We wish to thank Mr Wabbel for preparing the wires and Mr Tietsch for constructing the plugs and cages according to our plans. This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 688). Felix Fluri holds a fellowship of the Interdisziplin&#228;re Zentrum f&#252;r Klinische Forschung (IZKF), University Clinics W&#252;rzburg, Germany.

Animal experiments were approved by the University of W&#252;rzburg and the legal state authorities (Lower Franconia, approval number: 54-2531.01-102/13) and performed according to the recommendations for research in experimental stroke studies17 and the current Animal Research: Reporting of In Vivo Experiments Guidelines (http://www.nc3rs.org.uk/arrive-guidelines).
1. Anesthesia
<ol>
	<li>Check the anesthetic system to ensure adequate amounts of supply gas (oxygen) and isoflurane for the duration of the procedure. Connect the nosecone with the incisor bar of the stereotaxic instrument and put the incisor bar on -3.3 mm.</li>
	<li>Turn on the supply gas (2 L/min). Place the rat into a box and seal the top. Turn on the isoflurane vaporizer to 3.5%.</li>
	<li>When the rat is recumbent, switch the system so that the anesthetic gas flows to the nosecone which is fixed to the incisor bar.&#160;</li>
	<li>Remove the rat from the box chamber and shave the area between the ears and the eyes; using a cotton bud soaked with Jodosept PVP, swab the shaved area to remove any loose hair.</li>
	<li>Position the rat in the nosecone (Figure 1) and continue the anesthesia with isoflurane 2.5% in O2 (1 L/min). Check the level of anesthesia by pinching the interdigital area. If the rat is anesthetized adequately, the defensive reflexes are abolished (i.e., withdrawal of the foot).</li>
	<li>Monitor respiration and response to stimulation during procedure and adjust the vaporizer as needed.</li>
	<li>Apply vet ointment on eyes to prevent dryness while under anesthesia. Monitor and maintain body temperature at 37 &#177; 0.5 &#176;C by a feedback-controlled heating system.</li>
</ol>
2. Surgery
<ol>
	<li>Keep the surgical field sterile during the whole surgery. Once the surgeon&#180;s hands are sterile and the operating field is sterile, move only carefully and remember not to break sterility. This includes having also a sterile field (i.e.,&#160;sterile waterproof drapes) on which one may set down instruments.</li>
	<li>Inject 0.2 ml mepivacaine&#160;subcutaneously into the center of the shaved area. Mepivacaine is a local anesthetic that has a duration of action of up to 3 hr. It will further anesthetize the surgical area.&#160;</li>
	<li>Using a scalpel, make a midline incision starting between the ears and extending towards 2 cm. Ensure that the periosteum (shiny membrane under the skin) is also incised. Expose the skull with four clamps (Figure 2).</li>
	<li>Using a cotton bud, gently remove the periosteum until the coronal and sagittal sutures are exposed; thereafter, stanch the blood with cotton wool.</li>
	<li>Determine the coordinates of the bregma using a needle fixed at a probe holder, and then mark the tip of the needle with a black felt-tip pen. Using the anterior/posterior (AP), midline/lateral (ML) and dorsoventral (DV) drive screws, position the tip of the needle directly over the bregma.</li>
	<li>Take the AP and ML vernier scale readings: subtract 3.6 mm from the AP reading and 2.5 mm from the ML reading for electrode implantation into the right STN, or add 2.5 mm for electrode implantation into the left STN. This position will be marked by the dye of the felt-tip pen after lowering the tip of the needle to the surface of the skull.</li>
	<li>Clamp the dental drill onto the large probe holder of the stereotaxic instrument. Move the dental drill to the calculated area &#8211; i.e., the marked point on the skull. Looking through the microscope, drill a hole (diameter about 1 mm) through the skull until the dura is visible (the skull is about 1 mm thick). Retract the dura using micro-dissection forceps or a sterile needle. The dura is tough enough to destroy the tip of the electrode.</li>
	<li>Drill a hole with the dental drill in each frontal squama, and in the interparietal squama opposite to the electrode hole. Disconnect the probe holder from the stereotaxic instrument. Do not drill on a skull suture as venous vessels follow the sutures under the skull.</li>
	<li>Screw a bone screw into each of the five&#160;holes. Avoid threading the screws in too deep. For stainless-steel screws (M1.6), 2&#8211;3 turns of the screw will adequately hold the screw without putting pressure on the brain. The number of turns will depend on the pitch of the screw. Clamp the probe holder with the electrode in the micromanipulator (Figure 3).</li>
