Name: whisker-signaled eyeblink classical conditioning in head-fixed mice
Uploaded: 2016-03-30T14:00:00
Description: Watch this Scientific Journal Video about Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice at JoVE.com

This work was funded by the Department of Defense (W81XWH-13-01-0243) and the National Institutes of Health (R37 AG008796). We thank Alan Baker in Northwestern University&#39;s machine shop for building the head-fixed cylinder apparatus. We thank Dr. Shoai Hattori for his guidance in MATLAB and Solidworks. We thank Dr. John Power for the LabView software that controlled the experiment.

All procedures involving mice were performed in accordance with protocols approved by Northwestern University&#39;s Institutional Animal Care and Use Committee based on guidelines issued by the National Institute of Health.
1. The Cylinder (Figure 2A)
<ol>
	<li>Construct the cylinder as described by Chettih et al. and Heiney et al. from a long foam cylinder14-15. Cut a 10 cm length of cylinder and drill a hole through the center to fit the axle, a metal rod 12.7 ...

<ol>
	<li>Clark, R. E., Squire, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+conditioning+and+brain+systems:+the+role+of+awareness.">Classical conditioning and brain systems: the role of awareness.</a> Science. 280 (5360), 77-81 (1998).</li><li>Thompson, R. F., Kim, J. 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+systems+in+the+brain+and+localization+of+a+memory.">Memory systems in the brain and localization of a memory.</a> PNAS. 93 (24), 13438-13444 (1996).</li><li>Solomon, P. R., Vander Schaaf, E. R., Thompson, R. F., Weisz, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampus+and+trace+conditioning+of+the+rabbit's+classically+conditioned+nictitating+membrane+response.">Hippocampus and trace conditioning of the rabbit's classically conditioned nictitating membrane response.</a> Behav Neurosci. 100 (5), 729-744 (1986).</li><li>Moyer, J. R., Deyo, R. A., 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+trace+eye-blink+conditioning+in+rabbits.">Hippocampectomy disrupts trace eye-blink conditioning in rabbits.</a> Behav Neurosci. 104 (2), 243-252 (1990).</li><li>Weiss, C., Bouwmeester, H., Power, J. M., 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+lesions+prevent+trace+eyeblink+conditioning+in+the+freely+moving+rat.">Hippocampal lesions prevent trace eyeblink conditioning in the freely moving rat.</a> Behav Brain Res. 99 (2), 123-132 (1999).</li><li>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=Exploring+prefrontal+cortical+memory+mechanisms+with+eyeblink+conditioning.">Exploring prefrontal cortical memory mechanisms with eyeblink conditioning.</a> Behav Neurosci. 125 (3), 318-326 (2011).</li><li>Aiba, A., 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=Deficient+cerebellar+long-term+depression+and+impaired+motor+learning+in+mGluR1+mutant+mice.">Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice.</a> Cell. 79 (2), 377-388 (1994).</li><li>Skelton, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilateral+cerebellar+lesions+disrupt+conditioned+eyelid+responses+in+unrestrained+rats.">Bilateral cerebellar lesions disrupt conditioned eyelid responses in unrestrained rats.</a> Behav Neurosci. 102 (4), 586-590 (1988).</li><li>Takehara, K., Kawahara, S., Takatsuki, K., Kirino, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-limited+role+of+the+hippocampus+in+the+memory+for+trace+eyeblink+conditioning+in+mice.">Time-limited role of the hippocampus in the memory for trace eyeblink conditioning in mice.</a> Brain Res. 951 (2), 183-190 (2002).</li><li>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=Evoking+blinks+with+natural+stimulation+and+detecting+them+with+a+noninvasive+optical+device:+A+simple,+inexpensive+method+for+use+with+freely+moving+animals.">Evoking blinks with natural stimulation and detecting them with a noninvasive optical device: A simple, inexpensive method for use with freely moving animals.</a> J Neurosci Meth. 173, 108-113 (2008).</li><li>Royer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+timing,+rate+and+bursts+of+hippocampal+place+cells+by+dendritic+and+somatic+inhibition.">Control of timing, rate and bursts of hippocampal place cells by dendritic and somatic inhibition.</a> Nature. 15 (5), 769-775 (2012).</li><li>Goldey, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removable+cranial+windows+for+long-term+imaging+in+awake+mice.">Removable cranial windows for long-term imaging in awake mice.</a> Nature Protoc. 9 (11), 2515-2538 (2014).</li><li>Lovett-Barron, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+inhibition+in+the+hippocampus+supports+fear+learning.">Dendritic inhibition in the hippocampus supports fear learning.</a> Science. 343 (6173), 857-863 (2014).