The work was supported by grants from the Deustche Forschungsgemeinschaft DFG and the Leibniz-Institute for Neurobiology. We thank Maria-Marina Zempeltzi and Kathrin Ohl for technical assistance.

<ol>
	<li>Cohen, M. R., Newsome, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+electrical+microstimulation+has+revealed+about+the+neural+basis+of+cognition.">What electrical microstimulation has revealed about the neural basis of cognition.</a> Current Opinion in Neurobiology. 14 (2), 169-177 (2004).</li><li>Histed, M. H., Bonin, V., Reid, 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=Direct+activation+of+sparse,+distributed+populations+of+cortical+neurons+by+electrical+microstimulation.">Direct activation of sparse, distributed populations of cortical neurons by electrical microstimulation.</a> Neuron. 63 (4), 508-522 (2009).</li><li>Histed, M. H., Ni, A. M., Maunsell, J. H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+cortical+mechanisms+of+behavior+from+microstimulation+experiments.">Insights into cortical mechanisms of behavior from microstimulation experiments.</a> Progress in Neurobiology. 103, 115-130 (2013).</li><li>Bradley, D. 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=Visuotopic+mapping+through+a+multichannel+stimulating+implant+in+primate+V1.">Visuotopic mapping through a multichannel stimulating implant in primate V1.</a> Journal of Neurophysiology. 93, 1659-1670 (2005).</li><li>Scheich, H., Breindl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Animal+Model+of+Auditory+Cortex+Prostheses.">An Animal Model of Auditory Cortex Prostheses.</a> Audiology and Neurootology. 7 (3), 191-194 (2002).</li><li>Romo, R., Hern&#225;ndez, A., Zainos, A., Salinas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatosensory+discrimination+based+on+cortical+microstimulation.">Somatosensory discrimination based on cortical microstimulation.</a> Nature. 392, 387-390 (1998).</li><li>Douglas, R. J., Martin, K. A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+neuronal+circuits+in+the+neocortex.">Recurrent neuronal circuits in the neocortex.</a> Current Biology. 17 (13), 496-500 (2004).</li><li>Happel, M. F. K., Deliano, M., Handschuh, J., Ohl, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine-modulated+recurrent+corticoefferent+feedback+in+primary+sensory+cortex+promotes+detection+of+behaviorally+relevant+stimuli.">Dopamine-modulated recurrent corticoefferent feedback in primary sensory cortex promotes detection of behaviorally relevant stimuli.</a> The Journal of Neuroscience. 34 (4), 1234-1247 (2014).</li><li>Deliano, M., Scheich, H., Ohl, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+cortical+activity+after+intracortical+microstimulation+and+its+role+for+sensory+processing+and+learning.">Auditory cortical activity after intracortical microstimulation and its role for sensory processing and learning.</a> The Journal of Neuroscience. 29 (50), 15898-15909 (2009).</li><li>DeYoe, E. A., Lewine, J. D., Doty, 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=Laminar+variation+in+threshold+for+detection+of+electrical+excitation+of+striate+cortex+by+macaques.">Laminar variation in threshold for detection of electrical excitation of striate cortex by macaques.</a> Journal of Neurophysiology. 94 (5), 3443-3450 (2005).</li><li>Tehovnik, E. J., Slocum, W. M., Schiller, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delaying+visually+guided+saccades+by+microstimulation+of+macaque+V1:+spatial+properties+of+delay+fields.">Delaying visually guided saccades by microstimulation of macaque V1: spatial properties of delay fields.</a> The European Journal of Neuroscience. 22 (10), 2635-2643 (2005).</li><li>Wetzel, W., Wagner, T., Ohl, F. W., Scheich, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Categorical+discrimination+of+direction+in+frequency-modulated+tones+by+Mongolian+gerbils.">Categorical discrimination of direction in frequency-modulated tones by Mongolian gerbils.</a> Behavioural Brain Research. 91, 29-39 (1998).</li><li>Cain, C. K., LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escape+from+fear:+a+detailed+behavioral+analysis+of+two+atypical+responses+reinforced+by+CS+termination.">Escape from fear: a detailed behavioral analysis of two atypical responses reinforced by CS termination.</a> Journal of Experimental Psychology. Animal behavior processes. 33, 451-463 (2007).</li><li>Stark, H., Rothe, T., Deliano, M., Scheich, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+cortical+theta+activity+correlates+with+stages+of+auditory+avoidance+strategy+formation+in+a+shuttle-box.">Dynamics of cortical theta activity correlates with stages of auditory avoidance strategy formation in a shuttle-box.</a> Neuroscience. 151, 467-475 (2008).