Name: disrupting reconsolidation of fear memory in humans by a noradrenergic &#946;-blocker
Uploaded: 2014-12-18T14:00:00
Duration: 8 min 32 s
Description: Watch this Scientific Journal Video about Disrupting Reconsolidation of Fear Memory in Humans by a Noradrenergic Î²-Blocker at JoVE.com

This work is supported by a VICI grant (Merel Kindt) from the Netherlands Organization for Scientific Research.

Ethics statement: Our physician-approved protocol meets the requirements of the Ethical Committee of the Department of Psychology at the University of Amsterdam for testing human participants.
1. Attachment of the Fear Potentiated Startle and Shock Electrodes
<ol>
	<li>Ensure the experimental setting consists out of a sound-attenuated participants&#8217; room and a separated experimenter room.</li>
	<li>Use two computers with four screens for recording and monitoring physiological responses as well as for presenting the experimental script to the participants.</li>
	<li>Attach the double-sided adhesive collars to the EMG electrodes.</li>
	<li>Clean the participant&#8217;s skin below the right or left eye and on the forehead using an alcohol swab. Instruct the participants to keep their eyes closed to avoid potential eye irritation.</li>
	<li>Fill the center of the EMG electrodes with highly conductive electrolyte gel using a syringe. Do not try to fill the electrodes by applying small amounts of gel successively as this increases the risk of air bubbles within the gel, which may affect the recordings negatively.</li>
	<li>Attach one of the EMG electrodes below the lower eyelid of the right or left eye in line with the pupil in forward gaze. Attach the second one approximately 1 - 2 cm lateral to the first following the curvature of the lower eyelid. Attach a ground reference on the forehead approximately 1 cm below the hairline. Optimal placement of the electrodes relative to the muscle may require individual adjustments.</li>
	<li>Apply a drop of the conductive gel on the shock electrodes and attach them on the left upper wrist using nylon Velcro strap. Make sure the strap is not on too tight.</li>
	<li>Adjust the intensity of the 2-msec electric shock for each participant individually. Inform the participants that the shock should be &#8220;uncomfortable but not painful&#8221;.</li>
	<li>Manually trigger the shocks. Start at an intensity of 1 mA and continue triggering single shocks &#8212; gradually increasing the intensity in 2 or 3 mA increments &#8212; until the participant indicates that the shock is &#8220;uncomfortable but not painful&#8221;.</li>
	<li>Write down the final shock level and maintain this level throughout the remainder of the experiment.</li>
</ol>
2. Fear Acquisition - Day 1
Duration about 45 min.
<ol>
	<li>Instruct the participants about the delivery of shocks and the possible administration of propranolol HCl. Further inform them that propranolol HCl is used to treat hypertension. Obtain written informed consent.</li>
	<li>Screen the participants regarding their health and any conditions that would contra-indicate taking a single dose of 40 mg of propranolol HCl: pregnancy, seizure disorder, respiratory disorder, cardiovascular diseases, cardiovascular diseases in first-line relatives, diabetes, liver or kidney disorder, current use of an anti-depressant drug, or psychosis.</li>
	<li>Obtain blood pressures and heart rate measurements using an electronic sphygmomanometer. Ask the participants to remove their arm from the sleeves and put the inflatable cuff securely on the upper arm. Instruct the participants to relax and sit with their arm slightly bent on the same level as their heart and resting comfortably on a table or other flat surface. BP should be above 90/60 mmHg and heart rate above 60 BPM for inclusion in the experiment.</li>
	<li>Administer the State-Trait Anxiety Inventory42 and the Anxiety Sensitivity Index43 when a participant is medically cleared. Also administer the Spider Phobic Questionnaire44 when using pictures of spiders as the conditioned stimuli.</li>
	<li>Position the participants in front of a computer screen and attach the EMG and shock electrodes (see section 1 &#34;Attachment of the Fear Potentiated Startle and Shock Electrodes&#34;).</li>
	<li>Use two different learning procedures &#8212; see steps 2.6.1 and 2.6.2. Inform the participants about the two different pictures they will see on the computer screen. Instruct them to monitor the relationship between the pictures they are seeing and when a shock is received.
	<ol>
		<li>Non-Asymptotic Learning: Inform the participants that one of the pictures will be followed by the electric shock in most cases whereas the other will never be followed by the shock.</li>
		<li>Asymptotic Learning: Inform the participants which of the two pictures will always be followed by the electric shock and which picture will never be followed by the shock (i.e., instructed fear learning).</li>
	</ol>
	</li>
	<li>Inform the participants about the loud noises they will hear through the headphones. Order them to pay no attention to the noises.</li>
	<li>Instruct the participants to report the expectancy of an electric shock during the presentation of all pictures by shifting a cursor on continuous rating scales by using a mouse and to push the left mouse button within 5 sec following stimulus onset in order to confirm their expectancy. Ensure scales consist of 11-points labeled from &#8220;certainly no electric shock&#8221; through &#8220;uncertain&#8221; to &#8220;certainly an electric shock&#8221;. Present the scales continuously at the bottom of the computer screen in order to support the participants to focus their attention on the pictures. When the pictures disappear the cursor should automatically return to the &#8220;uncertain&#8221; position.</li>
	<li>Direct the participants to keep their eyes on the screen and sit as still as possible with both arms placed in a relaxed position with wrists up on the desk in front of them. Put on the headphones. Dim the lights and shut the door. Start the experiment from the other room.</li>
	<li>During fear acquisition one of the two pictures co-terminates with the shock: CS1, whereas the other one is never followed by the shock: CS2. Make use of fear relevant stimuli in order to strengthen the fear association during acquisition. For instance pictures of spiders: IAPS numbers 1200 - 1201.</li>
