This research was supported by a grant from the Ministry of Economy and Competitiveness (MINECO) of the Government of Spain, SAF2016-78898-C2-2-R.

This study was carried out in strict accordance with both Spanish regulations (Royal Decree 53/2013 - Law 32/2007) and European Union guidelines (Directive 2010/63/EU) on the care and use of animals in biomedical research.
1 . Preparation of the Rat
NOTE: We performed the experiments in male rats to avoid any hormonal changes.
<ol>
	<li>Anesthetize the animal using a mix of ketamine hydrochloride (30 mg/kg) and xylazine hydrochloride (5 mg/kg) injected intramuscularly; with this dose, the rat should be deeply anesthetized for around 1 h.</li>
	<li>Pinch the rat&#39;s toe; absence of a withdrawal reflex indicates that the animal is fully unconscious. If the rat responds to the pinch, give supplementary anesthesia at one third of the initial dose.</li>
	<li>Shave the scalp and disinfect the surgical area with povidone-iodine.</li>
	<li>Place the animal on a heating pad to maintain a temperature of 38 &#176;C and stabilize the animal&#39;s head in a stereotaxic frame using two ear bars and a bite bar. Use caution to avoid piercing the tympanic membrane with the ear bars.</li>
	<li>Protect the eyes by applying a drop of ophthalmic gel or serum saline to each eye.</li>
</ol>
2. Location of the AC in the Temporal Bone of the Rat
<ol>
	<li>Using a scalpel, make an incision along the midline to expose the skull and retract the periosteum covering the surface of the cranium.</li>
	<li>Use a sterile cotton tip to gently remove any blood covering the surface of the skull to visualize bregma, lambda, and interaural 0 according to the Paxinos and Watson atlas of the rat brain22.</li>
	<li>Make an incision in the temporal muscle near its dorsal insertion on the skull with a scalpel. Pull the muscle out using a needle and suture material, and fix the suture material to the stereotactic frame; this will expose the temporal bone. If bleeding occurs, rinse with cold sterile saline.</li>
	<li>Place a sterile straight needle in the stereotactic micromanipulator, making sure that it is fully secured.</li>
	<li>Slowly lower the needle until it is right above the surface of the skull, so that the tip of the needle is set to interaural 0. Set this point as zero, and determine coordinates from this point.</li>
	<li>Depending on the brain area of interest, vary the stereotactic coordinates. Determine these coordinates by utilizing the Paxinos and Watson atlas of the rat brain22. Once the coordinates are determined, move the needle to match those coordinates.</li>
	<li>Target the AC using the coordinates of the following four points: A: A/P = -5.8 mm, M/L = +/- 6.4 mm; B: A/P= -2.7 mm, M/L= +/- 6.4 mm; C: A/P= -2.7 mm, M/L= +/- 8.67 mm; D: A/P= -5.8 mm, M/L= +/- 8.67 mm. Lower the needle to right above the temporal bone to visualize each of these four points. Using a marker pen, mark the points on the temporal bone and connect them in order to draw a rectangle; the rectangle will serve as a guide to open of a window in the bone (Figure 1).</li>
</ol>
3. Surgical Exposure of the AC
<ol>
	<li>Open the window using an electric drill and a small drill bit (0.6 mm &#216;). Drill the perimeter of the rectangle at 8,000 rpm until the bone gives away. Cool the drilling surface by rinsing with cold sterile saline to prevent damage to subcortical structures. When the bone gives way, a drop in resistance can be detected. Be careful to not drill the brain.</li>
	<li>When the borders are loose, pull up the covering bone with fine forceps and store it in cold sterile saline.</li>
</ol>
4. Ablation of the AC
<ol>
	<li>Using a surgical microscope (10X), gently cut the meninges with a microsurgical knife and remove them using two fine-pointed forceps. If bleeding occurs, rinse with cold sterile saline.</li>
	<li>Gently aspirate the AC using a surgical suction device (pressure -0.24 bar) coupled to a sterile 20 G blunt tip needle. This point is critical and needs to be performed very carefully: aspirate only the six cortical layers and not the underlying white matter.</li>
	<li>Aspirate until the perforating arteries stop bleeding.</li>
	<li>When the aspiration is finished, cover the injured area with the extracted bone and apply an absorbable hemostatic gauze.</li>
	<li>Let the temporal muscle recover its original position, and then suture the skin using wound clips (9 mm). Apply antibiotic ointment (see the Table of Materials). Continue to apply ointment to the wound twice daily for three days. 
	NOTE: Each application consists of a thin layer applied on the wound.</li>
	<li>Inject buprenorphine subcutaneously in the back of the rat (0.05 mg/kg) as an analgesic 1 h after the surgery, and then every 8 h during 72 h.</li>
	<li>Keep the animal on the heating pad until it wakes up, and return it to its housing cage to recover.</li>