	<li>Using the AP, ML and DV drive screws, move the probe holder with the electrode until the tip is almost touching the bregma. Note the AP, ML and DV vernier scale readings at the bregma. When the readings are made, raise the electrode a few millimeters to prevent the electrode from scraping the skull during movement. To determine the coordinates of the position where the electrode has to be inserted into the hole, add 3.6 mm to the AP reading and add (or subtract) 2.5 mm to the ML reading.</li>
	<li>Using the AP and ML drive screws, move the electrode to the calculated position. At this point, the electrode tip should be situated directly over the drilled electrode hole. Then, by looking through the microscope, lower the electrode to the level of the dura (Figure 4). This level serves as zero-level in the DV direction. Thereafter, gently insert the tip of the electrode into the brain by looking through the microscope.</li>
	<li>Connect the electrode pin to the connector of the recording system. Put a Faraday cage (or substitute it with aluminum foil) over the rat in the stereotaxic instrument (Figure 5). Ground the stereotaxic instrument with the counterpoise of the room that is being worked in.</li>
	<li>Start the recording system. If available, also use a loudspeaker to obtain an acoustic signal of discharges/salves of single units during advancing the electrode.</li>
	<li>Slowly insert the electrode into the brain by recording the electric activity during advancing the electrode. At a depth of between 7.5 and 8.1 mm from the dura, the specific electric activity of the STN is usually detectable (Figure 6). The typical activity of neurons in the STN is characterized by an irregular firing pattern and a high firing rate (mean frequency: 40.9&#160;&#177; 12.9 Hz)18.</li>
	<li>During the recording, reduce anesthesia as much as possible (e.g., to 0.8&#8211;1.0%); low-anesthetized animals show a clearer electric brain activity.</li>
	<li>Swab away any blood or cerebrospinal fluid that was displaced at the surface of the skull when lowering the electrode.</li>
	<li>Mix up a small amount of dental cement and apply it around the electrode and around four&#160;of the five&#160;screws using a small spatula (Figure 7). The fifth&#160;screw will be used to fix the ground wire of the plug.</li>
	<li>Disconnect the electrode pin from the electrode holder and connector of the recording system when the dental cement is fixed.</li>
	<li>Unscrew the screw that was not fixed by dental cement. Put the plug on the electrode pin. Fix the ground wire of the plug with the fifth&#160;screw (Figure 8).</li>
	<li>Mix up dental cement and apply it around the plug. As the cement thickens, mold it around the plug to form a cap. Avoid sharp edges of the dental cement that may harm the animal and remove them during hardening (Figure 9A and B).</li>
	<li>Debride wound edges and close them with a suture at the front and behind the cap. Disinfect the wound edges.</li>
	<li>Connect the head plug to the wire that is fixed on a swivel. Remove the rat from the stereotaxic instrument.</li>
	<li>Apply tramadol (12.5 mg/kg, intraperitoneally) at the end of the intervention and then once daily for 2-3 days. Place the rat in a clean cage with thermal support, fix the swivel on this cage (Figure 10) and inspect it carefully for 1 hr.</li>
	<li>Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
</ol>
3. Stimulation
<ol>
	<li>Determine the resistance in the animal before stimulation using an impedance meter.</li>
	<li>Connect the plug of the swivel with a wire and the plugs at the other end of the wire with the current output and the output for the ground wire of the stimulator. Connect the stimulator with a computer in order to program the stimulator.</li>
	<li>Choose the parameters of stimulation in the program; for example, the parameters used in Parkinson&#8217;s disease are pulse length: 60 &#956;sec; frequency: 130 Hz. Stimulate the rat with an increasing current amplitude until dyskinesia are recognized. Reduce the electric intensity by 10&#8211;20% below the intensity that elicited dyskinesia or until neurologic signs disappear and the animal is comfortable. Monophasic rectangular pulses were used in this study.</li>
	<li>After completing the experiment, euthanize the animal with isoflurane: Adjust the isofurane flow rate or concentration to 5% or greater. Continue isoflurane exposure until 1 min after breathing stops.</li>
</ol>

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The authors declare that they have no competing financial interests.