	<li>Present both the CS1 and CS2 for 8 sec. Loud noises or startle probes &#8212; 40 msec and 104 dB &#8212; are presented 7 sec after CS onset; and for the CS1, these are followed by the 2-msec electric shock 500 msec later.</li>
	<li>Start the actual fear learning by presenting ten baseline startle probes to diminish initial startle reactivity:
	<ol>
		<li>Non-Asymptotic Learning: Present a total of 8 CS1 trials on a 75% reinforcement scheme: 6 CS1 trials are reinforced by a shock &#8212; the first and the fifth trial are unreinforced. Further present 8 CS2 trials as well as 8 NA trials during the inter-trial-intervals.</li>
		<li>Asymptotic Learning: Present a total of 3 CS1 trials on a 100% reinforcement scheme: all CS1 trials are reinforced by a shock. Further present 3 CS2 trials as well as 3 noise alone startle probes (NA) during the inter-trial-intervals.</li>
	</ol>
	</li>
	<li>Counterbalance the assignment of the pictures as CS1 and CS2 across participants. Randomize the order of trial type within blocks of CS1 - CS2 - NA such that no trial-type repeats more than two times. Vary the inter-trial-intervals&#160;&#8212; during which participants view a black screen &#8212; between 15 sec 20 sec and 25 sec with a mean of 20 sec.</li>
	<li>After completion of the experiment detach the electrodes and let the participants clean off the gel.</li>
	<li>Instruct the participants to judge the pleasantness of the shock on 11-point rating scales ranging from minus 5 (i.e., unpleasant) to 5 (i.e., pleasant).</li>
	<li>Explicitly instruct the participants to remember what they had learned during fear acquisition in order to augment retention of the CS-US contingency the following days as well as to prevent them from erroneously expecting a different contingency scheme during subsequent testing. Furthermore, to facilitate the salivary samples the next day, instruct the participants to refrain from exercise caffeine and alcohol during the 12 hr and to avoid food and drinks other than water as well as smoking and tooth brushing during the 2 hr prior to the next session.</li>
	<li>Clean the electrodes thoroughly with water after usage: get rid of all the gel without scratching any portion of the AgCl layer. After cleaning do not wipe off the water but use a different set of electrodes on the next participant and allow the other set to dry over time.</li>
</ol>
3. Memory Reactivation - Day 2
Duration about 105 min.
<ol>
	<li>Randomly assign the participants to either the propranolol HCl or placebo pill condition with the restriction that conditions are matched on STAI scores as close as possible given that trait anxiety determines the fear-reducing effects of disrupting reconsolidation45. Match the participants on SPQ scores as well when using pictures of spiders as the conditioned stimuli.</li>
	<li>Obtain blood pressures and heart rate measurements as well as salivary samples: instruct the participants just to place the swab in their mouths for about 3 min. Keep the Salivettes at minus 25&#176;C after removal.</li>
	<li>For the propranolol no-reactivation condition go to step 4.8.1.</li>
	<li>Position the participants in front of the computer screen and attach the EMG and shock electrodes (see section 1 &#34;Attachment of the Fear Potentiated Startle and Shock Electrodes&#34;). Report to the participants that the intensity of shock will remain the same.</li>
	<li>Inform the participants that the same two pictures will be presented on the computer screen and ask them to remember what they had learned the previous day &#8212; one of the pictures will again be followed by the electric shock in either most (i.e., non-asymptotic learning) or all (i.e., asymptotic learning) cases whereas the other will never be followed by the shock. Instruct the participants to report the expectancy of an electric shock during the presentation of all pictures. Put on the headphones and dim the lights. Shut the door and start the experiment from the other room.</li>
	<li>Begin the memory reactivation by presenting 10 baseline startle probes to diminish initial startle reactivity. Next present a single unreinforced CS1 trial followed by a single NA trial.</li>
	<li>Detach the participants from the experimental set-up and let them clean off the gel. Seat them behind the other table.</li>
	<li>Administer &#8220;double-blind&#8221; an oral dose of placebo pill or 40 mg of propranolol HCl. Pills should be blinded by a pharmacy or colleague independent of the study. Further a physician on call with knowledge of the protocol is considered necessary after drug administration.
	<ol>
		<li>Administer &#8220;single-blind&#8221; an oral dose of 40 mg of propranolol HCl for the propranolol no-reactivation condition.</li>
	</ol>
	</li>
	<li>Insert a resting period of about 90 min. Offer the participants magazines to read. Clean the electrodes in the meantime.</li>
	<li>Again obtain BP and heart rate measurements as well as salivary samples.</li>
</ol>
4. Test - Day 3
Duration about 45 min.
<ol>
	<li>Obtain BP and heart rate measurements.</li>
	<li>Position the participants in front of the computer screen and attach the EMG and shock electrodes. Again report to the participants that the intensity of shock will remain the same.</li>
	<li>Instruct the participants that the same two pictures will be presented on the computer screen. Again instruct the participants to report the expectancy of an electric shock during the presentation of all pictures. Put on the headphones and dim the lights. Shut the door and start the experiment from the other room.</li>
	<li>Start the experiment by presenting 10 baseline startle probes. During extinction present about 12 unreinforced CS1 and CS2 trials as well as 12 NA trials. After extinction participants get three unsignaled USs: reinstatement. Again present the participants with about 4 unreinforced CS1 and CS2 trials as well as 4 NA trials following the unsignaled USs: reinstatement testing.</li>
	<li>Detach the electrodes and let participants clean off the gel. Pay the participants a small amount for their participation in the experiment. Clean the electrodes thoroughly with water.</li>
</ol>