	<li>House animals individually to prevent cage mates from touching the sutured area, and provide some enrichment items. Change the sawdust daily to prevent infection, and carefully check that the animal recovers properly and does not show any signs of discomfort.</li>
</ol>
5. Tissue Collection
Caution: When handling paraformaldehyde (PFA), both solid and aqueous, wear personal protective equipment (PPE) and use a safety cabinet.
NOTE: Prepare 750 mL formaldehyde solution by dissolving 4% (w/v) PFA into 1x phosphate buffered solution (PBS) using heat (55 &#176;C). Filter the formaldehyde solution with filter paper. Prepare Ringer&#39;s Solution by dissolving 8.5 g of NaCl, 0.25 g of KCl, and 0.2 g NaHCO3in 1,000 mL of water (solution pH = 6.9).
<ol>
	<li>Let the animal survive for as long as it is needed for the study. When the research performed with the AC ablated rat is completed, terminally anaesthetize it by intra-peritoneal injection of 0.1 mL of sodium pentobarbital (60 mg/kg). Assess the depth of anesthesia by toe pinch and the absence of withdrawal reflex.</li>
	<li>When the animal is deeply anesthetized, perform an intracardiac perfusion23 of 125 mL of Ringer&#39;s Solution followed by 750 mL of formaldehyde solution by using a needle gauge of 1.8 mm of inside diameter.</li>
	<li>When the perfusion is finished, decapitate the rat at the first cervical vertebra.</li>
	<li>Use scissors to remove the skin and muscle from the head and expose the skull. Use scissors to cut and open the foramen magnum and remove the back of the skull.</li>
	<li>Make a transversal cut into the orbital bone using Spencer scissors, and use rongeurs to cut along the top edges of the skull to expose the brain. Be careful not to damage the brain.</li>
	<li>Once the brain is exposed, carefully remove the dura mater using fine-pointed forceps. Use a finger to gently scoop under and elevate the brain. Raise the brain and cut the nerves until it is free. Immerse the brain in formaldehyde solution and store it at 4 &#176;C for 24 h.</li>
</ol>
6. Localization of the AC Lesions
<ol>
	<li>After post-fixation, carefully place the brain into a sagittal rat brain matrix exposing the lateral surface of the brain.</li>
	<li>Place a camera 21 cm above the cortex surface using a camera holder, select the &#34;super macro shooting&#34; mode, and take a picture of the brain surface.</li>
	<li>Place the brain into a coronal rat brain matrix exposing the dorsal surface of the brain, and take another picture.</li>
	<li>Using an image editor program, open the images and scale them down 50% to make it easier to work with them. Identify the bregma, lambda, and interaural 0 references on the picture according to Paxinos and Watson coordinates19, and mark their position in the pictures (Figure 2). Draw the contour of the ablation over the lateral picture of the brain. Calculate the perimeter.</li>
	<li>Import the coordinate map where the primary (A1) and secondary regions (Dorsal and Ventral Cortices) of AC are located18 to the file of the editor program where you are working with the pictures. Click on the map and drag it to superimpose it to the lateral photograph of the ablated brain.
	<ol>
		<li>Make the bregma and lambda references of the coordinate map coincide with the bregma and lambda references identified in the picture of the lateral brain.</li>
		<li>Use the rhinal fissure as a reference to adjust the picture of the brain to the map, and make them coincide (Figure 2B).</li>
	</ol>
	</li>
	<li>Calculate the percentage of the lesion relative to the area occupied by the AC.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+down-regulation+of+sound-induced+c-Fos+protein+expression+in+the+inferior+colliculus+after+ablation+of+the+auditory+cortex.">Transient down-regulation of sound-induced c-Fos protein expression in the inferior colliculus after ablation of the auditory cortex.</a> Front Neuroanat. 4, 141 (2010).</li><li>Hunter, K. P., Willott, 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=Effects+of+bilateral+lesions+of+auditory+cortex+in+mice+on+the+acoustic+startle+response.">Effects of bilateral lesions of auditory cortex in mice on the acoustic startle response.</a> Physiol Behav. 54 (6), 1133-1139 (1993).</li><li>Lamas, V., Arevalo, J. C., Juiz, J. M., Merch&#225;n, M. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+the+auditory+cortex+results+in+changes+in+the+expression+of+neurotransmission-related+mRNAs+in+the+cochlea.">Ablation of the auditory cortex results in changes in the expression of neurotransmission-related mRNAs in the cochlea.</a> Hear Res. 346, 71-80 (2017).</li><li>Doron, N. N., Ledoux, J. E., Semple, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+the+tonotopic+core+of+rat+auditory+cortex:+physiological+evidence+for+a+posterior+field.">Redefining the tonotopic core of rat auditory cortex: physiological evidence for a posterior field.</a> J Comp Neurol. 453 (4), 345-360 (2002).</li><li>Polley, D. B., Read, H. L., Storace, D. a., Merzenich, M. 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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