</li><li>Ohl, F. W., Wetzel, W., Wagner, T., Rech, A., Scheich, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilateral+ablation+of+auditory+cortex+in+Mongolian+gerbil+affects+discrimination+of+frequency+modulated+tones+but+not+of+pure+tones.">Bilateral ablation of auditory cortex in Mongolian gerbil affects discrimination of frequency modulated tones but not of pure tones.</a> Learning &amp; Memory. 6 (4), 347-362 (1999).</li><li>Kurt, S., Ehret, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+discrimination+learning+and+knowledge+transfer+in+mice+depends+on+task+difficulty.">Auditory discrimination learning and knowledge transfer in mice depends on task difficulty.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (18), 8481-8485 (2010).</li><li>Happel, M. F. K., 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=Enhanced+cognitive+flexibility+in+reversal+learning+induced+by+removal+of+the+extracellular+matrix+in+auditory+cortex.">Enhanced cognitive flexibility in reversal learning induced by removal of the extracellular matrix in auditory cortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (7), 2800-2805 (2014).</li><li>Thomas, H., Tillein, J., Heil, P., Scheich, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+of+auditory+cortex+in+the+mongolian+gerbil+(Meriones+unguiculatus).+I.+Electrophysiological+mapping+of+frequency+representation+and+distinction+of+fields.">Functional organization of auditory cortex in the mongolian gerbil (Meriones unguiculatus). I. Electrophysiological mapping of frequency representation and distinction of fields.</a> The European journal of neuroscience. 5, 882-897 (1993).</li><li>Budinger, E., Heil, P., Scheich, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+organization+of+auditory+cortex+in+the+Mongolian+gerbil+(Meriones+unguiculatus).+III.+Anatomical+subdivisions+and+corticocortical+connections.">Functional organization of auditory cortex in the Mongolian gerbil (Meriones unguiculatus). III. Anatomical subdivisions and corticocortical connections.</a> European Journal of Neuroscience. 12, 2425-2451 (2000).</li><li>Langford, D. 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=Coding+of+facial+expressions+of+pain+in+the+laboratory+mouse.">Coding of facial expressions of pain in the laboratory mouse.</a> Nature Methods. 7 (6), 447-449 (2010).</li><li>Ohl, F. W., Scheich, H., Freeman, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Change+in+pattern+of+ongoing+cortical+activity+with+auditory+category+learning.">Change in pattern of ongoing cortical activity with auditory category learning.</a> Nature. 412 (6848), 733-736 (2001).</li><li>Scheich, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+semantics+of+learning+and+crossmodal+processing+in+auditory+cortex:+the+semantic+processor+concept.">Behavioral semantics of learning and crossmodal processing in auditory cortex: the semantic processor concept.</a> Hearing Research. 271 (1-2), 3-15 (2011).</li><li>Happel, M. F. K., Jeschke, M., Ohl, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+integration+in+primary+auditory+cortex+attributable+to+temporally+precise+convergence+of+thalamocortical+and+intracortical+input.">Spectral integration in primary auditory cortex attributable to temporally precise convergence of thalamocortical and intracortical input.</a> The Journal of Neuroscience. 30 (33), 11114-11127 (2010).</li><li>Ranck, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Which+elements+are+excited+in+electrical+stimulation+of+mammalian+central+nervous+system:+a+review.">Which elements are excited in electrical stimulation of mammalian central nervous system: a review.</a> Brain Research. 98, 417-440 (1975).</li><li>Clark, K. L., Armstrong, K. M., Moore, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+neural+circuitry+and+function+with+electrical+microstimulation.">Probing neural circuitry and function with electrical microstimulation.</a> Proceedings. Biological sciences / The Royal Society. 278 (1709), 1121-1130 (2011).</li><li>Ilango, A., Shumake, J., Wetzel, W., Scheich, H., Ohl, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+stimulation+of+lateral+habenula+during+learning:+frequency-dependent+effects+on+acquisition+but+not+retrieval+of+a+two-way+active+avoidance+response.">Electrical stimulation of lateral habenula during learning: frequency-dependent effects on acquisition but not retrieval of a two-way active avoidance response.</a> PloS one. 8 (6), e65684 (2013).</li><li>Weible, A. P., 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=Cortical+involvement+in+acquisition+and+extinction+of+trace+eyeblink+conditioning.">Cortical involvement in acquisition and extinction of trace eyeblink conditioning.</a> Behavioral Neuroscience. 114, 1058-1067 (2000).</li><li>Rothe, T., Deliano, M., Scheich, H., Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+task-relevant+conditioned+stimuli+from+background+stimuli+by+associative+learning.">Segregation of task-relevant conditioned stimuli from background stimuli by associative learning.</a> Brain Research. 1297, 143-159 (2009).</li></ol>