Authors have nothing to disclose.

In a series of studies we consistently demonstrate that, irrespective of gender, 40 mg of the noradrenergic beta-blocker propranolol either administered prior to or after memory reactivation effectively neutralized the conditioned fear responding (i.e., defensive startle response). None of the four mechanisms of relapse &#8212; reinstatement, renewal, spontaneous recovery and rapid reacquisition&#160;&#8212; were observed after disrupting reconsolidation with propranolol27-32,37. It is noteworthy that the fear-neutralizing effects were only observed for the defensive startle response, but neither for the threat expectancy rating nor for the skin conductance. The data for the electrodermal conditioning are not reported here as the general patterns for the SCR did not significantly deviate from the expectancy ratings. In human fear conditioning research, multiple indices of conditioned responding (e.g., US-expectancy, SCR, startle response, pupil dilation, neural activity) are usually obtained for reasons of cross-validation46. However, there is now convincing evidence that these different response levels do not necessarily act in concert and may even dissociate from each other3,27-32,34,37,47. Note that the startle response is an automatic defensive reflex, which is potentiated in response to a CS that is associated with a US of negative valence and can generally not be observed with USs of neutral or positive valence47,48 (e.g., vibrotactile stimulation or reaction time task). Hence, potentiation of the defensive startle reflex is a reliable and specific index of aversive conditioning34. In contrast, SCR conditioning can occur irrespective of the valence of the US47,48. Given that affective valence of the US does not modify SCR, electrodermal conditioning is a non-specific measure of anticipatory arousal. We therefore believe that the SCR is less suited as behavioral measure in human aversive conditioning research.
If we speculate on translating these findings into clinical practice, several issues and potential limitations are to be considered. First, it may be questioned whether a pharmacological manipulation by the noradrenergic beta-blocker is really necessary to disrupt the process of reconsolidation or whether a behavioral procedure aimed to interfere with reconsolidation would yield a similar neutralizing effect. Even though a one-session treatment of a low dose of propranolol is clearly nontoxic, an entirely behavioral procedure is always preferable over a pharmacological intervention. There is indeed an alternative method where extinction training is presented within the window of reconsolidation49. Several studies failed however to replicate these original findings by Schiller et al.29,50-52 but see 53,54. In addition to these conflicting results, another potential limitation of the extinction within reactivation procedure would be that in clinical practice the fear response is generally not extinguished in a one-session exposure treatment. For instance, imaginary exposure for patients with PTSD traditionally takes ten sessions before the fear subsides55. Thus, even acknowledging the obvious disadvantages of a simple pharmacological treatment in comparison to an entirely behavioral intervention, we believe that the noradrenergic manipulation of memory reconsolidation seems to be more feasible than extinction within the reconsolidation window.
A second issue concerns the optimal conditions to trigger memory reconsolidation. There is growing evidence that the mechanisms mediating the behavioral expression of fear are clearly dissociated from the mechanisms mediating the process of reconsolidation31,32,56-60. For instance, recent animal studies uncovered differential and dissociable receptors in the basolateral amygdala mediating the expression, destabilization and restabilization of previously conditioned fear memories61,62. A behavioral expression of fear memory is not only dissociated from processes mediating memory reactivation (i.e., access to a memory trace), it seems also not being indispensable for reconsolidation to occur61. As such, fear expression during memory reactivation is not informative on whether the memory trace enters a labile phase. Given that memory destabilization is a prerequisite for the noradrenergic beta-blocker to interfere with the restabilization process, an important question is how we can infer successful memory destabilization in clinical practice. A host of findings indicate that a crucial factor in inducing reconsolidation and in demarcating reconsolidation from either memory retrieval or the consolidation of a novel memory38,63 is the degree of prediction error induced during memory retrieval31,32. But given that no objective criterion is available to determine the optimal degree of prediction error in clinical practice, the current laboratory findings cannot easily be translated to treatment protocols.
Another challenge for translating the neuroscience literature into clinical practice concerns the ecological validity of the paradigm. The evidence for disrupting reconsolidation has mainly been shown in animals and humans for relatively new (one day old) and simple fear memories (i.e., tone shock; picture shock). It is not self-evident that disrupting reconsolidation of older, stronger and broader memory networks such as in patients with PTSD is as effective as it has been shown in the laboratory for cued fear conditioning. Also with respect to the dependent variable it is still unclear whether the observations from the animal and human laboratory studies generalize to patients with anxiety disorders. The fear reducing effects are thus far mainly demonstrated for the behavioral expression of aversive conditioning (i.e., freezing behavior in rodents or defensive startle reflex in humans), with only one exception where we demonstrated that also the subjective feelings of distress were significantly neutralized by noradrenergic blockade of memory reconsolidation37. It may be questioned whether these fear-reducing effects in the laboratory are indicative of the typical experiences of fear and avoidance behavior characteristic of patients with anxiety disorders. Future research should investigate whether the current findings indeed generalize to avoidance behaviors, one of the central symptoms of anxiety disorders.
In sum, although the Pavlovian aversive conditioning procedure is an excellent tool to study the basic mechanisms of fear learning and memory, we cannot easily translate the laboratory findings into clinical practice. The insights that we have been acquiring on the optimal, boundary and necessary conditions for memory reconsolidation should only be considered as a starting point for the development of reconsolidation-based treatments. On the other hand, the extensive research on extinction training has resulted in extinction-based exposure interventions, which still belong to the most effective treatments for anxiety disorders and other related disorders. Given that the noradrenergic blockade of memory reconsolidation overshadows the anxiolytic effect of extinction learning, disrupting reconsolidation points to a promising new intervention to effectively reduce excessive and irrational fears.

