This work is supported by a Baylor University Research Council grant and from a research grant from the Epilepsy Foundation.

The mice used in the following experiment were generated and housed at Baylor University at an ambient temperature of 22 &#176;C, with a 14&#160;hr light and 10&#160;hr dark (20:00 to 6:00 hr) diurnal cycle. The mice were given ad libitum access to food and water. All procedures to the mice were in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the animal protocol was approved by Baylor University Animal Care and Use Committee.
<p class="jove_step"...

<ol>
	<li>Powell, R. A., Honey, P. L., Symbaluk, D. 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> Introduction to learning and behavior. , .</li><li>Tsaltas, E., Preston, G. C., Gray, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+dorsal+bundle+lesions+on+serial+and+trace+conditioning.">The effects of dorsal bundle lesions on serial and trace conditioning.</a> Behav. Brain Res. 10, 361-374 (1983).</li><li>McAlonan, G. M., Dawson, G. R., Wilkinson, L. O., Robbins, T. W., Everitt, 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=The+effects+of+AMPA-induced+lesions+of+the+medial+septum+and+vertical+limb+nucleus+of+the+diagonal+band+of+Broca+on+spatial+delayed+non-matching+to+sample+and+spatial+learning+in+the+water+maze.">The effects of AMPA-induced lesions of the medial septum and vertical limb nucleus of the diagonal band of Broca on spatial delayed non-matching to sample and spatial learning in the water maze.</a> Eur. J. Neurosci. 7, 1034-1049 (1995).</li><li>Chowdhury, N., Quinn, J. J., Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+hippocampus+involvement+in+trace+fear+conditioning+with+long,+but+not+short,+trace+intervals+in+mice.">Dorsal hippocampus involvement in trace fear conditioning with long, but not short, trace intervals in mice.</a> Behav. Neurosci. 119, 1396-1402 (2005).</li><li>Quinn, J. J., Oommen, S. S., Morrison, G. E., Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-training+excitotoxic+lesions+of+the+dorsal+hippocampus+attenuate+forward+trace,+backward+trace,+and+delay+fear+conditioning+in+a+temporally+specific+manner.">Post-training excitotoxic lesions of the dorsal hippocampus attenuate forward trace, backward trace, and delay fear conditioning in a temporally specific manner.</a> Hippocampus. 12, 495-504 (2002).</li><li>Misane, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+involvement+of+the+dorsal+hippocampus+in+trace+fear+conditioning+in+mice.">Time-dependent involvement of the dorsal hippocampus in trace fear conditioning in mice.</a> Hippocampus. 15, 418-426 (2005).</li><li>Quinn, J. J., Loya, F., Ma, Q. D., Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+hippocampus+NMDA+receptors+differentially+mediate+trace+and+contextual+fear+conditioning.">Dorsal hippocampus NMDA receptors differentially mediate trace and contextual fear conditioning.</a> Hippocampus. 15, 665-674 (2005).</li><li>McEchron, M. D., Bouwmeester, H., Tseng, W., Weiss, C., Disterhoft, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampectomy+disrupts+auditory+trace+fear+conditioning+and+contextual+fear+conditioning+in+the+rat.">Hippocampectomy disrupts auditory trace fear conditioning and contextual fear conditioning in the rat.</a> Hippocampus. 8, 638-646 (1998).</li><li>Wiltgen, B. J., Sanders, M. J., Ferguson, C., Homanics, G. E., Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+fear+conditioning+is+enhanced+in+mice+lacking+the+delta+subunit+of+the+GABAA+receptor.">Trace fear conditioning is enhanced in mice lacking the delta subunit of the GABAA receptor.</a> Learn. Mem. 12, 327-333 (2005).</li><li>Davis, R. 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=Genetic+basis+for+susceptibility+to+noise-induced+hearing+loss+in+mice.">Genetic basis for susceptibility to noise-induced hearing loss in mice.</a> Hear. Res. 155, 82-90 (2001).</li><li>Zheng, Q. Y., Johnson, K. R., Erway, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+hearing+in+80+inbred+strains+of+mice+by+ABR+threshold+analyses.">Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses.</a> Hear. Res. 130, 94-107 (1999).</li><li>Moyer, J. R., Thompson, L. T., Disterhoft, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+eyeblink+conditioning+increases+CA1+excitability+in+a+transient+and+learning-specific+manner.">Trace eyeblink conditioning increases CA1 excitability in a transient and learning-specific manner.</a> 16, 5536-5546 (1996).</li><li>Leuner, B., Falduto, J., Shors, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+memory+formation+increases+the+observation+of+dendritic+spines+in+the+hippocampus.">Associative memory formation increases the observation of dendritic spines in the hippocampus.</a> J. Neurosci. 23, 659-665 (2003).</li><li>McEchron, M. D., Disterhoft, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+encoding+of+non-spatial+trace+conditioning.">Hippocampal encoding of non-spatial trace conditioning.</a> Hippocampus. 9, 385-396 (1999).</li><li>McLaughlin, J., Skaggs, H., Churchwell, J., Powell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medial+prefrontal+cortex+and+pavlovian+conditioning:+trace+versus+delay+conditioning.">Medial prefrontal cortex and pavlovian conditioning: trace versus delay conditioning.</a> Behav. Neurosci. 116, 37-47 (2002).</li><li>Runyan, J. D., Moore, A. N., Dash, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+prefrontal+cortex+in+memory+storage+for+trace+fear+conditioning.">A role for prefrontal cortex in memory storage for trace fear conditioning.</a> J. Neurosci. 24, 1288-1295 (2004).</li><li>Gilmartin, M. R., McEchron, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+neurons+in+the+medial+prefrontal+cortex+of+the+rat+exhibit+tonic+and+phasic+coding+during+trace+fear+conditioning.">Single neurons in the medial prefrontal cortex of the rat exhibit tonic and phasic coding during trace fear conditioning.