Name: Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats (Video) | JoVE Journal
Uploaded: 2017-06-22T15:00:00.000+00:00
Duration: 10 min 46 s
Description: Learn acute in vivo electrophysiological recordings of local field potentials and multi-unit activity from the hyperdirect pathway in anesthetized rats. This method enables simultaneous cortical and subcortical recordings for neuroscience research.

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5 minutes

We thank the Deutsche Forschungsgemeinschaft (DFG), KFO 247, for funding our study.

Experimental procedures were conducted in accordance with the German Animal Welfare Act (last revised in 2014) and European regulations (2010/63/EU). Experiments were approved by the local animal welfare authority (LaGeSo, Berlin), and conformed to local department and international guidelines.
NOTE: In the presented method two models of electrodes are used to record from the hyperdirect cortico-basal ganglia pathway that connects the primary motor cortex (M1) with the subthalamic nucleus (STN) and the substantia nigra pars reticulate (SNr). For epidural electrocorticogram (ECoG) recordings from the M1 custom-made low-impedance Ag/AgCl electrodes are used. The recordings from the STN and SNr are performed with commercially available high-impedance tungsten electrodes.
1. Construction of Epidural Ag/AgCl Epidural Electrodes
<ol>
	<li>Take an approx. 5 cm long strip of 99.99% pure silver wire with a diameter of 200 &#181;m and remove any coating if necessary.</li>
	<li>Hold the wire tip with the tip downwards into a flame of a lighter or candle until the tip starts to melt. Wait until the tip is ball-shaped and has a diameter of approximately 1 mm. Cut off the preformed electrode to a total length of 15 mm from the beginning of the ball-shaped tip to the wire end.</li>
	<li>Solder a precision connector to the wire end, which fits the used electrophysiological recording system. Cover the soldering point from the wire end to the connector with conductive silver varnish. This helps conductivity and results in a better signal quality.</li>
	<li>After the conductive varnish has dried, cover the soldering point with a 3 mm to 1 mm heat shrinking tube. Carefully use a watchmaker&#39;s hammer to flatten the ball-shaped tip to half the thickness.</li>
	<li>Put on examination gloves and take a lint-free cleaning cloth with 100% ethanol to remove any dirt and grease.</li>
	<li>Put the electrodes in a 15 mL centrifuge tube or cell culture tube and fill it up with household chlorine bleach (CAUTION; containing 2.8 g of sodium hypochloride per 100 g solvent) until the ball-shaped tip is fully covered. 
	CAUTION: Chlorine bleach is corrosive; always follow the manufacturer&#39;s safety instructions.</li>
	<li>Take the electrodes out after 23 min and flush them generously with distilled water. The successful application of a silver chloride layer appears as a homogenous purple change in color.</li>
	<li>Dry in air. After fully dried, take the electrodes with fine tweezers. With a fine paintbrush, apply liquid electrical insulation. Start on the wire directly behind the electrode tip and cover everything up to the heat shrinking tube. Let the insulation dry for at least 2 h.</li>
	<li>For quality control, perform a check of electrical conductivity with a multimeter. If available, perform impedance testing at 1 kHz using an appropriate impedance meter, while the electrode and test probe are put together in a 0.9% NaCl containing H2O solution without touching each other. Impedance values at 1 kHz should be approximately 8 k&#937;.</li>
</ol>
2. Affixing the Electrodes to a Stereotaxic Holder
NOTE: To record MUA and LFPs at the same time, use tungsten microwire electrodes with an impedance of 1.5 M&#937;. If the focus of the recordings is on high quality recordings of single units, choose microwire electrodes with a higher impedance (&#62;5 M&#937;). If the aim of the study is solely directed at LFPs, electrodes with lower impedances can be acceptable. For small structures, for which dorsoventral stereotaxic adjustments are often necessary, use pairs of electrodes with a suitable dorsoventral tip separation (in this case 250 &#181;m). Furthermore, this provides the advantage of a more local reference electrode, if needed. The stereotaxic coordinates are always measured from the lowermost electrode and are calculated in reference to the bregma.