Our brains are programmed to learn. Humans are well equipped with the ability to learn the potential dangers in life and perhaps even more important to learn the predictors of danger. Pavlovian aversive conditioning is an excellent tool to study associative fear learning not only in humans but also across a wide range of organisms1,2. This procedure involves presenting an innocuous biologically neutral conditioned stimulus (CS, e.g., a tone or picture) with a noxious or harmful unconditioned stimulus (US), typically a mild electrical shock. If the CS becomes a reliable predictor of the US, the CS will elicit species-typical conditioned behavioral responses (e.g., freezing in rats and potentiated startle reflex in humans), which are conceptualized as expressions of fear (see 3 for critical comments on this terminology). Not only does aversive conditioning research add to a better understanding of the molecular and cellular processes of associative fear learning and memory4 but it also provides a foundation for understanding the etiology and course of anxiety disorders5. Though it bears mentioning that anxiety disorders do not necessarily result from direct conditioning experiences such as traumatic events. They may also result from indirect or vicarious fear learning experiences6. But irrespective of the learning history, associative fear memory lies at the core of anxiety disorders. The Pavlovian conditioning paradigm has not only proven its utility in understanding the origin of anxiety disorders, it is also an excellent translational model to develop and advance treatment for anxiety disorders5.
In the laboratory the two most extensively studied procedures to reduce learned fear are (1) extinction and (2) disruption of reconsolidation. Even though extinction training &#8212; i.e., the repeated unreinforced re-exposure to the CS7,8 &#8212; is an effective anxiolytic strategy, animal and human fear conditioning studies reliably show that the conditioned fear response can easily return by re-exposure to unsignaled USs (i.e., reinstatement), a context change (i.e., renewal), or the passage of time (i.e., spontaneous recovery)7,9,10. A consensus has been building that extinction learning does not erase the original fear memory but instead reflects the formation of a new inhibitory memory. As a consequence, the fear memory may resurface resulting in a return of fear even after originally successful fear extinction. The various sources of relapse are still a major challenge for clinical practice.
An alternative approach to diminish conditioned fear responding is the more recently (re)discovered procedure of disrupting memory reconsolidation by pharmacological agents. This procedure is very promising as it not only diminishes conditioned fear responding but it even seems to erase associative fear memory, which might eventually solve the problem of relapse.
Memory reconsolidation refers to a two-phase process by which previously consolidated memories transfer to a transient destabilized state at retrieval and require a time-dependent restabilization to persist14-18. Gene transcription, protein and RNA synthesis are necessary for this restabilization and offer a window of opportunity for amnestic agents to affect the fear memory. Amnesia for learned fear has been demonstrated in animals by drugs (e.g., anisomycin) targeting the required protein synthesis14,19,20 or the release of neurotransmitters21,22. Protein synthesis may also be disturbed by the noradrenergic beta-blocker propranolol, which is supposed to inhibit the noradrenaline-stimulated CREB phosphorylation23-25. The fear reducing effects of propranolol have been demonstrated in animals and humans26-32. In a series of discriminative fear-conditioning studies, we consistently demonstrated that propranolol (40 mg) administered prior to or after memory reactivation effectively reduced the conditioned startle fear response and prevented the return of fear in healthy participants.
Proof of Principle
The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive days separated by at least 24 hr in order to (1) support (re)consolidation of the memories and (2) allow the drug to wash out before testing. On day 1 - the fear acquisition phase, healthy participants are exposed to a series of picture presentations. One picture stimulus (CS1+) is repeatedly paired with an aversive electric stimulus (US), resulting in the acquisition of a fear association, whereas another picture stimulus (CS2-) is never followed by an US. On day 2 - the memory reactivation phase, re-exposure to the unreinforced conditioned stimulus (CS1-) typically triggers a conditioned fear response. In this phase, we systemically administer 40 mg propranolol HCl, a &#946;-adrenergic receptor antagonist that indirectly targets the protein synthesis required for reconsolidation25. In view of the peak plasma concentrations of propranolol33, we administered an oral dose of propranolol 90 min prior to the reactivation of the fear memory in our first experiments. However, for a more optimal test of reconsolidation, the amnestic agent should be administered after the memory reactivation. Therefore, in our latter experiments we always administered propranolol after memory reactivation with very similar results. On day 3 - the test phase, the retention of the fear memory is tested 24 hr after the intervention (i.e., first test trial day 3), followed by an extinction procedure and situational triggers to test for the return of fear (i.e., reinstatement, renewal, spontaneous recovery, rapid reacquisition). For determining whether the effect of propranolol requires active retrieval of the fear memory, the drug should be administered to another fear-conditioned group without reactivation of the memory (i.e., propranolol no-reactivation condition)27,28. We used the potentiation of the startle reflex as a measure for conditioned fear responding and online US expectancy ratings as an index for contingency learning. The potentiation of the startle reflex is conceived as a specific and reliable index of fear34, subserved by the amygdala35. The most commonly used startle-eliciting stimulus is the &#8220;startle probe&#8221;, a loud noise that is presented through headphones during a stimulus or in the interval between two stimulus presentations (i.e., intertrial intervals)36. Stronger startle responses to the loud noise during the fear conditioned stimulus (CS1+) as compared to the control stimulus (CS2-) reflects the fearful state of the participant elicited by the feared stimulus (CS1+).
In a series of 10 consecutive experiments in independent samples 27-32,37 we consistently replicated our original finding where disrupting reconsolidation of fear memory by a beta-adrenergic blocker propranolol HCl effectively reduced the conditioned startle reflex and prevented the return of fear27. The observations that exposure to primary reinforcers (i.e., reinstatement), a change in context (i.e., renewal) or simple the passage of time (i.e., spontaneous recovery) did not lead to the re-emergence of conditioned fear responding as is generally observed after extinction training, support the superiority of disrupting reconsolidation over extinction learning. In addition to these retrieval techniques, reacquisition learning did not reveal any savings of the previously learned fear response. In sum, these findings suggest that disrupting reconsolidation of associative fear memory by propranolol effectively reduced the emotional expression of fear memory.
Furthermore, we tested several boundary and necessary conditions for disrupting reconsolidation: (1) The memory reactivation session seems procedurally similar to extinction training (i.e., unreinforced exposure), but it should involve less unreinforced trials than extinction training because noradrenergic beta-blockers (i.e., propranolol) may also interfere with the formation of extinction memory instead of targeting the original fear memory38,39; (2) Memory retrieval is not sufficient for memory reconsolidation18,31,40,41. Propranolol only reduced the conditioned fear responding when there was something to be learned during the retrieval session31,32. A discrepancy between what has already been learned and what can be learned on a given retrieval session (i.e., prediction error) seems to be necessary for inducing reconsolidation of associative fear memory32. The results indicate that the occurrence of a prediction error is a necessary condition for reconsolidation and provide a useful instrument for developing and optimizing reconsolidation-based treatments for patients suffering from anxiety disorders.

