using a specifically designed coding scheme including scores for communication, social support, warmth, enthusiasm, and play style, as well as frequency of behaviors like petting, praising, commands, and attention sounds. Exploratory Factor Analysis of the 20 variables measured revealed 3 factors, labeled as Owner Warmth, Owner Social Support, and Owner Control, which can be viewed as analogues to parenting style dimensions. The experimental procedure introduced here represents the first standardized measure of interaction styles of dog owners. The methodology presented here is a useful tool to investigate individual variation in the interaction style of pet dog owners that can be used to explain differences in the dog-human relationship, dogs&#39; behavioral outcomes, and dogs stress coping strategies, all crucial elements both from a theoretical and applied point of view.

This article presents eight different experimental tasks, mirroring the everyday life of dogs and owners, used to analyze how owners interact with their dogs in a standardized way. The tasks included both positive (e.g. play) and negative (potentially stressful) situations (e.g. physical restriction).

It has been suggested that the way in which owners interact with their dogs can largely vary and influence the dog-owner bond, but very few objective ...

Testing Animal Anxiety in Rats: Effects of Open Arm Ledges and Closed Arm Wall Transparency in Elevated Plus Maze Test

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral science, conflicting outcomes are often provided from different laboratories. To identify reasons for the different outcomes, we previously focused on arm features, which differ between laboratories, most notably the presence/absence of ledges at the sides of open arms and the transparency/opaqueness of closed arm walls. In a previous report, we used a custom designed container to compare rat behavior on different combinations of open and closed arm designs under otherwise identical experimental conditions, and showed that differences in arm features interfere with experimental outcomes. In brief, open arm ledges significantly increased anxiety-like behavior in rats, while transparent arms may also have decreased this behavior. Furthermore, we verified a higher detection sensitivity of the effect of an anxiolytic drug in a combination of no-ledged open arm + opaque walled closed arm compared with a combination of ledged open arm + transparent walled closed arm. In this report, we introduce our protocol for the elevated plus maze test, together with discussion of the key results from the previous report and our experimental experience. We believe this report will provide useful information for researchers who have employed or who plan to use the elevated plus maze in their studies.

Rats' anxiety-like behavior tested by the elevated plus maze varies highly depending on the testing environment. Here we show how apparatus settings, especially arm structure, affect behavior on the maze, and we explain a reliable protocol to detect effects of anxiolytic drugs. This report will contribute to anxiety research.

The elevated plus maze test is a behavioral test for assessing animal anxiety in rodents. Although this test is widely applied in the field of behavioral ...

An Objective and Child-friendly Assessment of Arm Function by Using a 3-D Sensor

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment of muscle functions is an essential and important, although challenging, prerequisite for successful clinical trials. Current clinical rating scales restrain the movement abnormalities to certain predefined coarse-grained individual items. The Kinect 3-D sensor has emerged as a low-cost and portable motion sensing technology used to capture and track people&#39;s movement in many medical and research fields. A novel approach using this 3-D sensor was developed and a game-like test was designed to objectively measure the upper limb function of patients with SMA. The prototype test targeted joint movement capability. While sitting in a virtual scene, the patient was instructed to extend, flex, and lift the whole arm in order to reach and place some objects. Both kinematic and spatiotemporal characteristics of upper limb movement were extracted and analyzed, e.g., elbow extension and flexion angles, hand velocity, and acceleration. The first study included a small cohort of 18 ambulant SMA patients and 19 age- and gender-matched healthy controls. A comprehensive analysis of arm movement was achieved; however, no significant difference between the groups were found due to the mismatch of patient&#39;s capability and the test difficulty. Based on this experience, a second version of the test consisting of a modified version of the first game with increased difficulties and a second game targeting muscle endurance were designed and implemented. The new test has not been conducted in any patient groups yet. Our work has demonstrated the potential capability of the 3-D sensor in assessing such muscle function and suggested an objective approach to complement the clinical rating scales.

An objective measure of muscle functions is challenging especially in children. Based on a commercially available digital 3-D sensor, a child-friendly gaming test was developed to assess upper limb function for clinical trials.