This study presents a step-by-step set of instructions for implanting a monopolar chronic electrode into the STN of rats. Although tungsten electrodes with low impedance are often used for DBS18,19, a monopolar electrode made of platinum/iridium (Pt/Ir) was employed that had an impedance of about 1 M&#937;. Pt/Ir electrodes are also used in patients with Parkinson&#8217;s disease because of their favorable properties: they demonstrate minimal erosion20 and do not produce relevant tissue damage21 if no high-charge densities are used. Since the aim of this study was a long-term stimulation set-up and, in order to achieve a translational approach, electrodes with the aforementioned specifications were applied in the present experiment. Histologic examination of brain slices showing the localization of the electrode tip corroborated the attenuated foreign-body response of Pt/Ir22 in this experiment.
In the present study, electrodes of Pt/Ir with an impedance of 1 M&#937; were used.&#160; Electrodes with lower, or even higher impedance, are only suitable for either recording cerebral activity or for stimulating cerebral structures, but not both. In contrast, an electrode impedance of 1 M&#937; , as used in our study, is suitable for both, recording the activity of deep brain areas and stimulating cerebral structures such as the STN. The major advantage of recording is the identification of the STN location in a short amount of time. Recording allows reliable localization of the STN, as our results have shown: histologic control yielded a high success rate of targeting the STN (8 of 10 animals). The electrode tip was implanted into the upper cell layers of the dorsal-lateral portion of the STN (DV: 7.7 mm) which is known to receive motor inputs mainly from the motor cortex23.
Using a wiring system for DBS may be limited by potential complications such as breaking of wires or a low degree of freedom of movement of animals. However, in our set-up, the wires were connected to swivels, which allowed the animals to move freely. Wireless in vivo stimulating systems (often fixed at the head or implanted into the trunk of the animal) are also limited by the requirement for batteries. As the batteries need to be small, the voltage is therefore low. When using 1 M&#937; electrodes, a high voltage is required to achieve the desired stimulus intensity and, in turn, results in larger batteries or frequent replacement of batteries. However, an advantage of the stimulus system used in our study is the large voltage compliance range of the stimulator and the option of constant current stimulation. In this mode the stimulator adjusts the voltage to changes in tissue impedance in order to provide a constant current output at the electrode. An impedance change is expected over the long-term course of DBS with the formation of a stable tissue-electrode interface, e.g., glial encapsulation of the electrode tip22.
In summary, the presented method of electrode implantation is technically simple to perform, reliable, and robust, allowing accurate and safe stimulation of the STN in rats without restricting the freedom of movement or even injuring the animal during the long-term course. With small modifications (e.g., using a plug with additional electrical outputs), this protocol is also applicable by implanting microelectrodes in both STNs or other cerebral structures, long-term recordings or both, stimulation of deep brain structures and recording activity of another cerebral region.