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:231px;">Name of Material - Equipment</td>
			<td style="width:335px;">Company</td>
			<td style="width:267px;">Catalog Number</td>
			<td style="width:487px;">Comments - Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 computers with 4 screens</td>
			<td>Dell</td>
			<td>Optiplex 9010</td>
			<td style="width:487px;">Recording and monitoring physiological responses. Presenting the experimental script.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amplifier</td>
			<td>Developed by B. Molenkamp - University of Amsterdam</td>
			<td></td>
			<td style="width:487px;">Designed around a Burr Brown INA101 amplifier and ISO103 isolation stage.&#160;</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;">VSSRP98&#160;</td>
			<td>Developed by B. Molenkamp - University of Amsterdam</td>
			<td></td>
			<td style="width:487px;">Physiological registration software. Record electromyography - EMG - activity using a bundled pair of electrodes wires connected to a front-end amplifier with an imput resistacne of 10 M&#937; and a bandwidth of DC-1500 Hz. Raw EMG signals are integrated in the amplifier. Integrated EMG signals are sampled at 1000 Hz and used for data analysis.</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td style="width:487px;">Analyzing data. Peak amplitudes are determined by taking the baseline 50 ms before probe onset to peak differences within 30 - 150 ms following probe onset and are recorded in microvolt.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Presentation</td>
			<td>NeuroBehavioral Systems Inc. - USA</td>
			<td></td>
			<td style="width:487px;">Stimulus presentation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Constant current stimulator&#160;</td>
			<td>Digitimer - UK</td>
			<td>DS7A</td>
			<td style="width:487px;">Generates electrical stimulation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shock electrodes</td>
			<td>Made by B. Molenkamp - University of Amsterdam</td>
			<td></td>
			<td style="width:487px;">Ag electrodes of 20 mm * 25 mm with fixed inter-electrodes mid-distance of 45 mm.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Headphones</td>
			<td>Sennheiser Electronic GmbH &#38; CO - Germany</td>
			<td>HD 25-1 II</td>
			<td style="width:487px;">Presentation of startle probse and background noises.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EMG electrodes</td>
			<td>Made by B. Molenkamp - University of Amsterdam</td>
			<td></td>
			<td style="width:487px;">Three 7 mm sintered Ag-AgCl electrodes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Double-sided adhesive collars</td>
			<td>MedCaT - the Netherlands</td>
			<td>848125</td>
			<td style="width:487px;">13-mm x 5-mm. For attaching the EMG electrodes to the skin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conductive gel</td>
			<td>Signa Gel - Parker Laboratories Inc. - USA&#160;</td>
			<td>224.550.011</td>
			<td style="width:487px;">Facilitates conduction from the skin to both the EMG and shock electrodes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol swabs&#160;</td>
			<td>Sanadep 0.5 % - Microtek Medical - the Netherlands</td>
			<td>3053800</td>
			<td style="width:487px;">For cleaning the skin of the participant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sphygmomanometer</td>
			<td>Omron Healthcare Europe B.V. - the Netherlands</td>
			<td>M4-I HEM-752-E</td>
			<td style="width:487px;">Measuring blood pressures and heart rate.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton salivettes</td>
			<td>Sarstedt - Germany</td>
			<td>511.534</td>
			<td style="width:487px;">Obtaining salivary samples.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved tip syringe</td>
			<td>Monoject - Covidien - USA</td>
			<td>412</td>
			<td style="width:487px;">Applies gel to EMG and shock electrodes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propranolol HCl - 40 mg and placebo pills&#160;</td>
			<td>Huygens Apotheek - the Netherlands</td>
			<td></td>
			<td style="width:487px;">Pills should be identical in exterior.</td>
		</tr>
	</tbody>
</table>