The authors have nothing to disclose.

This protocol describes a method of simultaneous site-specific ICMS and multi-channel electrophysiological recordings in a learning animal by using a two-way aversive foot-shock controlled shuttle-box system. The protocol emphasizes technical key concepts for such combination and points out the importance of grounding the animal only via its common ground electrode, leaving the gridfloor at a floating voltage. Here, auditory shuttle-box learning was applied to Mongolian gerbils as learning-related plastic reorganizations...

A fundamental aim of behavioral neuroscience is to establish specific links between neuronal structural and functional properties, learning, and perception. Neural activity associated with perception and learning can be studied by electrophysiological recording of action potentials and local field potentials in various brain structures at multiple sites. Whereas electrophysiological recordings provide correlative associations between neural activity and behavior, direct electrical intracortical microstimulation (ICMS) for over a century has been the most direct method for testing causal relationships of excited populations of neurons and their behavioral and perceptual effects1&#8211;3. Many studies have demonstrated that animals are able to make use of various spatial and temporal properties of electrical stimuli in perceptual tasks depending on the stimulation site within for instance retinotopic4, tonotopic5, or somatotopic6 regions in the cortex. Propagation of electrically evoked activity in the cortex is mainly determined by the layout of axonal fibers and their distributed synaptic connectivity2 that, in cortex, is clearly layer-dependent7. The resultant polysynaptic activation evoked by ICMS is henceforth much more wide-spread than direct effects of the electrical field2,8,9. This explains why thresholds of perceptual effects elicited by intracortical microstimulation can be strongly layer-dependent8,10,11 and site-dependent9. A recent study demonstrated in detail that stimulation of upper layers yielded more wide-spread activation of corticocortical circuits in mainly supragranular layers, while stimulation of deeper layers of cortex result in a focal, recurrent corticoefferent intracolumnar activation. Parallel behavioral experiments revealed that the latter has much lower perceptual detection thresholds8. Therefore, the advantage of site-specific ICMS as conditioned stimuli was exploited in combination with electrophysiological recordings to causally relate specific cortical circuit activations8 to behavioral measures of learning and perception in the shuttle-box.
The two-way shuttle-box paradigm is a well-established laboratory apparatus to study avoidance learning12. A shuttle-box consists of 2 compartments separated by a hurdle or doorway. A conditioned stimulus (CS) that is represented by a suitable signal like a light or sound, is contingently followed by an aversive unconditioned stimulus (US), as for instance a foot shock over a metal grid floor. Subjects can learn to avoid the US by shuttling from one shuttle-box compartment to the other in response to the CS. Shuttle-box learning involves a sequence of distinguishable learning phases13,14: First, subjects learn to predict the US from the CS by classical conditioning and to escape from the US by instrumental conditioning, as the US is terminated upon shuttling. In a next phase, subjects learn to avoid the US altogether by shuttling in response to the CS&#160;before US onset (avoidance reaction). Generally, shuttle-box learning involves classical conditioning, instrumental conditioning, as well as goal-directed behavior depending on learning phase14.