Progressive and irreversible muscle atrophy characterizes Spinal Muscular Atrophy (SMA) and other similar muscle disorder diseases. Objective assessment ...

Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall improvement of patients with major depressive disorder (MDD). Therefore, the purpose of this study was to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and then evaluate the anti-depression effect of Xiaoyaosan. Mice were randomly divided into four groups: a control group, a model group, a treatment group with Xiaoyaosan, and a treatment group with fluoxetine. All mice were individually kept in cages, and depression was induced in the mice by exposing them to several designed manipulations of CUMS for 21 days, as described in the protocol. Mice in the control group and model group received 0.5 mL of distilled water, while mice in the treatment groups received either Xiaoyaosan (0.25 g/kg/day) or fluoxetine (2.6 mg/kg/day). The drugs used in the study were given intragastrically daily during the entire three weeks. To estimate the depressive-like behaviors, a series of parameters including the coat state, body weight, open field test score, and sucrose preference test score were recorded. Data analysis showed that behaviors of model mice were significantly changed compared to behaviors of mice in the control group, which were improved by the treatment of Xiaoyaosan and fluoxetine. The current findings demonstrated the anti-depression effects of Xiaoyaosan on the behaviors of CUMS-induced mice and revealed that compounds from the Xiaoyaosan prescription may be worthwhile for treating depression, considering their beneficial effects on depressive-like behaviors.

Here, we present a protocol to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and evaluate the anti-depression effect of Xiaoyaosan.

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall ...

An Instrumented Pull Test to Characterize Postural Responses

Impairment of postural reflexes, termed postural instability, is a common and disabling deficit in Parkinson's disease. To assess postural reflexes, clinicians typically employ the pull test to grade corrective responses to a backward perturbation at the shoulders. However, the pull test is prone to issues with reliability and scaling (score/4). Here, we present an instrumented version of the pull test to more precisely quantify postural responses. Akin to the clinical test, pulls are manually administered except pull force is also recorded. Displacements of the trunk and feet are captured by a semi-portable motion tracking system. Raw data represent distance traveled (in millimeter units), making subsequent interpretation and analysis intuitive. The instrumented pull test also detects variabilities influencing pull test administration, such as pull force, thereby identifying and quantifying potential confounds that can be accounted for by statistical techniques. The instrumented pull test could have application in studies seeking to capture early abnormalities in postural responses, track postural instability over time, and detect responses to therapy.

Impairment of postural reflexes, termed postural instability, is difficult to quantify. Clinical assessments such as the pull test suffer issues with reliability and scaling. Here, we present an instrumented version of the pull test to objectively characterize postural responses.

Impairment of postural reflexes, termed postural instability, is a common and disabling deficit in Parkinson's disease. To assess postural reflexes, ...

The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli may facilitate breeding selections and identify animals that are better suited to specific management strategies. Multiple tests have been developed to evaluate cattle temperament (e.g., exit velocity, chute score, pen score, open field test, startle test, bovine zero maze), but each of these tests evaluates the animal&#39;s response to different stimuli (e.g., isolation, novel environment, startle, willingness to enter an enclosed area). Cattle temperament has been observed to be relatively stable over time. However, the evaluation of temperament has the potential to be influenced by current conditions, previous experiences, and observer bias. Many of these temperament tests have been improperly categorized as fear tests and have also been criticized for being subjective. This paper provides a framework for standardizing behavioral tests for cattle and suggests that these different evaluations assess different aspects of the animal&#39;s overall temperament.

Here, we present a protocol to conduct a variety of behavioral tests in cattle that have been designed to evaluate emotions. A battery of behavioral tests (open field test, startle test, bovine zero maze, exit velocity, pen score, and chute score) were conducted to evaluate different components of animal temperament.

Animal temperament is complex and has implications for productivity and economic profitability. Quantifying an animal&#39;s response to differing stimuli ...

Validating a Murine Model of Angelman Syndrome Using Behavioral Tests

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

Author Spotlight: A Battery of Highly Reproducible Behavioral Tests to Validate an Angelman Syndrome Murine Model 

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...