Deep brain stimulation (DBS) is a treatment based on the delivery of electrical impulses via implanted electrodes to specific cerebral structures, such as the internal globus pallidus1, the subthalamic nucleus (STN)2&#8211;4 or the ventral intermediate thalamus5. In the last two decades, this treatment has been established as a powerful therapeutic tool for Parkinson&#39;s disease1&#8211;4, dystonia6 and tremor7, and is also used to modulate chronic pain7, psychiatric disorders (i.e., obsessive&#8211;compulsive disorder8, major depression9) or intractable epilepsy10,11. Furthermore, DBS might, in the future, become a treatment option for refractory arterial hypertension12 or orthostatic hypotension13.
The physiological mechanisms underlying the effects of DBS remain poorly understood. Studies in anesthetized rodents have provided insight into neural responses to high-frequency stimulation that mimic clinically applied DBS14. However, these studies not only lack behavioral corroboration of the DBS effect but also result in considerable variability depending on the stimulation parameters applied14.
To investigate more concisely the behavioral effects and underlying mechanisms of DBS in conscious rodents, a stimulation set-up is needed that fulfills specific requirements. DBS is mostly used as a long-term therapy (e.g., Parkinson&#39;s disease, chronic pain). Thus, the stimulation set-up in rodents should be designed so that the unit consists of an electrode with a plug, as well as a wire from the plug to an external stimulator; and this unit should be lightweight but unbreakable when fixed onto the skull. Furthermore, freedom of movement is indispensable for rats during stimulation over a prolonged period. The target structures of DBS are small; for example, the STN in rats has a length of 1.2 mm and a volume of 0.8 mm3,15. Therefore, electrodes must be designed such that the nucleus is not lesioned during insertion and targeting needs to be precise. As most DBS studies conducted in rodents have used landmark based stereotactic insertion of the electrode to the target structure, the error rate can be relatively high, even when using the coordinates according to Paxinos and Watson16. This results in a larger number of animals needed to reach a statistically meaningful result.
In the present study an electrode implantation technique is introduced, that targets the STN with high accuracy by using a microrecording system while advancing the electrode. In addition, a stimulation system is presented that does not only allow a high degree of mobility for the stimulated animal but also guarantees continuous stimulation via secure fixation of the stimulation wire (which is protected by a stainless-steel spring) onto the head of the rat.

<table><tbody><tr><td>Pt/Ir electrode</td><td>FHC Inc.</td><td>UE</td><td>Custom-made: Specification: UEPSEGSECN1M</td></tr><tr><td>Plugs</td><td>GT Labortechnik (Arnstein/Germany)</td><td></td><td>Custom-made</td></tr><tr><td>Pin header</td><td>DISTRELEC</td><td>143-95-324</td><td>single-row, 90&amp;deg; 1x3 datamate, Type M80-8420342</td></tr><tr><td>Socket</td><td>DISTRELEC</td><td>143-95-621</td><td>single-row,straight 2 mm pole no.1 x 3 datamate, Type M80-8400342</td></tr><tr><td>Stainless steel spring</td><td>Plastics ONE</td><td>SS0102</td><td>Part-#: .120 X .156 Spring ID (mm): 3.0&amp;nbsp; Spring OD (mm): 4.0</td></tr><tr><td>Dental cement/Paladur</td><td>Heraeus Kulzer</td><td>64707938</td><td>Liquid, 500 ml</td></tr><tr><td>Dental cement/Paladur</td><td>Heraeus Kulzer</td><td>64707954</td><td>Powder, rose, 500 g</td></tr><tr><td>Head screw</td><td>Hummer &amp;amp; Reiss</td><td>V2ADIN84 M1.6x3</td><td></td></tr><tr><td>Jodosept PVP</td><td>Vetoquinol</td><td></td><td>435678/E04</td></tr><tr><td>Mepivacain 1%</td><td>AstraZeneca</td><td></td><td>PZN03338515</td></tr><tr><td>Epinephrine</td><td>Sanofi-Aventis</td><td></td><td>PZN00176118</td></tr><tr><td>Tramadolhydrochloride</td><td>Rotexmedica</td><td></td><td>38449.00.00</td></tr></tbody></table>

microelectrode guided implantation of electrodes into the subthalamic nucleus of rats for long-term deep brain stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