disrupting reconsolidation of fear memory in humans by a noradrenergic &#946;-blocker

The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive days. On day 1 - the fear acquisition phase, healthy participants are exposed to a series of picture presentations. One picture stimulus (CS1+) is repeatedly paired with an aversive electric stimulus (US), resulting in the acquisition of a fear association, whereas another picture stimulus (CS2-) is never followed by an US. On day 2 - the memory reactivation phase, the participants are re-exposed to the conditioned stimulus without the US (CS1-), which typically triggers a conditioned fear response. After the memory reactivation we administer an oral dose of 40 mg of propranolol HCl, a &#946;-adrenergic receptor antagonist that indirectly targets the protein synthesis required for reconsolidation by inhibiting the noradrenaline-stimulated CREB phosphorylation. On day 3 - the test phase, the participants are again exposed to the unreinforced conditioned stimuli (CS1- and CS2-) in order to measure the fear-reducing effect of the manipulation. This retention test is followed by an extinction procedure and the presentation of situational triggers to test for the return of fear. Potentiation of the eye blink startle reflex is measured as an index for conditioned fear responding. Declarative knowledge of the fear association is measured through online US expectancy ratings during each CS presentation. In contrast to extinction learning, disrupting reconsolidation targets the original fear memory thereby preventing the return of fear. Although the clinical applications are still in their infancy, disrupting reconsolidation of fear memory seems to be a promising new technique with the prospect to persistently dampen the expression of fear memory in patients suffering from anxiety disorders and other psychiatric disorders.

Manipulation check propranolol: Contrary to pill placebo, both the systolic and diastolic BP as well as the salivary alpha amylase should decrease 90 min after the propranolol intake during memory reactivation, which indicates that the drug manipulation exerted its intended physiological effect. BP and salivary alpha amylase should again return to baseline levels at the test on day 3.
US expectancy ratings: With the current protocol you may not expect any effects of the propranolol treatment on the US expectancy ratings. Irrespective of the pharmacological treatment, the differential US expectancy ratings should increase during fear acquisition and decrease during extinction learning 48 hr later. Moreover the presentation of the unsignaled USs is expected to result in a return of the differential expectancy ratings (Figure&#160;1A-C).
Prerequisite for reconsolidation: In order to trigger the process of reconsolidation, the memory trace should firstly be destabilized as a necessary condition for the restabilization phase. For fear memory destabilization, an expectation of threat at the moment of reactivation (i.e., intact CS1-US expectancy) is necessary31 though not sufficient. Pursuing on the idea that the function of reconsolidation is to update the memory trace to an ever-changing environment, the memory reactivation should also involve a prediction error (PE: a mismatch between what is expected and what actually happens). Given that the US expectancy ratings remain unaffected by the propranolol manipulation, changes in threat expectations from acquisition to test (PE) may serve as an independent indicator of memory destabilization (see 32 for more details). But only when the memory reactivation follows asymptotic learning, these changes in threat expectation may be observed. In case of partially reinforced non-asymptotic learning, a shift in threat expectancies is not necessary for memory destabilization. Hence a retrieval trial may also trigger reconsolidation without a change in expectancies after non-asymptotic learning.
Startle fear responding: Successful fear acquisition on day 1 is demonstrated by an increase in differential startle fear responding (i.e., CS1 vs. CS2) from the first to the last trial of acquisition (Figure&#160;1D-F). Higher startle fear responding to the stimulus that was previously coupled with the shock (CS1) compared to noise alone during memory reactivation on day 2 further indicates that the fear memory is well consolidated.
During the test phase on day 3 you may expect intact startle fear responding in the placebo group: startle responding to the CS1 should be higher compared to the CS2 at the beginning of test. Extinction training subsequently reduces the differential startle responding (i.e., CS1 vs. CS2). Moreover the presentation of the unsignaled USs should result in a return of fear responding to the CS1 compared to the CS2 (Figure&#160;1D).
Contrary to pill placebo, the administration of propranolol HCl is expected to result in an elimination of the differential startle fear responding (i.e., CS1 vs. CS2) at the beginning of the test phase on day 3. Furthermore you may expect that the unsignaled USs will not recover the startle fear responding (Figure&#160;1E).
Note that propranolol should not have any fear-reducing effects when the pill is administered without memory reactivation on day 2 (Figure&#160;1F).
<img alt="Figure 1" src="/files/ftp_upload/52151/52151fig1highres.jpg" width="700px" /> 
Figure 1. US expectancy ratings to the CS1 and CS2 trials and startle fear responses to the CS1, CS2 and noise alone (NA) trials during acquisition, memory reactivation, extinction and test for the placebo (A,D), propranolol (B,E), and propranolol no- reactivation (C,F) groups. R-CS1- refers to an unreinforced reactivation trial. <a href="https://www.jove.com/files/ftp_upload/52151/52151fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Disrupting Reconsolidation of Fear Memory in Humans by a Noradrenergic &#946;-Blocker