A successful brain surgery hinges upon two factors: keeping the animal alive during and after the procedure, and accurately locating the area of interest. Ensuring that the rat is deeply anesthetized during the surgery (testing the withdrawal reflex), and receives adequate analgesics and non-ototoxic antibiotics should help survival. Additionally, the rat should be kept on a heating pad until it wakes from anesthesia to avoid hypothermia. Suturing will decrease the susceptibility to infection, and proper technique is vital: Animals will pick at their wound clips, so they should be implanted tight enough to prevent removal without placing too much tension on the wound.
To accurately locate the AC (or any other cortical area), it is important to determine the position of bregma, lambda, and interaural 0 to use them as references to calculate the limits of the targeted region. Any mistake in calculating the coordinates will result in the partial ablation of AC or the undesired aspiration of other surrounding areas. Thus, the needle tip should only touch the bone at interaural 0, and then translate the antero-posterior and medio-lateral coordinates according to what is described in this protocol.
In this manuscript, we have also described how to surgically expose and ablate the AC. There are three critical steps: the drilling process, the opening and removal of the meninges, and the ablation by aspiration. Drilling should be performed at a low speed with minimum pressure, as a high drilling speed generates heat that can affect nearby subcortical structures. However, maintaining a low speed and cooling the drilling area with cold sterile saline should prevent any damage. In addition, minimum pressure is essential to avoid a sudden break of the skull and subsequent injury to the underlying cortex. The opening and removal of the meninges that cover the AC should be performed carefully to avoid breaking the blood vessels. If bleeding occurs, the early and late prognosis is generally unfavorable and it is questionable whether such an animal meets the inclusion criteria for a reliable study. We recommend euthanasia in this case. Finally, the aspiration (probably the most difficult aspect in performing an effective lesion), must be restricted to the gray matter. There are two indicators that can help detect the presence of the white matter: (1) a change in the color contrast, as the white matter is brighter than the grey matter; and (2) the cessation of bleeding from the perforating arteries.
After any manipulation performed in the brain, it is necessary to assess the localization, size, and extension of the procedure made in the cortex for the subsequent analysis and validation of data obtained from the animal. In this manuscript, we detail how to localize the ablation performed in the cortex using a coordinate map previously described by our group18. This map was constructed using anatomical references obtained from serial section reconstructions of histological sections, correlated with the Paxinos and Watson atlas of the rat brain22. Accordingly, the map differentiates between the primary (A1) and secondary cortices (Dorsal and Ventral) of the AC. The main advantage of this coordinate map is that it allows the rapid localization of the lesion by superimposing a picture taken from the lateral surface of the brain placed in a sagittal brain matrix. Another advantage is that laboratories with less experience in anatomy can use the map by adapting it to their animal models. It is only necessary to set the distances between bregma, lambda, and interaural 0 references in a control perfused brain, and scale the map up or down accordingly. Use the Rhinal Fissure as reference to adjust the picture of the brain to the map. The depth of the ablation cannot be determined in this coordinate map, so it should be determined in brain histological sections.
The combination of stereotactic methods with the surgical exposure of the AC are basic methods that could be easily adapted by any investigator who wishes to target the AC in the rat. This might be for an acute experiment or one that requires implantation of permanent devices. Moreover, the surgical ablation of the AC has been previously used as a model to study the effects of chronic cortical deprivation in hearing. AC ablations could also be used to study the effects that unilateral AC ablations exert in other cortical areas, or serve as a model of stroke. Thus, the experimental designs described here are useful methods that can be applied individually or in combination to a wide range of experimental designs.