The shuttle-box procedure can be set up easily and generally produces robust behavior after a few daily training sessions15&#8211;17. In addition to simple avoidance conditioning (detection), the shuttle-box can be further used to study stimulus discrimination by employing go/nogo paradigms. Here, animals are trained to avoid the US by a conditioned response (CR) (go behavior; shuttle into opposite compartment) in response to a go-stimulus (CS+) and by nogo behavior (stay in the current compartment; no CR) in response to a nogo-stimulus (CS-). Parallel microstimulation and recording of neural activity with high-density multielectrode arrays allow to study the physiological mechanisms underlying successful learning. Several technical details that are fundamental for the successful combinations of shuttle-box training, ICMS and parallel electrophysiology, will be discussed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Teflon-insulated stainless steel wire</td>
			<td>California Fine Wire</td>
			<td></td>
			<td>diam. 50&#181;m w/ isolation</td>
		</tr>
		<tr>
			<td>Pin connector system&#160;</td>
			<td>Molex Holding GmbH</td>
			<td>510470200</td>
			<td>1.25 mm pitch PicoBlade</td>
		</tr>
		<tr>
			<td>TEM grid Quantifoil</td>
			<td>Science Services</td>
			<td>EQ225-N27</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental acrylic Paladur</td>
			<td>Heraeus Kulzer</td>
			<td>64707938</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand-held drill OmniDrill35</td>
			<td>WPI&#160;</td>
			<td>503599</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 500mg/10ml</td>
			<td>Ratiopharm GmbH</td>
			<td>7538837</td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun 2%, 25ml</td>
			<td>Bayer Vital GmbH</td>
			<td>5066.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium-Chloride 0.9%, 10ml</td>
			<td>B.Braun AG&#160;</td>
			<td>PRID00000772</td>
			<td></td>
		</tr>
		<tr>
			<td>Lubricant KY-Jelly</td>
			<td>Johnson &#38; Johnson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shuttle-box E10-E15</td>
			<td>Coulbourn Instruments</td>
			<td>H10-11M-SC</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulus generator MCS STG 2000</td>
			<td>Multichannel Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plexon Headstage cable 32V-G20</td>
			<td>Plexon Inc.</td>
			<td>HSC/32v-G20</td>
			<td></td>
		</tr>
		<tr>
			<td>Plexon Headstage&#160; 32V-G20</td>
			<td>Plexon Inc.</td>
			<td>HST/32v-G20</td>
			<td></td>
		</tr>
		<tr>
			<td>PBX preamplifier 32 channels</td>
			<td>Plexon Inc.</td>
			<td>32PBX box</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel Acquisition System</td>
			<td>Plexon Inc.</td>
			<td>MAP 32/HLK2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostate CM3050 S</td>
			<td>Leica Microsystems GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Signal processing Card Ni-Daq</td>
			<td>National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lab StandardTM Stereotaxic Instruments</td>
			<td>Stoelting Co.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Audio attenator g.pah</td>
			<td>g.pah Guger technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cresyl violet acetate</td>
			<td>Roth GmbH</td>
			<td>7651.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Roticlear&#160;</td>
			<td>Roth GmbH</td>
			<td>A538.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate</td>
			<td>Roth GmbH</td>
			<td>6779.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrat(II) trihydrate</td>
			<td>Roth GmbH</td>
			<td>7974.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-sodium hydrogen phospahte dihydrate</td>
			<td>Merck</td>
			<td align="right">1,065,801,000</td>
			<td></td>
		</tr>
		<tr>
			<td>ICM Impedance Conditioning Module</td>
			<td>FHC</td>
			<td>55-70-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Temperarture Controler</td>
			<td>World Precision Instruments</td>
			<td>ATC2000</td>
			<td></td>
		</tr>
	</tbody>
</table>

combined shuttle-box training with electrophysiological cortex recording and stimulation as a tool to study perception and learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

This section illustrates a representative example of shuttle-box learning in a Mongolian gerbil. The subject was trained to discriminate the ICMS site between two stimulation electrodes implanted 700 &#181;m apart from each other in auditory cortex (Figures 1 and 2). Stimulation arrays can be customized in different spatial designs (Figure 1). Here, discrimination of the two ICMS sites was learned within 3 training sessions with presentation of 30 CS+ and CS- each (Figure 3A-C</s...