</a> Behav. Neurosci. 119, 1496-1510 (2005).</li><li>Crow, T., Xue-Bian, J. J., Siddiqi, V., Kang, Y., Neary, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+mitogen-activated+protein+kinase+by+one-trial+and+multi-trial+classical+conditioning.">Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning.</a> J. Neurosci. 18, 3480-3487 (1998).</li><li>Martin, K. C., 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=MAP+kinase+translocates+into+the+nucleus+of+the+presynaptic+cell+and+is+required+for+long-term+facilitation+in+Aplysia.">MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia.</a> Neuron. 18, 899-912 (1997).</li><li>Crestani, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+fear+conditioning+involves+hippocampal+alpha5+GABA(A)+receptors.">Trace fear conditioning involves hippocampal alpha5 GABA(A) receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 8980-8985 (2002).</li><li>Crestani, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+GABAA-receptor+clustering+results+in+enhanced+anxiety+and+a+bias+for+threat+cues.">Decreased GABAA-receptor clustering results in enhanced anxiety and a bias for threat cues.</a> Nat. Neurosci. 2, 833-839 (1999).</li><li>Moore, M. D., 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=Trace+and+contextual+fear+conditioning+is+enhanced+in+mice+lacking+the+alpha4+subunit+of+the+GABA(A)+receptor.">Trace and contextual fear conditioning is enhanced in mice lacking the alpha4 subunit of the GABA(A) receptor.</a> Neurobiol. Learn. Mem. 93, 383-387 (2010).</li><li>Cushman, J. D., Moore, M. D., Jacobs, N. S., Olsen, R. W., Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+pharmacogenetic+analysis+on+the+role+of+the+alpha4+GABA(A)+receptor+subunit+in+the+ethanol-mediated+impairment+of+hippocampus-dependent+contextual+learning.">Behavioral pharmacogenetic analysis on the role of the alpha4 GABA(A) receptor subunit in the ethanol-mediated impairment of hippocampus-dependent contextual learning.</a> Alcohol Clin. Exp. Res. 35, 1948-1959 (2011).</li><li>Raybuck, J. D., Lattal, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+dissociation+of+amygdala+and+hippocampal+contributions+to+trace+and+delay+fear+conditioning.">Double dissociation of amygdala and hippocampal contributions to trace and delay fear conditioning.</a> PLoS ONE. 6, (2011).</li><li>Kwapis, J. L., Jarome, T. J., Schiff, J. C., Helmstetter, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+consolidation+in+both+trace+and+delay+fear+conditioning+is+disrupted+by+intra-amygdala+infusion+of+the+protein+synthesis+inhibitor+anisomycin.">Memory consolidation in both trace and delay fear conditioning is disrupted by intra-amygdala infusion of the protein synthesis inhibitor anisomycin.</a> Learn. Mem. 18, 728-732 (2011).</li><li>Gilmartin, M. R., Kwapis, J. L., Helmstetter, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+and+contextual+fear+conditioning+are+impaired+following+unilateral+microinjection+of+muscimol+in+the+ventral+hippocampus+or+amygdala,+but+not+the+medial+prefrontal+cortex.">Trace and contextual fear conditioning are impaired following unilateral microinjection of muscimol in the ventral hippocampus or amygdala, but not the medial prefrontal cortex.</a> Neurobiol. Learn. Mem. 97, 452-464 (2012).</li><li>Baysinger, A. N., Kent, B. A., Brown, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscarinic+receptors+in+amygdala+control+trace+fear+conditioning.">Muscarinic receptors in amygdala control trace fear conditioning.</a> PLoS ONE. 7, (2012).</li><li>Wanisch, K., Tang, J., Mederer, A., Wotjak, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+fear+conditioning+depends+on+NMDA+receptor+activation+and+protein+synthesis+within+the+dorsal+hippocampus+of+mice.">Trace fear conditioning depends on NMDA receptor activation and protein synthesis within the dorsal hippocampus of mice.</a> Behav. Brain. 157, 63-69 (2005).</li><li>Smith, D. R., Gallagher, M., Stanton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+differences+and+nonassociative+effects+in+mouse+trace+fear+conditioning.">Genetic background differences and nonassociative effects in mouse trace fear conditioning.</a> Learn. Mem. 14, 597-605 (2007).</li><li>Rudy, J. W., O'Reilly, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contextual+fear+conditioning,+conjunctive+representations,+pattern+completion,+and+the+hippocampus.">Contextual fear conditioning, conjunctive representations, pattern completion, and the hippocampus.</a> Behav. Neurosci. 113, 867-880 (1999).</li><li>Wiltgen, B. J., Sanders, M. J., Anagnostaras, S. G., Sage, J. R., Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context+fear+learning+in+the+absence+of+the+hippocampus.">Context fear learning in the absence of the hippocampus.</a> J. Neurosci. 26, 5484-5491 (2006).</li><li>Reijmers, L. G., Perkins, B. L., Matsuo, N., Mayford, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+a+stable+neural+correlate+of+associative+memory.">Localization of a stable neural correlate of associative memory.</a> Science. 317, 1230-1233 (2007).</li><li>Huerta, P. T., Sun, L. D., Wilson, M. A., Tonegawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+temporal+memory+requires+NMDA+receptors+within+CA1+pyramidal+neurons.">Formation of temporal memory requires NMDA receptors within CA1 pyramidal neurons.</a> Neuron. 25, 473-480 (2000).</li><li>Jacobs, N. S., Cushman, J. D., Fanselow, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+accurate+measurement+of+fear+memory+in+Pavlovian+conditioning:+Resolving+the+baseline+issue.">The accurate measurement of fear memory in Pavlovian conditioning: Resolving the baseline issue.</a> J. Neurosci. Methods. 190, 235-239 (2010).</li></ol>