Implanting an electrode into the STN of a rat using a recording system &#8211; as presented here &#8211; is an effective and accurate procedure for DBS that takes approximately 1 hr per animal. This model is a fairly minor procedure: out of 10 rats subjected to surgery, all survived the intervention. Twenty-four hr after intervention, the state of each rat was monitored and no animal achieved more than 1 of 3 points according to the severity code. During the period of continuous stimulation (14 days, 24 hr a day), no wire detached, broke or was bitten through. None of the 10 rats lost the cap of dental cement nor did they get hurt by the equipment during the phase of stimulation. The impedance measured in these 10 animals before stimulation was 353 &#177; 101 k&#937; . Rats were stimulated at a frequency of 130 Hz and a pulse width of 60 &#181;sec. The mean stimulus intensity was 60 &#181;A, which was set at 20% below the intensity threshold of orofacial or contralateral forepaw dyskinesia, thereby preventing problems with feeding or locomotion during the period of stimulation.
Fourteen days after intervention and continuous stimulation, all 10 rats were euthanized by decapitation after deep anesthesia and brains were rapidly harvested. In a rat brain matrix, a 2 mm thick brain block encompassing the STN was cut and immediately frozen at &#8211;80 &#176;C. These brain blocks were cut in coronal sections (8 &#181;m thick). Each section was stained with hematoxylin &#38; eosin to visualize the position where the tip of the electrode was located, as well as to detect sings of inflammation or scar tissue due to the electrode. The success rate for localizing the electrode in the STN was 8 of 10 animals. In these 8 rats, the tip of the implanted electrode was situated in the STN, as shown histologically. Figure 11 illustrates the electrode location in the STN. A small lesion developed after continuous stimulation was found in all rats. This lesion was surrounded by a small number of inflammatory cells (Figure 11).
<img alt="Figure 1" src="/files/ftp_upload/53066/53066fig1.jpg" /> 
Figure 1. Fixation of the head in the stereotaxic instrument. The rat is fixed by the ear bars of the stereotaxic frame, as well as by the gas anesthesia mask. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53066/53066fig2.jpg" /> 
Figure 2. Exposing the skull. After a midline incision, the skin and periosteum are rolled to the wound edges and kept away from the surgical area using four clamps. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53066/53066fig3.jpg" /> 
Figure 3. Fixation of the electrode in a probe holder. Using forceps, the pin of the electrode is inserted in the plug and fixed with a probe holder. The plug is connected with the recording apparatus via a wire. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53066/53066fig4.jpg" /> 
Figure 4.&#160;Insertion of the electrode in the brain. After determining the exact AP and ML coordinates of the subthalamic nucleus, the tip of the electrode is advanced to the level of the punctured dura and the dorsoventral vernier scale reading is taken. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53066/53066fig5.jpg" /> 
Figure 5. Protection of electric interference. A Faraday cage (or, alternatively, aluminum foil) is put over the rat in the stereotaxic instrument and the instrument, as well as the animal, is grounded. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53066/53066fig6.jpg" /> 
Figure 6. Recording of brain activity. The subthalamic nucleus (STN) shows an irregular firing pattern and a high firing rate (mean frequency: 40.9&#160;&#177; 12.9 Hz)18. Before entering the STN, the electrode passes a relatively silent region, which is consistent with the zona incerta; the vertical size of this area measures about 0.5&#8211;1 mm. Thereafter, the number of spikes increases, indicating that the insertion into the STN is complete. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/53066/53066fig7.jpg" /> 
Figure 7. Fixation of the electrode. When the subthalamic nucleus is identified by means of recording, the electrode is fixed by applying dental cement around the electrode shank and the screws. This allows unplugging of the connector from the electrode pin without shifting the position of the electrode. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/53066/53066fig8.jpg" /> 
Figure 8. Attaching the plug to the electrode pin. The plug for the connector of the stimulator is attached to the electrode pin. The ground wire, which is soldered onto the plug, is fixed with a screw onto the skull. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/53066/53066fig9.jpg" /> 
Figure 9. Fixation of the plug. (A) Frontal and (B) Lateral views. Dental cement is applied around the plug and a cap is formed; sharp edges should be avoided. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/53066/53066fig10.jpg" /> 
Figure 10. Connection of the rat to the stimulator. A swivel was joined into the circuit to prevent the wire from becoming tangled. A stainless-steel spring protects the wire if the rat starts to bite the wire. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" src="/files/ftp_upload/53066/53066fig11.jpg" /> 
Figure 11. Brain section through the subthalamic nucleus (STN) (hematoxylin &#38; eosin staining). (A) Overview, magnification 2.5. A solid line surrounds the STN. A small lesion is visible where the electrode tip was located during a 14 day period of stimulation. It is of note that there is no penetration canal of the electrode visible (shank diameter: 125 &#956;m), indicating that the electrode is conserving the tissue. (B) Image detail from picture A (box), magnification 100. A small number of inflammatory cells was detectable around the lesion due to the reaction of the brain tissue to the electrode tip. Arrow: indicating an example of an inflammatory cell. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53066/53066fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation at JoVE.com