Watch this Scientific Journal Video about Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning at JoVE.com

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

combined-shuttle-box-training-with-electrophysiological-cortex

Research

JoVE Journal

Behavior

10.2K Views.  Leibniz Institute for Neurobiology, Magdeburg, Germany.  למידת הימנעות הסעות-תיבה-מבוססת היטב במדעי המוח התנהגותיים. פרוטוקול זה מתאר כיצד ניתן לשלב הסעות תיבה לומדת במכרסמים עם microstimulation אתר ספציפי החשמלי intracortical (ICMS) ובו זמנית בchronical הקלטות vivo ככלי ללמוד היבטים רבים של למידה ותפיסה. 

Video: Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.

Mouse models have become increasingly popular in studies of behavioral neuroscience. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. This protocol describes the Pasta Matrix Reaching Task, which is a skilled motor task for use in mouse models of stroke.

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models ...

Stimulating the Lip Motor Cortex with Transcranial Magnetic Stimulation

Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception. Researchers have used single-pulse and repetitive TMS to stimulate the lip representation in the motor cortex. The excitability of the lip motor representation can be investigated by applying single TMS pulses over this cortical area and recording TMS-induced motor evoked potentials (MEPs) via electrodes attached to the lip muscles (electromyography; EMG). Larger MEPs reflect increased cortical excitability. Studies have shown that excitability increases during listening to speech as well as during&#160;viewing speech-related movements. TMS can be used also to disrupt the lip motor representation. A 15-min train of low-frequency sub-threshold repetitive stimulation has been shown to suppress motor excitability for a further 15-20 min. This TMS-induced disruption of the motor lip representation impairs subsequent performance in demanding speech perception tasks and modulates auditory-cortex responses to speech sounds. These findings are consistent with the suggestion that the motor cortex contributes to speech perception. This article describes how to localize the lip representation in the motor cortex and how to define the appropriate stimulation intensity for carrying out both single-pulse and repetitive TMS experiments.

Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception.&#160; This article describes how to record motor evoked potentials (MEPs) from the lip muscles and how to disrupt the motor lip representation using repetitive TMS.

Transcranial magnetic stimulation (TMS) has proven to be a useful tool in investigating the role of the articulatory motor cortex in speech perception. ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Investigating Motor Skill Learning Processes with a Robotic Manipulandum

Skilled reaching tasks are commonly used in studies of motor skill learning and motor function under healthy and pathological conditions, but can be time-intensive and ambiguous to quantify beyond simple success rates. Here, we describe the training procedure for reach-and-pull tasks with ETH Pattus, a robotic platform for automated forelimb reaching training that records pulling and hand rotation movements in rats. Kinematic quantification of the performed pulling attempts reveals the presence of distinct temporal profiles of movement parameters such as pulling velocity, spatial variability of the pulling trajectory, deviation from midline, as well as pulling success. We show how minor adjustments in the training paradigm result in alterations in these parameters, revealing their relation to task difficulty, general motor function or skilled task execution. Combined with electrophysiological, pharmacological and optogenetic techniques, this paradigm can be used to explore the mechanisms underlying motor learning and memory formation, as well as loss and recovery of function (e.g. after stroke).

A paradigm is presented for training and analysis of an automated skilled reaching task in rats. Analysis of pulling attempts reveals distinct subprocesses of motor learning.

Skilled reaching tasks are commonly used in studies of motor skill learning and motor function under healthy and pathological conditions, but can be ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Using a Virtual Store As a Research Tool to Investigate Consumer In-store Behavior

People&#39;s responses to products and/or choice environments are crucial to understanding in-store consumer behaviors. Currently, there are various approaches (e.g., surveys or laboratory settings) to study in-store behaviors, but the external validity of these is limited by their poor capability to resemble realistic choice environments. In addition, building a real store to meet experimental conditions while controlling for undesirable effects is costly and highly difficult. A virtual store developed by virtual reality techniques potentially transcends these limitations by offering the simulation of a 3D virtual store environment in a realistic, flexible, and cost-efficient way. In particular, a virtual store interactively allows consumers (participants) to experience and interact with objects in a tightly controlled yet realistic setting. This paper presents the key elements of using a desktop virtual store to study in-store consumer behavior. Descriptions of the protocol steps to: 1) build the experimental store, 2) prepare the data management program, 3) run the virtual store experiment, and 4) organize and export data from the data management program are presented. The virtual store enables participants to navigate through the store, choose a product from alternatives, and select or return products. Moreover, consumer-related shopping behaviors (e.g., shopping time, walking speed, and number and type of products examined and bought) can also be collected. The protocol is illustrated with an example of a store layout experiment showing that shelf length and shelf orientation influence shopping- and movement-related behaviors. This demonstrates that the use of a virtual store facilitates the study of consumer responses. The virtual store can be especially helpful when examining factors that are costly or difficult to change in real life (e.g., overall store layout), products that are not presently available in the market, and routinized behaviors in familiar environments.