</li><li>Chettih, S. N., McDougle, S. D., Ruffolo, L. I., Medina, 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=Adaptive+timing+of+motor+output+in+the+mouse:+the+role+of+movement+oscillations+in+eyelid+conditioning.">Adaptive timing of motor output in the mouse: the role of movement oscillations in eyelid conditioning.</a> Front in Integ Neurosci. 5 (72), (2011).</li><li>Heiney, S. A., Wohl, M. P., Chettih, S. N., Ruffolo, L. I., Medina, 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=Cerebellar-Dependent+Expression+of+Motor+Learning+during+Eyeblink+Conditioning+in+Head-Fixed+Mice.">Cerebellar-Dependent Expression of Motor Learning during Eyeblink Conditioning in Head-Fixed Mice.</a> J Neurosci. 34 (45), 14845-14853 (2014).</li><li>Siegel, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trace+Eyeblink+Conditioning+in+Mice+is+Dependent+upon+the+Dorsal+Medial+Prefrontal+Cortex,+Cerebellum,+and+Amygdala:+Behavioral+Characterization+and+Functional+Circuity.">Trace Eyeblink Conditioning in Mice is Dependent upon the Dorsal Medial Prefrontal Cortex, Cerebellum, and Amygdala: Behavioral Characterization and Functional Circuity.</a> eNeuro. , (2015).</li><li>Galvez, R., Weiss, C., Cua, S., Disterhoft, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+precisely+timed+stimulation+of+mouse+whiskers+in+a+freely+moving+preparation:+application+for+delivery+of+the+conditioned+stimulus+in+trace+eyeblink+conditioning.">A novel method for precisely timed stimulation of mouse whiskers in a freely moving preparation: application for delivery of the conditioned stimulus in trace eyeblink conditioning.</a> J Neurosci Meth. 177 (2), 434-439 (2009).</li><li>Gruart, A., S&#225;nchez-Campusano, R., Fern&#225;ndez-Guiz&#225;n, A., Delgado-Garc&#236;a, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Differential+and+Timed+Contribution+of+Identified+Hippocampal+Synapses+to+Associative+Learning+in+Mice.">A Differential and Timed Contribution of Identified Hippocampal Synapses to Associative Learning in Mice.</a> Cereb Cortex. , (2014).</li><li>Weiss, 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=Impaired+Eyeblink+Conditioning+and+Decreased+Hippocampal+Volume+in+PDAPP+V717F+Mice.">Impaired Eyeblink Conditioning and Decreased Hippocampal Volume in PDAPP V717F Mice.</a> Neurobiol Dis. 11 (3), 425-433 (2002).</li><li>Galvez, R., Weiss, C., Weible, A. P., 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=Vibrissa-signaled+eyeblink+conditioning+induces+somatosensory+cortical+plasticity.">Vibrissa-signaled eyeblink conditioning induces somatosensory cortical plasticity.</a> J Neurosci. 26 (22), 6062-6068 (2006).</li><li>Galvez, R., Weible, A. P., 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=Cortical+barrel+lesions+impair+whisker-CS+trace+eyeblink+conditioning.">Cortical barrel lesions impair whisker-CS trace eyeblink conditioning.</a> Learn &amp; Memory. 14 (1), 94-100 (2007).</li><li>Johnson, K. R., Zheng, Q. Y., 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=A+Major+Gene+Affecting+Age-Related+Hearing+Loss+Is+Common+to+at+Least+Ten+Inbred+Strains+of+Mice.">A Major Gene Affecting Age-Related Hearing Loss Is Common to at Least Ten Inbred Strains of Mice.</a> Genomics. 70 (2), 171-180 (2000).</li><li>Tseng, W., Guan, R., Disterhoft, J. F., Weiss, C. <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+is+hippocampally+dependent+in+mice.">Trace eyeblink conditioning is hippocampally dependent in mice.</a> Hippocampus. 14 (1), 58-65 (2004).</li><li>Joachimsthaler, B., Brugger, D., Skodras, A., Schwarz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spine+loss+in+primary+somatosensory+cortex+during+trace+eyeblink+conditioning.">Spine loss in primary somatosensory cortex during trace eyeblink conditioning.</a> J Neurosci. 35 (9), 3772-3781 (2015).</li><li>Boele, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+and+extracerebellar+involvement+in+mouse+eyeblink+conditioning:+the+ACDC+model.">Cerebellar and extracerebellar involvement in mouse eyeblink conditioning: the ACDC model.</a> Front in Cell Neurosci. 3 (19), (2010).</li><li>Koekkoek, S. K. E., Den Ouden, W. L., Perry, G., Highstein, S. M., De Zeeuw, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+kinetic+and+frequency-domain+properties+of+eyelid+responses+in+mice+with+magnetic+distance+measurement+technique.">Monitoring kinetic and frequency-domain properties of eyelid responses in mice with magnetic distance measurement technique.</a> J Neurophysiol. 88 (4), 2124-2133 (2002).</li><li>Ward, R. L., Flores, L. 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=Infragranular+barrel+cortex+activity+is+enhanced+with+learning.">Infragranular barrel cortex activity is enhanced with learning.</a> J Neurophysiol. 108 (5), 1278-1287 (2012).</li></ol>