<ol>
	<li>Take a standard stereotaxic electrode holder with an acrylic block and clamp and lay it securely on a flat surface in the field of view of a surgical microscope.</li>
	<li>Loosely fix the first pair of electrodes to the acrylic block of the holder with pieces of adhesive tape (3 mm x 8 mm) using fine tweezers. The electrodes should protrude the acrylic block approx. 12 mm.</li>
	<li>Carefully fix the second bipolar electrode next to the first electrode. For targeting the structures of the hyperdirect pathway, the distance should be 2 mm (Figure 1). For most standard stereotaxic electrode holders, this is the adjacent recess. Use a caliper gauge to verify. Different networks can be approached the same way. For this, the acrylic block may be turned to a certain degree.</li>
	<li>Adjust the second pair of electrodes by sliding it carefully to a position, in which the most ventral tip is approximately 200 &#181;m recessed compared to the first electrode (Figure 1). Do this under microscopic vision. For this, use a 30 G cannula (outer diameter 300 &#181;m) to better estimate the distance.</li>
	<li>Press on the adhesive tape, and then secure with the holder&#39;s metal clamp.</li>
</ol>
3. Surgery
<ol>
	<li>For electrophysiological recordings, use urethane (CAUTION) for anesthesia. 
	Caution: Urethane is toxic and carcinogenic, so always adhere to the safety regulations and data sheet given by the substance&#39;s manufacturer.</li>
	<li>Prepare a solution of 200 mg/mL urethane in 0.9% NaCl medical saline solution.</li>
	<li>Administer a total of 1.3 g/kg bodyweight urethane intraperitoneally (IP). Depending on the rat strain it might be reasonable to split the dose into two doses with a 15 min interval in between the injections in order to enhance the safety of the anesthesia.</li>
	<li>Check the depth of anesthesia by using pedal-withdrawal reflex and other suitable reflexes. If the anesthesia is not deep enough to perform surgery, inject 0.15 g/kg bodyweight of urethane IP and wait another 15 min.</li>
	<li>Apply an eye ointment to prevent corneal dehydration.</li>
	<li>Constantly monitor respiratory rate and pedal withdrawal reflex during anesthesia. Use a small animal heating pad with temperature control to ensure that a physiological body temperature is maintained throughout the surgery. Before the start electrophysiological recordings, change to a non-electrical alternative (e.g. sodium acetate head pad).</li>
	<li>Shave the fur alongside the dorsal side of the head to achieve a clean surgical field. Disinfect around the incision site with appropriate surgical disinfectant. Fix the animal in the stereotactic frame.</li>
	<li>Perform a 2-cm long incision of the scalp in sagittal direction with a scalpel. Use a scalpel to slightly scrape off the skull aponeurosis and disinfect the skull. Use cotton buds soaked in 3% H2O2 to remove any remaining tissue.</li>
	<li>Use an electrocauter or thermocauter to control bleeding, if necessary. Stop bleedings from the skull bone and hypoderm, if the bleeding does not stop spontaneously after 1-2 min and hinders sight onto the skull.</li>
	<li>Adjust the incisor bar until the head is positioned in flat skull position, which means the bregma and lambda as stereotaxic reference points are in the same plane. This is most important to achieve high surgical precision. Use a standard stereotaxic rat alignment tool, calibrate the designated tip to the bregma under microscopic vision and adjust the incisor bar until the designated points for the bregma and lambda on the tool touch the skull at the same time. 
	NOTE: A view from one side with focused light from the other may help to determine this condition. Alternatively, take a stereotaxic holder with a fine cannula and measure the dorsoventral coordinates of Bregma and lambda under microscopic vision. Adjust the incisor bar until the dorsoventral coordinates of Bregma and lambda are the same.</li>
	<li>Use a stereotaxic holder with cannula, calibrate to the bregma and then calculate the position of all drill holes on the skull. Using the stereotaxic holder, mark the positions of the holes to be drilled either by carefully scratching the skull or by using a surgical color marker. The coordinates for this depend on the targets, coordinates with reference to the bregma are given for the hyperdirect pathway in Table 1, including the suggested coordinates for cerebellar reference electrodes.</li>
	<li>Using a microdrill carefully drill all holes. For the STN and SNr, drill a common hole (approximately 2 mm x 3 mm in size). All other drill holes should have a diameter of about 1 mm.</li>
	<li>Take two fine cannulas (at least 27 G) and bend their tips to form a hooked shape, using a hard surface or tweezers. Use these to remove any debris from the drill holes, and carefully cut and remove the dura mater in the common STN/SNr hole.</li>
	<li>Flush the drill holes with physiologic saline. Apply a drop of physiologic saline every 15 min to the drill holes to prevent the brain and dura from drying-out.</li>
	<li>Take a microdrill and matching stainless steel micro-screw (e.g. a M 1.2 mm x 2 mm screw), drill a hole and screw in a micro-screw in between the drill holes of the reference epidural electrodes above cerebellum, do the same for the M1 epidural electrodes.</li>
	<li>Slide the self-built Ag/AgCl epidural electrodes into the drill holes for the reference electrodes and M1 electrodes. Guide the electrode tip with fine tweezers and slide it directly below the skull bone into the drill hole.</li>
	<li>Fix all epidural electrodes with two-component dental acrylic. Make sure not to cover the bregma point nor affect the common STN/SNr hole.</li>
	<li>Insert the prepared holder with the tungsten microwire electrodes into the stereotaxic frame.</li>
	<li>Calibrate the most ventral electrode, which is intended to target the STN, to the bregma. Adjust to the calculated position above the common STN/SNr hole and lower the electrodes down to the brain under microscopic vision. Make sure that the tungsten microwire electrodes go inside the brain smoothly.</li>
</ol>
4. Electrophysiological Mapping and Recordings
NOTE: For this step, a Faraday cage and a multi-channel electrophysiological recording system with recording software capable of online-filtering and online spike-sorting is necessary. Preferably use a system that works with a preamplifier positioned near the head of the animals to keep electrical noise and artifacts to an absolute minimum. Besides the tungsten microwire electrodes, at least one epidural and one reference electrode are necessary to perform recordings of the hyperdirect pathway. It is recommended to insert epidural and reference electrodes pairwise without them touching each other, this helps in case of malfunctioning and allows for different types of referencing in data analysis.
