Name: preparation of an awake mouse for recording neural responses and injecting tracers
Uploaded: 2012-06-26T12:00:00
Description: Watch this Scientific Journal Video about Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers at JoVE.com

Supported by NSF grant 0920060, NIH grant DC004395, NHMRC grant 1009482, NSW Office of Science and Medical Research, and the Garnett Passe and Rodney Williams Memorial Foundation.

1. Head-restraint Overview
<ol>
	<li>To assemble a custom-built head-post, insert a 1/16" stainless steel roll-pin perpendicularly into a hole drilled in a 3/32" stainless steel rod to form a cross. The vertical piece of the head-post should be about 20 mm and the horizontal cross piece about 15 mm. Tap one end of the vertical rod to accept a #1-72 screw. (Fig. 1).</li>
	<li>During surgery and recording, the head-post is secured in a custom-built brass or aluminum mounting bar<...

<ol>
	<li>Evans, E. F., Nelson, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+responses+of+single+neurones+in+the+cochlear+nucleus+of+the+cat+as+a+function+of+their+location+and+the+anaesthetic+state.">The responses of single neurones in the cochlear nucleus of the cat as a function of their location and the anaesthetic state.</a> Exp. Brain Res. 17, 402-427 (1973).</li><li>Joris, P. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+classes+in+the+dorsal+cochlear+nucleus+and+its+output+tract+in+the+chloralose-anesthetized+cat.">Response classes in the dorsal cochlear nucleus and its output tract in the chloralose-anesthetized cat.</a> J. Neurosci. 18, 3955-3966 (1998).</li><li>Populin, 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=Anesthetics+change+the+excitation/inhibition+balance+that+governs+sensory+processing+in+the+cat+superior+colliculus.">Anesthetics change the excitation/inhibition balance that governs sensory processing in the cat superior colliculus.</a> J. Neurosci. 25, 5903-5914 (2005).</li><li>Young, E. D., Brownell, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+to+tones+and+noise+of+single+cells+in+dorsal+cochlear+nucleus+of+unanesthetized+cats.">Responses to tones and noise of single cells in dorsal cochlear nucleus of unanesthetized cats.</a> J. Neurophysiol. 39, 282-300 (1976).</li><li>Donoghue, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+properties+of+neurons+in+two+parts+of+the+primary+motor+cortex+of+the+awake+rat.">Contrasting properties of neurons in two parts of the primary motor cortex of the awake rat.</a> Brain Res. , 333-3173 (1985).</li><li>Westby, G. W., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+floating+microwire+technique+for+multichannel+chronic+neural+recording+and+stimulation+in+the+awake+freely+moving+rat.">A floating microwire technique for multichannel chronic neural recording and stimulation in the awake freely moving rat.</a> J. Neurosci. Methods. 76, 123-133 (1997).</li><li>Suga, N., O'Neill, W. E., Manabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+neurons+sensitive+to+combinations+of+information-bearing+elements+of+biosonar+signals+in+the+mustache+bat.">Cortical neurons sensitive to combinations of information-bearing elements of biosonar signals in the mustache bat.</a> Science. 200, 778-781 (1978).</li><li>O'Neill, W. E., Suga, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+range-sensitive+neurons+in+the+auditory+cortex+of+the+mustache+bat.">Target range-sensitive neurons in the auditory cortex of the mustache bat.</a> Science. 203, 69-73 (1979).</li><li>Suga, N., O'Neill, W. E., Manabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harmonic-sensitive+neurons+in+the+auditory+cortex+of+the+mustache+bat.">Harmonic-sensitive neurons in the auditory cortex of the mustache bat.</a> Science. 203, 270-274 (1979).</li><li>Portfors, C. V., Felix, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2nd+Spectral+integration+in+the+inferior+colliculus+of+the+CBA/CaJ+mouse.">2nd Spectral integration in the inferior colliculus of the CBA/CaJ mouse.</a> Neuroscience. , 136-1159 (2005).</li><li>Felix, R. A., Portfors, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory,+inhibitory+and+facilitatory+frequency+response+areas+in+the+inferior+colliculus+of+hearing+impaired+mice.">Excitatory, inhibitory and facilitatory frequency response areas in the inferior colliculus of hearing impaired mice.</a> Hear Res. 228, 212-229 (2007).</li><li>Portfors, C. V., Jonson, K. G., Roberts, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over-representation+of+species-specific+vocalizations+in+the+awake+mouse+inferior+colliculus.">Over-representation of species-specific vocalizations in the awake mouse inferior colliculus.</a> Neuroscience. 162, 486-500 (2009).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2007).</li></ol>

The advantage of the head-post restraint system for electrophysiological and neuroanatomical experiments in mouse is that experiments can be conducted with unanesthetized, awake mice, eliminating potential response contamination due to anesthetic drugs. In addition, the set up can be used over multiple days to allow for more efficient animal use. 
Most components for the head-post are available off-the-shelf. Only the head-post and mounting apparatus are custom-made, both of which are relativ...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Etch gel for cleaning skull prior to dental cement</td>
 <td>Henry Schein</td>
 <td>101-5396</td>
 <td>12/pk with 1.2 mL syringes</td>
 </tr>
 <tr>
 <td>Primer for dental cement</td>
 <td>Kerr Optibond</td>
 <td>25881</td>
 <td>8 mL bottle</td>
 </tr>
 <tr>
 <td>Adhesive for dental cement</td>
 <td>Kerr Optibond</td>
 <td>25882</td>
 <td>8 mL bottle</td>
 </tr>
 <tr>
 <td>Applicators for dental cement</td>
 <td>Kerr Optibond</td>
 <td>24680</td>
 <td>200/pk</td>
 </tr>
 <tr>
 <td>Dental Cement</td>
 <td>Charisma, Heraeus Kulzer</td>
 <td>66000085</td>
 <td>4gm Syringe Refill</td>
 </tr>
 <tr>
 <td>Tungsten Rod (Ground Pin)</td>
 <td>A-M Systems</td>
 <td>717200</td>
 <td>0.010" diameter</td>
 </tr>
 <tr>
 <td>Economy UV Curing Light</td>
 <td>Henry Schein</td>
 <td>CU-80</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Head-post</td>
 <td>Built in-house</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mounting bar</td>
 <td>Built in-house</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mounting block</td>
 <td>Built in-house</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Stereotaxic Frame</td>
 <td>David Kopf Instruments</td>
 <td>902</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mouse and Neonatal Rat Adaptor</td>
 <td>Stoelting</td>
 <td>51625</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Deltaphase Isothermal Pad</td>
 <td>Braintree Scientific, Inc.</td>
 <td>39DP</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

preparation of an awake mouse for recording neural responses and injecting tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Watch this Scientific Journal Video about Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers at JoVE.com

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

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