Classical eyeblink conditioning is a form of associative learning that is a useful tool for understanding the neural substrates underlying learning and memory. Previous methods employed for eyeblink conditioning in rodents such as mice involved a chamber that allowed for the animal to move about freely. A head-fixed preparation for eyeblink conditioning in mice, using the apparatus described by Chettih et al. and Heiney et al. and most recently utilized in light-evoked trace eyeblink conditioning in Sie...

Eyeblink conditioning is a form of Pavlovian conditioning and a model system for investigating the neural mechanisms of associative learning and memory. It has been investigated in various species, including humans, rabbits, cats, rats, and mice. The paradigm involves the presentation of two paired stimuli: a neutral conditioned stimulus (CS; e.g., a tone, a flash of light, or whisker stimulation), and a salient unconditioned stimulus (US; e.g., an air puff to the eye, or periorbital shock). The US elicits an unconditioned, reflexive eyeblink response (i.e., UR). Eventually, after several presentations of the paired CS-US, the subject learns to associate the CS with the US. This learning manifests itself in the form of a conditioned response (CR), an eyeblink elicited by the CS alone that precedes the presentation of the US.
Eyeblink conditioning in the trace form includes a stimulus-free interval of a few hundred milliseconds that separates the CS and the US (Figure 1). Trace conditioning is a form of declarative learning since it requires awareness of the stimulus contingencies1. The temporal gap requires the animal to keep a neural &#39;trace&#39; of the CS in forebrain regions such as the hippocampus in order for the US and the CS to become associated1-6. Along with the forebrain regions, trace conditioning is also dependent on the cerebellum7.
Eyeblink conditioning is, therefore, a useful paradigm for the investigation of the multiple facets of memory, including acquisition, consolidation, and retrieval. During eyeblink conditioning, a control group of animals is presented with unpaired stimuli in random order to test for pseudoconditioning or sensitized responses to the CS that may be caused by US presentation alone rather than a learned CS-US association.
A commonly used apparatus for the investigation of eyeblink conditioning in rodents is a chamber in which the rodents are allowed to move about freely during the training process8-10. With this type of apparatus, a tether is normally attached to a headpiece that is affixed to the rodent&#39;s skull. The tether allows for the delivery of the US (and sometimes the CS) and for transmitting the animal&#39;s response to those stimuli (i.e., the eyeblink response)10. The tether itself may be modified based on the type of stimuli delivered and how the eyeblink response is recorded.
The reason for using &#34;freely-moving&#34; tethered mice for eyeblink conditioning is that mice struggle against restraint. Though other species may be more amenable to restraint, the major advantage of using mice in eyeblink conditioning experiments is that the majority of available genetically modified mutant strains are mouse strains. In addition to struggling, complete restraint of mice results in acute distress. A head-fixed mouse preparation that minimizes stress would increase the physiological information that can be obtained during eyeblink conditioning. For example, this system would allow imaging of cortical neurons with 2-photon microscopy11.
Head-fixed preparations have been used in previous experiments for optical imaging of the cortex through removable cranial implants, in vivo electrophysiological recordings of the rodent brain with tetrode arrays, two-photon calcium imaging, and also as a platform for eyeblink conditioning in mice11-16.
In the head-fixed system, reliable stimulation and recordings are ensured without complete restraint of the mouse (Figure 2). A headpiece like the one used in the freely moving system is affixed to the mouse&#39;s skull. During training, the headpiece is affixed to a connector that is attached to bars over a cylindrical treadmill in order to stabilize the rodent&#39;s head (Figure 2A). The cylindrical treadmill allows the mouse to rest comfortably, but if the mouse so wishes, also allows it to run or to walk. With the use of this system, mice can be trained with a whisker vibration as the CS and a mild periorbital electrical shock as the US (Figure 1). The US is delivered through wires surgically placed underneath the skin lateral to the eye. The CS is delivered via a comb that is attached to a 2-layer rectangular bending actuator (Figure 2B). The comb and bending actuator are then attached to a magnetic base that is moved to the proper position during training and is adjusted for optimal delivery for each individual animal. The comb is positioned to straddle the selected whiskers. During delivery of the CS, a signal is sent to the bending actuator that displaces the comb and leads to vibration of the whiskers17.
Other stimuli such as a tone or a flash of light have been used as effective conditioned stimuli in mice in the past16,18,19. The reason whisker stimulation is chosen for the CS in this experimental paradigm is the dependence of murine animals on their vibrissae for somatosensory information input during exploration. Whisker stimulation has been shown to be a reliable and effective CS20. Furthermore, given the well-established and organized cortical substrate of the vibrissae system (i.e., the barrel cortex), whisker stimulation as the CS provides an elegant tool for mapping cortical changes and plasticity associated with learning eyeblink conditioning20,21. A head-fixed system allows for the precise stimulation of selected whiskers to compare responses between stimulated neurons and neurons receiving inputs from non-stimulated whiskers. Finally, many strains of mice exhibit age-related hearing loss as relatively young adults22, and eyelid closure during the conditioned blink alters a visual CS (although a visual CS does ameliorate issues with startle responses16). Whisker stimulation is not affected by either of these complications.
Presented here are unique and important modifications upon other head-fixed preparations for eyeblink conditioning, including methods for CS and US delivery, and the acquisition of the eyeblink response. The reliability of this apparatus and the training paradigm in eyeblink conditioning is demonstrated by learning curves from conditioned mice and a relatively flat learning curve from pseudoconditioned control animals (Figure 7A).