The rat is one of the most commonly used animal models in auditory neuroscience. The robustness of its behavior makes it able to work for hundreds of trials per day. Its sensitivity and spectral acuity for hearing1,2, and the anatomical and functional organization of its central system, comparable to other mammals3, make the rat a suitable animal model for analyzing a wide range of research topics in auditory neuroscience. The rat auditory cortex (AC), in particular, has been the subject of several anatomical and physiological studies that have tried to understand its structure, organization, and role in sound processing3. Nowadays, the AC has become popular among neuroscientists interested in experience-dependence plasticity, auditory perception, the synaptic basis of receptive field organization, and the cortical control of the sound processing in the subcortical auditory nuclei4,5,6,7,8,9. To address the challenges that these new approaches pose, procedures that can accurately locate and surgically expose the AC will expedite research efforts. Stereotactic techniques make it easy to localize specific regions within the brain without physiology testing. Although brain size varies slightly between animals, the location of any brain area can be determined using stereotactic coordinates set from landmarks on the skull of the rat brain.
The restricted ablation of the AC is the surgical removal of the sensory region of the cortex most directly related with hearing. In contrast to other methods used to block the activity of the AC, such as cooling or local lidocaine injections10,11,12, the surgical ablation of the AC results in the chronic loss of function. Thus, AC ablations are more suitable for studying the long-term effects of cortical deprivation, as well as the subsequent phenomena of lesion plasticity. The combination of stereotactic methods with surgical ablations of the AC has been used successfully to study the physiological, behavioral, and molecular effects of cortical control deprivation13,14,15,16,17,18,19. For example, a rat model with bilateral AC ablations has been used to study the effects of cortical ablation in the auditory startle reflex and auditory brainstem responses (ABR)16. Recently, we have compared the effects that unilateral versus bilateral ablations of the rat AC produce in ABR thresholds, amplitudes, and latencies at different time points after the injury18. In addition, the rat model of restrictive AC ablation has also been used to study the effect of corticofugal pathway degeneration in the inferior collicus13,14,15 and the inner ear17,19. After such a manipulation is performed in the brain, it is necessary to assess the localization, size, and extension of the lesions made in the cortex. Although very useful, the main limitation of tonotopic maps based on neuronal responses20,21 are the electrophysiological techniques required to locate the auditory fields in the rat brain. Since not all laboratories have the necessary equipment and/or expertise to make such recordings, we constructed a coordinate map based on the transfer of the cytoarchitectural limits of the AC to an image of the brain&#39;s surface18. This map can be very useful to locate the AC without physiology testing.
The present protocol describes a method for the stereotactically guided location, surgical exposure, and ablation of the AC in rats. It also describes how to use our coordinate map18 to easily localize the extension of the lesion over a picture of the surface of the ablated brains.