Disrupting reconsolidation is a promising approach to dampen the behavioral expression of fear memory in patients with anxiety disorders or posttraumatic stress disorder. In a series of human fear conditioning studies we showed that disrupting reconsolidation by the noradrenergic &#946;-blocker propranolol is very effective in erasing conditioned fear responding.

The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive ...

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Research

JoVE Journal

Behavior

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.

This article presents a protocol for a contextual and cued fear conditioning test using a video analyzing system to assess fear learning and memory in mice.

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

Studying Food Reward and Motivation in Humans

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as hedonics, motivation, and goal value in more objective ways. This is particularly relevant for the understanding of overeating and obesity as well as their potential treatments. In this paper are described a set of measures of food-related motivation using handgrip force as a motivational measure. These methods can be used to examine changes in food related motivation with metabolic (satiety) and pharmacological manipulations and can be used to evaluate interventions targeted at overeating and obesity. However to understand food-related decision making in the complex food environment it is essential to be able to ascertain the reward goal values that guide the decisions and behavioral choices that people make. These values are hidden but it is possible to ascertain them more objectively using metrics such as the willingness to pay and a method for this is described. Both these sets of methods provide quantitative measures of motivation and goal value that can be compared within and between individuals.

This article describes a set of methods for the measurement of food related motivation and food related goal values in humans.

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as ...

A Cognitive Paradigm to Investigate Interference in Working Memory by Distractions and Interruptions

Goal-directed behavior is often impaired by interference from the external environment, either in the form of distraction by irrelevant information that one attempts to ignore, or by interrupting information that demands attention as part of another (secondary) task goal. Both forms of external interference have been shown to detrimentally impact the ability to maintain information in working memory (WM). Emerging evidence suggests that these different types of external interference exert different effects on behavior and may be mediated by distinct neural mechanisms. Better characterizing the distinct neuro-behavioral impact of irrelevant distractions versus attended interruptions is essential for advancing an understanding of top-down attention, resolution of external interference, and how these abilities become degraded in healthy aging and in neuropsychiatric conditions. This manuscript describes a novel cognitive paradigm developed the Gazzaley lab that has now been modified into several distinct versions used to elucidate behavioral and neural correlates of interference, by to-be-ignored distractors versus to-be-attended interruptors. Details are provided on variants of this paradigm for investigating interference in visual and auditory modalities, at multiple levels of stimulus complexity, and with experimental timing optimized for electroencephalography (EEG) or functional magnetic resonance imaging (fMRI) studies. In addition, data from younger and older adult participants obtained using this paradigm is reviewed and discussed in the context of its relationship with the broader literatures on external interference and age-related neuro-behavioral changes in resolving interference in working memory.

A novel cognitive paradigm is developed to elucidate behavioral and neural correlates of interference by to-be-ignored distractors versus interference by to-be-attended interruptors during a working memory task. In this manuscript, several variants of this paradigm are detailed, and data obtained with this paradigm in younger/older adult participants is reviewed.

Goal-directed behavior is often impaired by interference from the external environment, either in the form of distraction by irrelevant information that ...

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study demonstrates a quantitative sensory testing protocol using techniques to measure tactile sensation and pain threshold for pressure and heat using portable and easily accessed equipment. These techniques and equipment are ideal for new laboratories and clinics where cost is a concern or a limiting factor. We demonstrate measurement techniques for the following: cutaneous mechanical sensitivity on the arms and legs (von-Frey filaments), radiant and contact heat sensitivity (with both threshold and qualitative assessments using the Visual Analog Scale (VAS)), and mechanical pressure sensitivity (algometer, with both threshold and the VAS). The techniques and equipment described and demonstrated here can be easily purchased, stored, and transported by most clinics and research laboratories around the world. A limitation of this approach is a lack of automation or computer control. Thus, these processes can be more labor intensive in terms of personnel training and data recording than the more sophisticated equipment. We provide a set of reliability data for the demonstrated techniques. From our description, a new laboratory should be able to set up and run these tests and to develop their own internal reliability data.

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to obtain.

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study ...