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			<td height="21" style="height:21px;width:436px;">Exervo TeraNova Foam Roller 36&#34; x 6&#34;&#160;</td>
			<td style="width:357px;">Amazon</td>
			<td style="width:181px;">B002ONUM0E</td>
			<td style="width:377px;">For cylinder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Plexiglas</td>
			<td style="width:357px;"></td>
			<td></td>
			<td>Custom-made; 1 cm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">Metal Rods (12.7mm diameter)</td>
			<td style="width:357px;"></td>
			<td></td>
			<td>Custom-made</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:436px;">4-40 machine screw (.25 in long)</td>
			<td style="width:357px;">Amazon Supply</td>
			<td>&#160;B00F33Q8QO</td>
			<td>For cylinder</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Classic Design Hair Comb</td>
			<td style="width:357px;">Conair</td>
			<td style="width:181px;">93505WG-320</td>
			<td>For whisker stimulation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:436px;">2-Layer Rectangular Bending Actuator</td>
			<td style="width:357px;">Piezo Systems</td>
			<td>T220-A4-303X&#160;</td>
			<td>For whisker stimulation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:436px;">Solder and Flux Kit</td>
			<td style="width:357px;">Piezo Systems</td>
			<td>MSF-003-NI</td>
			<td>For whisker stimulation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:436px;">Magnetic Base</td>
			<td style="width:357px;">Thor Labs</td>
			<td style="width:181px;">MB175</td>
			<td>For whisker stimulation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:436px;">Threaded rod for magnetic base</td>
			<td style="width:357px;"></td>
			<td style="width:181px;"></td>
			<td>Custom-made</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:436px;">Strips based on 221 series nylon strip connectors from Electronic Connector Corp.</td>
			<td style="width:357px;"></td>
			<td style="width:181px;"></td>
			<td>Custom-made, based on Weiss and Disterhoft, 2008</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:436px;">TO-220 Style Transistor</td>
			<td style="width:357px;">Amazon Supply</td>
			<td>B0002ZPZYO&#160;</td>
			<td>For connector; for the wings</td>
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			<td height="20" style="height:20px;width:436px;">Relia-Tac Sockets</td>
			<td style="width:357px;">Electronic Connector Corp.</td>
			<td style="width:181px;">220-S02</td>
			<td>For connector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:436px;">Relia-Tac Pins</td>
			<td style="width:357px;">Electronic Connector Corp.</td>
			<td style="width:181px;">220-P02</td>
			<td>For headpiece</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:436px;">0-80 stainless steel machine screw (1 in. long)</td>
			<td>Amazon Supply</td>
			<td>B000FN68EE</td>
			<td>Locking Screw</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:436px;">0-80 stainless steel machine screw hex nut (5/32 in. thick)</td>
			<td>Amazon Supply</td>
			<td>B000N2TK7Y</td>
			<td>Locking Screw Head</td>
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			<td height="20" style="height:20px;width:436px;">Loctite Super Glue-Liquid</td>
			<td>Loctite</td>
			<td>1365896</td>
			<td>Cyanoacrylic glue; for the locking screw</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:436px;">Quick Setting Epoxy</td>
			<td style="width:357px;">Ace Hardware</td>
			<td>18613</td>
			<td>For connector and whisker stimulation system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:436px;">Ethernet Cable Wires</td>
			<td style="width:357px;"></td>
			<td></td>
			<td>Ethernet cable can be taken apart to use the individual wires for the connector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:436px;">Polyimide coated stainless steel wires (2 in. long, .005 in. diameter)</td>
			<td style="width:357px;">PlasticsOne</td>
			<td>005sw/2.0 37365 S-S&#160;</td>
			<td>For headpiece, EMG and shock wires</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:436px;">Stainless steel uncoated wire (.005 in. diameter)</td>
			<td style="width:357px;">AM Systems</td>
			<td>792800</td>
			<td>For headpiece, ground wires</td>
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			<td height="20" style="height:20px;width:436px;">Tenma Variable Autotransformer</td>
			<td style="width:357px;">Tenma</td>
			<td>72-110</td>
			<td>For the whisker stimulation; rheostat to adjust current to the bending actuator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:436px;">Amplifier</td>
			<td style="width:357px;">A-M Systems</td>
			<td>1700</td>
			<td>Amplifier for filtering and amplifying EMG signals</td>
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			<td height="20" style="height:20px;width:436px;">WPI A385R stimulus isolator</td>
			<td style="width:357px;">World Precision Instruments</td>
			<td>31405</td>
			<td>For the electrical shock</td>
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		<tr height="18">
			<td height="18" style="height:18px;width:436px;">Isothesia (Isoflurane)</td>
			<td style="width:357px;">Henry Schein: Animal Health</td>
			<td style="width:181px;">50031</td>
			<td>For surgery; anesthesia</td>
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whisker-signaled eyeblink classical conditioning in head-fixed mice