<table>
	<tbody>
		<tr>
			<td>Stereotaxic frame</td>
			<td>David Kopf Ins.</td>
			<td>900</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>WILD M650 Heerbrugg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>DAGA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dental micromotor</td>
			<td>W&#38;H elco</td>
			<td>5118</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond burr</td>
			<td>B Braun</td>
			<td>GD021R</td>
			<td>0.6 mm</td>
		</tr>
		<tr>
			<td>Surgical suction device</td>
			<td>Atmos</td>
			<td>Atmoforte E2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Merial</td>
			<td></td>
			<td>30 mg/kg</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td></td>
			<td>5 mg/kg</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>SM-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Lawton</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodine</td>
			<td>Meda</td>
			<td>Betadine</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile saline serum</td>
			<td>B.Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20G sterile needle</td>
			<td>Terumo Neolus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture material</td>
			<td>B.Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic Ointment</td>
			<td>Quadriderm (Betametasona, Gentamicina, Clotrimazol) - Schering-Plough</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>dimeda</td>
			<td>10.331.12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical needles</td>
			<td>World Precision Instruments</td>
			<td>501940</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Indivior UK</td>
			<td>Buprex</td>
			<td>0.01-0.05 mg/kg</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>dimeda</td>
			<td>08.120.15</td>
			<td></td>
		</tr>
		<tr>
			<td>Spencer scissor</td>
			<td>dimeda</td>
			<td>08.804.14</td>
			<td></td>
		</tr>
		<tr>
			<td>Rongeurs</td>
			<td>Lawton</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical knife</td>
			<td>MSP</td>
			<td>7503</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbable hemostatic gauze</td>
			<td>Surgicel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Saggital rat Brain Matrix</td>
			<td>Activational systems Inc.</td>
			<td>RBM-1000DV / RBM 4000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Vetoquinol</td>
			<td></td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Olympus 5.1 MP</td>
			<td>C-5060 wide zoom</td>
			<td>lens F2.8-4.8</td>
		</tr>
		<tr>
			<td>Wound clips</td>
			<td>Reflex 9</td>
			<td></td>
			<td>9 mm</td>
		</tr>
		<tr>
			<td>Canvas 12</td>
			<td>ACD Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>needle gauge</td>
			<td></td>
			<td></td>
			<td>diameter 1.8 mm</td>
		</tr>
		<tr>
			<td>Separatory funnel</td>
			<td>labbox</td>
			<td>11409</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>GluA2 primer Forward</td>
			<td>GeneBank</td>
			<td>NM_017261</td>
			<td>CGGCAGCTCAGCTAAAAACT</td>
		</tr>
		<tr>
			<td>GluA2 primer Reverse</td>
			<td>GeneBank</td>
			<td>NM_017261</td>
			<td>TTGTAGCTGGTGGCTGTTGA</td>
		</tr>
		<tr>
			<td>GluA3 primer Forward</td>
			<td>GeneBank</td>
			<td>NM_032990</td>
			<td>ATTGCTGATGGTGCAATGAC</td>
		</tr>
		<tr>
			<td>GluA3 primer Reverse</td>
			<td>GeneBank</td>
			<td>NM_032990</td>
			<td>TTTGCATTGTCGCAAGTCTC</td>
		</tr>
	</tbody>
</table>

stereotactically-guided ablation of the rat auditory cortex, and localization of the lesion in the brain