There have been several studies that have elucidated the neural circuitry that underlies trace fear conditioning. Trace fear conditioning is believed to involve the CA1 of the hippocampus12-14. There is also evidence that the medial prefrontal cortex (mPFC) plays a large role in trace eye-blink conditioning15, and the mPFC has been found to be involved in trace fear conditioning. One study found that mPFC neurons provide sustained activity during the trace period, thus providing a structure that can...

In fear conditioning a neutral stimulus (NS) is paired with an aversive unconditioned stimulus (US). The NS is normally a tone and becomes a conditioned stimulus (CS) through repeated pairings with the US. The CS can then elicit a conditioned response (CR), such as freezing, in the absence of the aversive US. A commonly used fear conditioning protocol is delay conditioning. In this protocol the onset of the NS and the US is contiguous or with some overlap in stimulus presentation. Even though delay fear conditioning is one of the most commonly used types of temporal associative conditioning, there are several other types of associative conditioning temporal arrangements: simultaneous conditioning, backward conditioning, and trace conditioning1. In trace fear conditioning there is a stimulus-free interval between the NS and the US of several seconds resulting in a &#34;trace&#34; period.
Several studies have reported deficits in trace fear conditioning when neurotoxic lesions are produced in the structures that input into the hippocampus2-5 or when pharmacological agents are used to block receptor function in the hippocampus. Lesion to the hippocampus results in deficits in trace conditioning and contextual conditioning, but does not impair delay fear conditioning8. There are several benefits to using trace fear conditioning. The fear conditioning protocol can be achieved over a three-day testing period and allows for hippocampal-dependent memory that is not spatially dependent. Trace fear conditioning can be used as a complementary test to the Morris water maze, novel object recognition test, or other maze tests in investigating hippocampal-dependent memory.

<table border="0" cellpadding="0" cellspacing="0" width="741">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">FreezeFrame</td>
			<td style="width:143px;">Coulbourn</td>
			<td style="width:119px;"></td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">30% Isopropanol</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td style="width:263px;">Purchase 90% isopropanol and dilute it down to 30%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% Ethanol</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Amp-meter</td>
			<td style="width:143px;">Med-Associates</td>
			<td style="width:119px;">ENV-420</td>
			<td style="width:263px;">Windows XP, Vista, and 7 Compatible (32-bit only)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital Sound Level Meter</td>
			<td style="width:143px;"></td>
			<td style="width:119px;">33-2055</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Vanilla Extract</td>
			<td style="width:143px;">McCormick Pure Vanilla Extract</td>
			<td style="width:119px;"></td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sticky Notes</td>
			<td style="width:143px;">Post-it</td>
			<td style="width:119px;"></td>
			<td style="width:263px;">3X3 inch</td>
		</tr>
	</tbody>
</table>

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.

For the representative results we present data from control C57BL/6J adult mice that received the neutral stimulus pairings with the unconditioned stimulus (shock condition) compared to mice that received the neutral stimulus but did not receive the unconditioned stimulus (no shock condition). It is important to run this condition when first setting up this behavioral test to determine whether the protocol has been performed correctly.
The data in Figure 1 represent the traini...

Watch this Scientific Journal Video about Trace Fear Conditioning in Mice at JoVE.com

Trace Fear Conditioning in Mice

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 ...

Research

JoVE Journal

Behavior

32.5K 次观看.  Baylor University. 在接下来的实验中，我们描述了一个协议，用于在小鼠体内微量恐惧条件。这种类型的联想记忆，包括分隔中性刺激和非条件刺激一丝时期。 