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive repertoire of scientific techniques available to study learning and memory at the cellular level, it is ideal to have a stable cranial platform. Because mice do not readily tolerate restraint, they are usually trained while moving about freely in a chamber. Conditioned stimulus (CS) and unconditioned stimulus (US) information are delivered and eyeblink responses recorded via a tether connected to the mouse's head. In the head-fixed apparatus presented here, mice are allowed to run as they desire while their heads are secured to facilitate experimentation. Reliable conditioning of the eyeblink response is obtained with this training apparatus, which allows for the delivery of whisker stimulation as the CS, a periorbital electrical shock as the US, and analysis of electromyographic (EMG) activity from the eyelid to detect blink responses.

8-10 week old male C57Bl6/J mice were trained on trace eyeblink conditioning on the head-fixed cylindrical treadmill apparatus. 8 mice were trained with paired CS-US presentations (conditioned group) and 9 mice were trained with unpaired CS and US presentations (pseudoconditioned group).
Example EMG recordings of a conditioned response from a conditioned mouse are shown in Figures 3 and 4. EMG r...

Watch this Scientific Journal Video about Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice at JoVE.com

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive ...

The overall goal of this protocol is to use genetic advances in mice, and our understanding of the whisker system, to investigate the neurobiological basis of eyeblink conditioning, a translatable paradigm for studying learning and memory. This method can help answer mechanistic questions in learning and memory by using calcium imaging and electrophysiology in genetically manipulated mice while stimulating specific whiskers during conditioning. The main advantage of this technique is the ability to secure the heads of mice while minimizing stress, by allowing the mice to ambulate at their own pace on a cylindrical treadmill.The implications of this technique extend toward potential treatments for memory impairments, including those associated with normal aging and Alzheimer's disease. Preparation of the cylinder and the whisker stimulation system are each covered in the text protocol. This section covers preparation of the headpiece.The headpiece strip is a 3D printed seven hole strip modeled from the Amphenol 221 series nylon strip that is no longer manufactured commercially. From the 3D printed piece, tap the first hole in the strip for 0-80 by one inch machine screw and leave one hole empty after the first hole. Next, using a vice, put five gold-plated pins in the remaining five holes.Push the pins through the narrower holes in the plastic. Next, using a thermal stripper, strip a half centimeter of polyimide coating. Then, solder the stripped end to one of the pins where the pin has an opening.Cut the soldered wire to between six and seven millimeters and strip about two millimeters of the wire. Now, repeat this process to attach four wires in total to the five pins. The first two wires will record eyelid EMG responses while the second two will deliver electrical stimulation to the periorbital region.To the fifth pin, solder an uninsulated five centimeter stainless steel wire to serve as an electrical ground. After making all the connections, check each of them with a multimeter to ensure their electrical continuity. Begin with a fully anesthetized animal, confirmed by a toe pinch.Inject a dose of buprenorphine hydrochloride as an analgesic. Then apply a small amount of ophthalmic ointment to the corneas and shave the top of the animal's head. Next, secure the animal's head to a stereotaxic frame and disinfect the scalp with three alternating scrubs of povidone iodine and alcohol.With a number 10 or 15 scalpel blade, make an incision along the midline of the scalp, exposing the skull from the front of the eyes to past the interparietal bone. Retract the flaps of the skin with six microclips, exposing as much of the skull as possible, including the sides and back. Next, use the scalpel to scrape the periostium off the skull.Then clean the top of the skull with 3%hydrogen peroxide three times. Once the skull has dried, drill two holes with a size 34 inverted cone bur, being careful not to damage the brain. Place one hole in front of bregma, and place the other