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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+results+of+bilateral+pallidal+stimulation+in+Parkinson&#8217;s+disease.">Long-term results of bilateral pallidal stimulation in Parkinson&#8217;s disease.</a> Annals of Neurology. 55 (6), 871-875 (2004).</li><li>Odekerken, V. J., van Laar, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subthalamic+nucleus+versus+globus+pallidus+bilateral+deep+brain+stimulation+for+advanced+Parkinson&#8217;s+disease+(NSTAPS+study):+a+randomised+controlled+trial.">Subthalamic nucleus versus globus pallidus bilateral deep brain stimulation for advanced Parkinson&#8217;s disease (NSTAPS study): a randomised controlled trial.</a> The Lancet Neurology. 12 (1), 37-44 (2013).</li><li>Benabid, A. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+basal+ganglia+in+rats,+marmosets,+macaques,+baboons,+and+humans:+Volume+and+neuronal+number+for+the+output,+internal+relay,+and+striatal+modulating+nuclei.">Comparison of the basal ganglia in rats, marmosets, macaques, baboons, and humans: Volume and neuronal number for the output, internal relay, and striatal modulating nuclei.</a> The Journal of Comparative Neurology. 445 (3), 238-255 (2002).</li><li>Paxinos, G., Watson, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The rat brain in stereotaxic coordinates. , (2007).</li><li>Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bench+to+bedside:+the+quest+for+quality+in+experimental+stroke+research.">Bench to bedside: the quest for quality in experimental stroke research.</a> Journal of Cerebral Blood Flow, &amp; Metabolism. 26 (12), 1465-1478 (2006).</li><li>Maesawa, S., Kaneoke, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stimulation+of+the+subthalamic+nucleus+in+hemiparkinsonian+rats:+neuroprotection+of+dopaminergic+neurons.">Long-term stimulation of the subthalamic nucleus in hemiparkinsonian rats: neuroprotection of dopaminergic neurons.</a> Journal of Neurosurgery. 100 (4), 679-687 (2004).</li><li>Spieles-Engemann, A. 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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 damage2...

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 border="0" cellpadding="0" cellspacing="0">
	<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&#176; 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.1x3 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&#160; 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, 500g</td>
		</tr>
		<tr>
			<td>Head screw</td>
			<td>Hummer &#38; 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 wir...

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, ...

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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

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

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 ...

Cannula Implantation into the Cisterna Magna of Rodents

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to the skull or the brain parenchyma. In anesthetized rodents, the exposure of the dura mater by blunt dissection of the neck muscles allows the insertion of a cannula into the cisterna magna (CM). The cannula, composed either by a fine beveled needle or borosilicate capillary, is attached via a polyethylene (PE) tube to a syringe. Using a syringe pump, molecules can then be injected at controlled rates directly into the CM, which is continuous with the subarachnoid space. From the subarachnoid space, we can trace CSF fluxes by convective flow into the perivascular space around penetrating arterioles, where solute exchange with the interstitial fluid (ISF) occurs. CMc can be performed for acute injections immediately following the surgery, or for chronic implantation, with later injection in anesthetized or awake, freely moving rodents. Quantitation of tracer distribution in the brain parenchyma can be performed by epifluorescence, 2-photon microscopy, and magnetic resonance imaging (MRI), depending on the physico-chemical properties of the injected molecules. Thus, CMc in conjunction with various imaging techniques offers a powerful tool for assessment of the glymphatic system and CSF dynamics and function. Furthermore, CMc can be utilized as a conduit for fast, brain-wide delivery of signaling molecules and metabolic substrates that could not otherwise cross the blood brain barrier (BBB).

Here we describe a protocol to perform cisterna magna cannulation (CMc), a minimally invasive way to deliver tracers, substrates and signaling molecules into the cerebrospinal fluid (CSF). Combined with different imaging modalities, CMc enables glymphatic system and CSF dynamics assessment, as well as brain-wide delivery of various compounds.

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...