The rat auditory cortex (AC) is becoming popular among auditory neuroscience investigators who are interested in experience-dependence plasticity, auditory perceptual processes, and cortical control of sound processing in the subcortical auditory nuclei. To address new challenges, a procedure to accurately locate and surgically expose the auditory cortex would expedite this research effort. Stereotactic neurosurgery is routinely used in pre-clinical research in animal models to engraft a needle or electrode at a pre-defined location within the auditory cortex. In the following protocol, we use stereotactic methods in a novel way. We identify four coordinate points over the surface of the temporal bone of the rat to define a window that, once opened, accurately exposes both the primary (A1) and secondary (Dorsal and Ventral) cortices of the AC. Using this method, we then perform a surgical ablation of the AC. After such a manipulation is performed, it is necessary to assess the localization, size, and extension of the lesions made in the cortex. Thus, we also describe a method to easily locate the AC ablation postmortem using a coordinate map constructed by transferring the cytoarchitectural limits of the AC to the surface of the brain.The combination of the stereotactically-guided location and ablation of the AC with the localization of the injured area in a coordinate map postmortem facilitates the validation of information obtained from the animal, and leads to a better analysis and comprehension of the data.

We performed a stereotactically guided location, surgical exposure, and unilateral ablation of the AC in three Wistar rats. The localization of the lesion confirmed that ablations performed in the three rats encroached the major subdivisions of the AC (primary, dorsal, and ventral cortices), and comprised a range of 80 to 100% of the total AC area (Figure 2B).
The protocol described here to perform restrictive AC ablations has previously been used in our laboratory to study the long-term effects of cortical control deprivation in the subcortical auditory nuclei, as well as the subsequent phenomena of plasticity. In these studies, the protocol of AC ablations was validated by applying physiological (ABR), behavioral (startle responses, prepulse inhibition; PPI), and molecular (DNA microarrays, qPCR, and Western Blot) methods13,14,15,16,17,18,19. Here, to demonstrate the efficacy of our protocol, we let the three AC ablated rats survive for one week, and collected the cochleae during the tissue collection step to study the changes in the expression of the most relevant AMPA subunits present in the adult cochlea, GluA2 and GluA3, by qPCR. The comparison between the transcripts from AC ablated rats and sham control animals where all the surgery process but not the cortical ablation was performed showed a down-regulation for GluA2, and an up-regulation for GluA3 in both cochleae (Figure 3), which is in agreement with our previous study19.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56429/56429fig1.jpg" /> 
Figure 1: Images of the rat temporal bone at three different surgical steps. (A) Transfer of the stereotaxic coordinates of the AC to the temporal bone. The coordinates of the four points are: A: A/P= -5.8 mm, M/L= +/- 6.4 mm; B: A/P= -2.7 mm, M/L= +/- 6.4 mm; C: A/P= -2.7 mm, M/L= +/- 8.67 mm; D: A/P= -5.8 mm, M/L= +/- 8.67 mm. (B) The coordinates are used as a reference to draw a rectangle on the surface of the temporal bone that will guide the opening of a window. (C) Shows the window opened in the bone after drilling. The meninges with blood vessels can be observed on the surface of the brain. R: rostral, D: dorsal. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56429/56429fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56429/56429fig2.jpg" /> 
Figure 2: Procedure for locating lesions in the rat brain. (A) Photograph of the dorsal surface of an AC ablated brain with stereotactically implanted needles in Lambda and Bregma (according to Paxinos and Watson coordinates19). Dotted lines mark the position of Bregma and Interaural 0 in the 9 cm x 9 cm grid, as well as in the dorsal surface of the brain. (B) Photograph of the lateral surface of the ablated brain superimposed to the coordinate map of the AC. The perimeter of the lesion is labeled in red in the picture. The perimeter of the AC area is labeled in black in the map. In this example, the percentage of AC ablation with respect to the total area occupied by the AC is 84.79%. AC: auditory cortex, IA: inter aural, FR: Rhinal Fissure. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56429/56429fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56429/56429fig3.jpg" /> 
Figure 3: Changes in mRNA levels of AMPA receptor subunits GluA2 and GluA3 after unilateral AC ablations 7 days post-lesion. Results are presented as the mean &#177; standard deviation of the fold change. Changes for GluA2 transcripts are represented in blue. Changes for GluA3 transcripts are presented in red. A significant decrease in GluA2 and an increase in GluA3 is observed in both cochleae (ipsi- and contralateral to the ablation) relative to sham controls with no cortical ablations at 7 days after the surgery; this is in agreement with our previous results19. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56429/56429fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain at JoVE.com

Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain

We describe a method for the stereotactically-guided location, exposure, and ablation of the auditory cortex in rats. The localization of the ablation is assessed using a coordinate map postmortem.

The rat auditory cortex (AC) is becoming popular among auditory neuroscience investigators who are interested in experience-dependence plasticity, ...

stereotactically-guided-ablation-rat-auditory-cortex-localization

Nanyang Technological University

Research

JoVE Journal

Behavior

11.5K Views.  University of Salamanca. We describe a method for the stereotactically-guided location, exposure, and ablation of the auditory cortex in rats. The localization of the ablation is assessed using a coordinate map postmortem.

Video: Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

Infant Auditory Processing and Event-related Brain Oscillations

Rapid auditory processing and acoustic change detection abilities play a critical role in allowing human infants to efficiently process the fine spectral and temporal changes that are characteristic of human language. These abilities lay the foundation for effective language acquisition; allowing infants to hone in on the sounds of their native language. Invasive procedures in animals and scalp-recorded potentials from human adults suggest that simultaneous, rhythmic activity (oscillations) between and within brain regions are fundamental to sensory development; determining the resolution with which incoming stimuli are parsed. At this time, little is known about oscillatory dynamics in human infant development. However, animal neurophysiology and adult EEG data provide the basis for a strong hypothesis that rapid auditory processing in infants is mediated by oscillatory synchrony in discrete frequency bands. In order to investigate this, 128-channel, high-density EEG responses of 4-month old infants to frequency change in tone pairs, presented in two rate conditions (Rapid: 70 msec ISI and Control: 300 msec ISI) were examined. To determine the frequency band and magnitude of activity, auditory evoked response averages were first co-registered with age-appropriate brain templates. Next, the principal components of the response were identified and localized using a two-dipole model of brain activity. Single-trial analysis of oscillatory power showed a robust index of frequency change processing in bursts of Theta band (3 - 8 Hz) activity in both right and left auditory cortices, with left activation more prominent in the Rapid condition. These methods have produced data that are not only some of the first reported evoked oscillations analyses in infants, but are also, importantly, the product of a well-established method of recording and analyzing clean, meticulously collected, infant EEG and ERPs. In this article, we describe our method for infant EEG net application, recording, dynamic brain response analysis, and representative results.

High-density electroencephalography (dEEG) is being used increasingly to study brain development and plasticity in the early years of life. Here we present an application of sophisticated analysis techniques that builds on traditional EEG recording to understand the oscillatory dynamics of rapid auditory processing in the infant brain.

Rapid auditory processing and acoustic change detection abilities play a critical role in allowing human infants to efficiently process the fine spectral ...

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 ...

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 ...

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

Using Eye Movements Recorded in the Visual World Paradigm to Explore the Online Processing of Spoken Language

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded via an eye tracker as the participant produces or comprehends a spoken language describing the concurrent visual world. This paradigm has high versatility, as it can be used in a wide range of populations, including those who cannot read and/or who cannot overtly give their behavioral responses, such as preliterate children, elderly adults, and patients. More importantly, the paradigm is extremely sensitive to fine grained manipulations of the speech signal, and it can be used to study the online processing of most topics in language comprehension at multiple levels, such as the fine grained acoustic phonetic features, the properties of words, and the linguistic structures. The protocol described in this article illustrates how a typical visual world eye tracking study is conducted, with an example showing how the online processing of some semantically complex statements can be explored with the visual world paradigm.