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

microelectrode-guided-implantation-electrodes-into-subthalamic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

19.5K Views.  University Hospital Wuerzburg. A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Video: Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through implanted electrodes. Deep brain stimulation was first implemented as a therapeutic modality by Benabid et al. in the late 1980s, when he used this technique to stimulate the ventral intermediate nucleus of the thalamus for the treatment of tremor 1. Currently, the procedure is used to treat patients who fail to respond adequately to medical management for diseases such as Parkinson's, dystonia, and essential tremor. The efficacy of this procedure for the treatment of Parkinson's disease has been demonstrated in well-powered, randomized controlled trials 2. Presently, the U.S. Food and Drug Administration has approved DBS as a treatment for patients with medically refractory essential tremor, Parkinson's disease, and dystonia. Additionally, DBS is currently being evaluated for the treatment of other psychiatric and neurological disorders, such as obsessive compulsive disorder, major depressive disorder, and epilepsy. 
DBS has not only been shown to help people by improving their quality of life, it also provides researchers with the unique opportunity to study and understand the human brain. Microelectrode recordings are routinely performed during DBS surgery in order to enhance the precision of anatomical targeting. Firing patterns of individual neurons can therefore be recorded while the subject performs a behavioral task. Early studies using these data focused on descriptive aspects, including firing and burst rates, and frequency modulation 3. More recent studies have focused on cognitive aspects of behavior in relation to neuronal activity 4,5. This article will provide a description of the intra-operative methods used to perform behavioral tasks and record neuronal data with awake patients during DBS cases. Our exposition of the process of acquiring electrophysiological data will illuminate the current scope and limitations of intra-operative human experiments.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through ...

Medicine

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people.  There is a need ...

Behavior

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 ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help delineate the epileptic region to resect. In long-term applications such as brain-computer interfaces, these epicortical electrodes are used to record the movement intention of the brain, to control the robotic limbs of paralyzed patients. However, current stiff electrode grids do not answer the need for high-resolution brain recordings and long-term biointegration. Recently, conformable electrode arrays have been proposed to achieve long-term implant stability with high performance. However, preclinical studies for these new implant technologies are needed to validate their long-term functionality and safety profile for their translation to human patients. In this context, porcine models are routinely employed in developing medical devices due to their large organ sizes and easy animal handling. However, only a few brain applications are described in the literature, mostly due to surgery limitations and integration of the implant system on a living animal.
Here, we report the method for long-term implantation (6 months) and evaluation of soft ECoG arrays in the minipig model. The study first presents the implant system, consisting of a soft microfabricated electrode array integrated with a magnetic resonance imaging (MRI)-compatible polymeric transdermal port that houses instrumentation connectors for electrophysiology recordings. Then, the study describes the surgical procedure, from subdural implantation to animal recovery. We focus on the auditory cortex as an example target area where evoked potentials are induced by acoustic stimulation. We finally describe a data acquisition sequence that includes MRI of the whole brain, implant electrochemical characterization, intraoperative and freely moving electrophysiology, and immunohistochemistry staining of the extracted brains.
This model can be used to investigate the safety and function of novel design of cortical prostheses; mandatory preclinical study to envision translation to human patients.

Subdural Soft Electrocorticography ECoG Array Implantation and Long-Term Cortical Recording in Minipigs

Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help ...

Bioengineering

Implanting a Wireless Bidirectional Microelectrode System in a Rat Brain

Source: Melo-Thomas, L., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/56299">A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats.</a> J. Vis. Exp. (2017)
The video demonstrates a procedure for implanting a wireless bidirectional multichannel electrode unit into a rat brain to record and stimulate multichannel neural networks.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Summary

Explore More Videos

Chapters in this video

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

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

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

Deep Brain Stimulation with Simultaneous fMRI in Rodents

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

Implanting a Wireless Bidirectional Microelectrode System in a Rat Brain