This paper describes the use of a desktop virtual store to create virtual shopping environments to investigate in-store consumer behavior. A description of the protocol to build and run experiments, example results from an experiment concerning store layout, and important considerations when conducting virtual store experiments are presented.

People&#39;s responses to products and/or choice environments are crucial to understanding in-store consumer behaviors. Currently, there are various ...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

Paradigms of Lower Extremity Electrical Stimulation Training After Spinal Cord Injury

Skeletal muscle atrophy, increased adiposity and reduced physical activity are key changes observed after spinal cord injury (SCI) and are associated with numerous cardiometabolic health consequences. These changes are likely to increase the risk of developing chronic secondary conditions and impact the quality of life in persons with SCI. Surface neuromuscular electrical stimulation evoked resistance training (NMES-RT) was developed as a strategy to attenuate the process of skeletal muscle atrophy, decrease ectopic adiposity, improve insulin sensitivity and enhance mitochondrial capacity. However, NMES-RT is limited to only a single muscle group. Involving multiple muscle groups of the lower extremities may maximize the health benefits of training. Functional electrical stimulation-lower extremity cycling (FES-LEC) allows for the activation of 6 muscle groups, which is likely to evoke greater metabolic and cardiovascular adaptation. Appropriate knowledge of the stimulation parameters is key to maximizing the outcomes of electrical stimulation training in persons with SCI. Adopting strategies for long-term use of NMES-RT and FES-LEC during rehabilitation may maintain the integrity of the musculoskeletal system, a pre-requisite for clinical trials aiming to restore walking after injury. The current manuscript presents a combined protocol using NMES-RT prior to FES-LEC. We hypothesize that muscles conditioned for 12 weeks prior to cycling will be capable of generating greater power, cycle against higher resistance and result in greater adaptation in persons with SCI.

Spinal cord injury is a traumatic medical condition that may result in elevated risks of chronic secondary metabolic disorders. Here, we presented a protocol using surface neuromuscular electrical stimulation-resistance training in conjunction with functional electrical stimulation-lower extremities cycling as a strategy to ameliorate several of these medical problems.

Skeletal muscle atrophy, increased adiposity and reduced physical activity are key changes observed after spinal cord injury (SCI) and are associated with ...

Continuous Theta Burst Stimulation of the Posterior Medial Frontal Cortex to Experimentally Reduce Ideological Threat Responses

Decades of behavioral science research have documented functional shifts in attitudes and ideological adherence in response to various challenges, but little work to date has illuminated the neural mechanisms underlying these dynamics. This paper describes how continuous theta burst transcranial magnetic stimulation may be employed to experimentally assess the causal contribution of cortical regions to threat-related ideological shifts. In the example protocol provided here, participants are exposed to a threat prime-an explicit reminder of their own inevitable death and bodily decomposition-following a downregulation of the posterior medial frontal cortex (pMFC) or a sham stimulation. Next, disguised within a series of distracter tasks, participants' relative degree of ideological adherence is assessed-in the present example, with regard to coalitional prejudice and religious belief. Participants for whom the pMFC has been downregulated exhibit less coalitionally biased responses to an immigrant critical of the participants' national in-group, and less conviction in positive afterlife beliefs (i.e., God, angels, and heaven), despite having recently been reminded of death. These results complement prior findings that continuous theta burst stimulation of the pMFC influences social conformity and sharing and illustrate the feasibility of investigating the neural basis of high-level social cognitive shifts using transcranial magnetic stimulation.

Threats reliably evoke shifts in high-level ideological investment, but little work to date has explored the neural mechanisms underlying these dynamics. This paper describes how continuous theta burst transcranial magnetic stimulation may be employed to test the contribution of the posterior medial frontal cortex (and/or other regions) to threat-related ideological shifts.

Decades of behavioral science research have documented functional shifts in attitudes and ideological adherence in response to various challenges, but ...

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.