A Real-world What-Where-When Memory Test

Episodic memory is a complex memory system which allows recall and mental re-experience of previous episodes from one&#39;s own life. Real-life episodic memories are about events in their spatiotemporal context and are typically visuospatial, rather than verbal. Yet often, tests of episodic memory use verbal material to be recalled (word lists, stories). The Real-World What-Where-When memory test requires participants to hide a total of 16 different objects in 16 different locations over two temporal occasions, 2 h apart. Another two hours later, they are then asked to recall which objects (What) they had hidden in which locations (Where) and on which of the two occasions (When). In addition to counting the number of correctly recalled complete what-where-when combinations, this task can also be used to test real-world spatial memory and object memory. This task is sensitive to normal cognitive aging, and correlates well with performance on other episodic memory tasks, while at the same time providing more ecological validity and being cheap and easy to run.

A Real-world What-Where-When Memory Test

The Real-World What-Where-When memory test is a novel episodic memory test, in which participants need to recall which objects have been hidden in which locations on which of two distinct occasions. It is easy to run and is sensitive to normal cognitive aging.

Episodic memory is a complex memory system which allows recall and mental re-experience of previous episodes from one&#39;s own life. Real-life episodic ...

Reducing State Anxiety Using Working Memory Maintenance

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working Memory (WM) and the threat of shock paradigms. In the Sternberg WM paradigm, subjects are required to maintain a series of letters in the WM for a brief interval and respond by identifying whether the position of a given letter in the series matches a numerical prompt. In the threat of shock paradigm, subjects are exposed to alternating blocks where they are either at risk of receiving unpredictable presentations of a mild electric shock or are safe from the shock. Anxiety is probed throughout the safe and threat blocks using the acoustic startle reflex, which is potentiated under threat (Anxiety-Potentiated Startle (APS)). By conducting the Sternberg WM paradigm during the threat of shock and probing the startle response during either the WM maintenance interval or the intertrial interval, it is possible to determine the effect of WM maintenance on APS.

This protocol demonstrates how to measure anxiety-potentiated startle during the Sternberg Working Memory paradigm.

The purpose of this protocol is to explain how to examine the relationship between working memory processes and anxiety by combining the Sternberg Working ...

Frame-by-Frame Video Analysis of Idiosyncratic Reach-to-Grasp Movements in Humans

Prehension, the act of reaching to grasp an object, is central to the human experience. We use it to feed ourselves, groom ourselves, and manipulate objects and tools in our environment. Such behaviors are impaired by many sensorimotor disorders, yet our current understanding of their neural control is far from complete. Current technologies for investigating human reach-to-grasp movements often utilize motion tracking systems that can be expensive, require the attachment of markers or sensors to the hands, impede natural movement and sensory feedback, and provide kinematic output that can be difficult to interpret. While generally effective for studying the stereotypical reach-to-grasp movements of healthy sighted adults, many of these technologies face additional limitations when attempting to study the unpredictable and idiosyncratic reach-to-grasp movements of young infants, unsighted adults, and patients with neurological disorders. Thus, we present a novel, inexpensive, and highly reliable yet flexible protocol for quantifying the temporal and kinematic structure of idiosyncratic reach-to-grasp movements in humans. High speed video cameras capture multiple views of the reach-to-grasp movement. Frame-by-frame video analysis is then used to document the timing and magnitude of pre-defined behavioral events such as movement start, collection, maximum height, peak aperture, first contact, and final grasp. The temporal structure of the movement is reconstructed by documenting the relative frame number of each event while the kinematic structure of the hand is quantified using the ruler or measure function in photo editing software to calibrate 2 dimensional linear distances between two body parts or between a body part and the target. Frame-by-frame video analysis can provide a quantitative and comprehensive description of idiosyncratic reach-to-grasp movements and will enable researchers to expand their area of investigation to include a greater range of naturalistic prehensile behaviors, guided by a wider variety of sensory modalities, in both healthy and clinical populations.

This protocol describes how to use frame-by-frame video analysis to quantify idiosyncratic reach-to-grasp movements in humans. A comparative analysis of reaching in sighted versus unsighted healthy adults is used to demonstrate the technique, but the method can also be applied to the study of developmental and clinical populations.

Prehension, the act of reaching to grasp an object, is central to the human experience. We use it to feed ourselves, groom ourselves, and manipulate ...

Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders such as post-traumatic stress disorder (PTSD). To understand the biological mechanisms of fear dysregulation in PTSD, it is important to start with a valid animal model of the disorder. This protocol describes the methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of PTSD, in both rats and mice. SEFL was developed to recapitulate critical aspects of PTSD, including long-term sensitization of fear learning caused by an acute stressor. SEFL uses aspects of Pavlovian fear conditioning but produces a distinct and robust sensitized fear response far greater than normal conditional fear responses. The trauma procedure involves placing a rodent in a conditioning chamber and administering 15 unsignaled shocks randomly distributed over 90 minutes (for rat experiments; for mouse experiments, 10 unsignaled shocks randomly distributed over 60 minutes are used). On day 2, rodents are placed in a novel conditioning context where they receive a single shock; then, on day 3 they are placed back in the same context as on day 2 and tested for changes in freezing levels. Rodents that previously received the trauma display enhanced levels of freezing on the test day compared to those that received no shocks on the first day. Thus, with this model, a single highly stressful experience (the trauma) produces extreme fear of the stimuli associated with the traumatic event.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

Fear behaviors are important for survival, but disproportionately high levels of fear can increase the vulnerability for developing psychiatric disorders ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation&#8217;s behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the ...