hole in front of lambda, left of the midline when conditioning the right eye, or vice versa.These holes should be drilled carefully to fit the screws, and great effort should be made to not damage the brain. Into the holes, place screws for the electrical connections. Each full turn will lower the screw 0.28 millimeters, so two full turns is sufficient.Next, create several small divits on the skull in order to increase the surface area for the cement. From the completed headpiece, wind the ground wire in a figure eight configuration around the two screws. Leave some slack in the ground wire so the headpiece can be positioned properly.Now, apply cold luting cement. Coat the skull and the screws liberally and allow the cement to dry. Once the cement is dried, position the headpiece vertically, with the pins standing up above the skull.Secure the headpiece with a holder attached to an arm from the frame. The holder must have five gold-plated sockets to accept the pins of the headpiece. Position the two EMG wires on the muscularis orbicularis oculi above the eye socket.Then, position the two stimulating wires. Put the stripped ends under the skin two to four millimeters directly caudal to the eye. Do not let their ends touch each other.If the wires are so long that they could scratch the eye of the animal, trim them back or bend them back. Some bare wire must remain exposed. Now, cement the base of the wires to the skull with a little more luting cement and allow it to dry.Use about half the volume previously used. To proceed, remove the microclips and reposition the skin. Allow the skin to settle naturally to prevent tension on the skin that would distort the eyelid inhibiting blinking and thus ruin the experiment.Once the skin is settled, remove the headpiece holder and seal the exposed area with dental cement. Avoid dripping cement on the eyes, or the pins of the headpiece. Then use a cotton swab dampened with dental cement solvent to smooth and manipulate partially cured cement as needed.Once the cement has dried completely, remove the animal from the stereotaxic frame and allow it to recover on a warm surface before returning it to its home cage. Start habituating mice after they have had at least five days of recovery time. To secure the mouse, briefly restrain it by the tail to pinch it behind the shoulders.Then lift the mouse from under the abdomen and torso. Next, attach a connector to the headpiece and turn the locking screw. Then, place the mouse gently on the cylinder and hold it still while using two screws to fasten the connector to the bars above the cylinder.Then release the mouse. In the first two sessions, let the mouse habituate to the cylinder for the same length of time as a conditioning session. During the habituation, record the spontaneous blink rate.Also, pre-expose the mice to the whisker vibration stimulator without applying electrical stimulation. With the piezo system close to the conditioned side, slip the teeth of the comb over individual whiskers. Select the same whiskers for every session.Then proceed with classical conditioning training as presented in the text. Eight to ten week old male C57 Black 6J mice were trained with trace eyeblink conditioning in the headfixed cylindrical treadmill. EMG recordings of each trial were taken.The EMGs were rectified and integrated with a 10 millisecond time constant, then averaged across all the trials. The evolution of conditioned repsonses over 10 training sessions shows that gradually the response got larger in amplitude and the response latency became shorter. This evolution is not seen in the responses from the psuedoconditioned mice, where the stimuli are presented in random order.The evolution of well timed, and thus well learned, conditioned responses can also be seen in the histograms of the time to the peak of the response following CS onset. After watching this video, you should have a good understanding of how to prepare the headpiece, surgically implant the headpiece on a mouse, and place the mouse on the cylindrical treadmill, for headfixed eyeblink conditioning. Your mice should exhibit a good learning curve across days as they acquire the conditioning.Once mastered, this technique can be done in one hour if it is performed properly. While attempting this procedure, it's important to remember to remain vigilant in observing any distress from the animals. Appropriate steps should be taken in order to minimize stress to the animals.