The visual world paradigm monitors participants' eye movements in the visual workspace as they are listening to or speaking a spoken language. This paradigm can be used to investigate the online processing of a wide range of psycholinguistic questions, including semantically complex statements, such as disjunctive statements.

In a typical eye tracking study using the visual world paradigm, participants' eye movements to objects or pictures in the visual workspace are recorded ...

An Instrumented Pull Test to Characterize Postural Responses

Impairment of postural reflexes, termed postural instability, is a common and disabling deficit in Parkinson's disease. To assess postural reflexes, clinicians typically employ the pull test to grade corrective responses to a backward perturbation at the shoulders. However, the pull test is prone to issues with reliability and scaling (score/4). Here, we present an instrumented version of the pull test to more precisely quantify postural responses. Akin to the clinical test, pulls are manually administered except pull force is also recorded. Displacements of the trunk and feet are captured by a semi-portable motion tracking system. Raw data represent distance traveled (in millimeter units), making subsequent interpretation and analysis intuitive. The instrumented pull test also detects variabilities influencing pull test administration, such as pull force, thereby identifying and quantifying potential confounds that can be accounted for by statistical techniques. The instrumented pull test could have application in studies seeking to capture early abnormalities in postural responses, track postural instability over time, and detect responses to therapy.

Impairment of postural reflexes, termed postural instability, is difficult to quantify. Clinical assessments such as the pull test suffer issues with reliability and scaling. Here, we present an instrumented version of the pull test to objectively characterize postural responses.

Impairment of postural reflexes, termed postural instability, is a common and disabling deficit in Parkinson's disease. To assess postural reflexes, ...

Transcranial Direct Current Stimulation for Online Gamers

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that applies a weak electric current to the scalp to modulate neuronal membrane potentials. Compared to other brain stimulation methods, tDCS is relatively safe, simple, and inexpensive to administer.
Since excessive online gaming can negatively affect mental health and daily functioning, developing treatment options for gamers is necessary. Although tDCS over the dorsolateral prefrontal cortex (DLPFC) has demonstrated promising results for various addictions, it has not been tested in gamers. This paper describes a protocol and a feasibility study for applying repeated tDCS over the DLPFC and neuroimaging to examine the underlying neural correlates in gamers.
At baseline, individuals who play online games report average weekly hours spent on games, complete questionnaires on addiction symptoms and self-control, and undergo brain 18F-&#64258;uoro-2-deoxyglucose positron emission tomography (FDG-PET). The tDCS protocol consists of 12 sessions over the DLPFC for 4 weeks (anode F3/cathode F4, 2 mA for 30 min per session). Then, a follow-up is conducted using the same protocol as the baseline. Individuals who do not play online games receive only baseline FDG-PET scans without tDCS. Changes of clinical characteristics and asymmetry of regional cerebral metabolic rate of glucose (rCMRglu) in the DLPFC are examined in gamers. In addition, asymmetry of rCMRglu is compared between gamers and non-gamers at baseline.
In our experiment, 15 gamers received tDCS sessions and completed baseline and follow-up scans. Ten non-gamers underwent FDG-PET scans at the baseline. The tDCS reduced addiction symptoms, time spent on games, and increased self-control. Moreover, abnormal asymmetry of rCMRglu in the DLPFC at baseline was alleviated after tDCS.
The current protocol may be useful for assessing treatment efficacy of tDCS and its underlying brain changes in gamers. Further randomized sham-controlled studies are warranted. Moreover, the protocol can be applied to other neurological and psychiatric disorders.

We present a protocol and a feasibility study for applying transcranial direct current stimulation (tDCS) and neuroimaging assessment in online gamers.

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that applies a weak electric current to the scalp to modulate ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...