whisker-signaled-eyeblink-classical-conditioning-in-head-fixed-mice

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 ...

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. Higher cognitive functions that require precise control over sensorimotor responses such as decision-making and attentional modulation, however, are typically assessed in nonhuman primates. Despite the richness of primate behavior that allows multiple variants of these functions to be studied, the rodent model remains an attractive, cost-effective alternative to primate models. Furthermore, the ability to fully automate operant conditioning in rodents adds unique advantages over the labor intensive training of nonhuman primates while studying a broad range of these complex functions.
Here, we introduce a protocol for operantly conditioning rats on performing working memory tasks. During critical epochs of the task, the protocol ensures that the animal's overt movement is minimized by requiring the animal to 'fixate' until a Go cue is delivered, akin to nonhuman primate experimental design. A simple two alternative forced choice task is implemented to demonstrate the performance. We discuss the application of this paradigm to other tasks.

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

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 ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Classical Short-Delay Eyeblink Conditioning in One-Year-Old Children

Classical eyeblink conditioning (EBC) refers to the learned association between a conditioned stimulus (an auditory tone) and an unconditioned stimulus (a puff of air to the cornea). Eyeblink conditioning is often used experimentally to detect abnormalities in cerebellar-dependent learning and memory that underlies this type of associative learning. While experiments in adults and older children are relatively simple to administer using commercial equipment, eyeblink conditioning in infants is more challenging due to their poor compliance, which makes correct positioning of the equipment difficult. To achieve conditioning in one-year-old infants, a custom-made or an adapted commercial system can be used to deliver the air puff to the infant's cornea. The main challenge lies in successfully detecting and classifying the behavioral responses. We report that automated blink detection methods are unreliable in this population, and that conditioning experiments should be analyzed using frame-by-frame analysis of supplementary video camera recordings. This method can be applied to study developmental changes in eyeblink conditioning and to examine whether this paradigm can detect children with neurological disorders.

This protocol describes an eyeblink conditioning paradigm appropriate for experiments with one-year-old infants. Commercial or custom-made equipment can be used to deliver the stimuli, and data collection and analysis should be performed on the video recordings.

Classical eyeblink conditioning (EBC) refers to the learned association between a conditioned stimulus (an auditory tone) and an unconditioned stimulus (a ...

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

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 ...

Operant Conditioning Task to Measure Song Preference in Zebra Finches

An operant conditioning paradigm is used to test the song preference of female zebra finches. Finches are placed in a two-chambered cage with a connecting opening and indicate their preference for a song by landing on a perch within each chamber. By interrupting the infrared beam from a photoelectric sensor above each perch, the bird activates the playback of a song through a speaker located on each side of the cage. Freely available software is used to trigger the song playback from each perch. To determine the song preference of each animal, her chamber preference is first identified by triggering no song playback when she lands on each perch. This chamber preference is then compared to her song preference. A minimum activity threshold is set to ensure the preference is real. Using this method, we show that paired females prefer the song of their partner. This method was used to understand the contribution of dopamine to the formation and maintenance of song preference.

We describe a technique to evaluate song preference in zebra finches. Females are placed in a two-chambered cage and song preference is measured by the number of times she triggers the playback of one song by landing on a perch within one chamber, compared with triggering a different song in the second chamber. Perch landings are counted using infrared sensors.

An operant conditioning paradigm is used to test the song preference of female zebra finches. Finches are placed in a two-chambered cage with a connecting ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...