<ol>
	<li>Put a mobile Faraday cage above the stereotaxic frame. If only a stationary Faraday cage is available, carefully move the stereotaxic frame into the Faraday cage while making sure deep brain electrodes are not lowered into the brain until the stereotaxic frame is in its final position.</li>
	<li>Connect the electrodes to the headstage of the electrophysiological setup. Make sure the reference electrodes are connected to appropriate reference channels.</li>
	<li>Set up the recording software: Bandpass filter (0.05-8,000 Hz) and amplify (gain 1,500-2,000x) the raw data signal. Use an online LFP and spike filter with appropriate settings (bandpass filter 0.05-250 Hz for LFPs, bandpass filter 300-8,000 Hz for MUA). For all filters, use a butterworth-type filter.</li>
	<li>Set up a spike threshold, if applicable, for online spike sorting. Most recording software allows for setting up of a spike threshold, which is an amplitude value above which a signal is marked as a spike by the software. This threshold can either be determined mathematically as a factor or standard deviation of the mean amplitude of the filtered spike signal, or can preferably be determined by visual inspection of data segments &#60;500 ms and set up as a line above the signal noise in a graphical user interface. 
	NOTE: The intention in setting up a spike threshold is to count spikes and sort units in order to provide information on how many neurons are presently recorded and how their spikes are shaped.</li>
	<li>Slowly lower the tungsten microwire electrodes to 1 mm dorsal of the target, which is STN for hyperdirect pathway. Wait for the signal to stabilize if necessary.</li>
	<li>For the electrophysiological mapping, advance the electrodes ventrally in steps of 100 &#181;m. At each step, evaluate the firing pattern, firing rate and shape of spikes. Compare those with the typical examples given in Figure 2. Commonly, dense nuclei show fast and continuous spiking over several dorsoventral steps, whereas fiber-rich structures show low firing rates and less homogenous spike activity in subsequent ventral steps.</li>
	<li>For the hyperdirect pathway, make sure that the most ventral electrode is inside STN. 
	NOTE: The STN is reached when a considerable increase in MUA is detected ventral of the Zona incerta. Ventral to the STN, spiking stops almost completely because the electrode has reached the internal capsule. When the most ventral electrode is in STN, the configuration of the tungsten microelectrodes ensures the posterior, second electrode is in SNr. Dorsoventral fine-tuning in small increments might be necessary to record typical MUA in STN and SNr at the same time. Note that the frequency of MUA depends on the number of neurons actually recorded and on the level brain activation.</li>
	<li>Once the electrodes are in the desired structures, set up online filtering and spike-sorting (see Figure 4), and then start the recording of the data. Typical examples for the different cortical synchronization states that can be identified in the LFP recordings are shown in Figure 3.</li>
</ol>
5. End of Experiment
<ol>
	<li>When recordings are done, slowly raise the electrodes out of the brain and instantaneously flush them with physiological saline. The electrodes can be reused after thorough flushing and visual inspection. Discharge bend electrodes.</li>
	<li>Euthanize the animals by an IP injection of an overdose of urethane (2.5 g/kg bodyweight). 
	NOTE: Urethane should only be used for final procedures.</li>
	<li>If histological verification of the electrode position or other histological staining procedures is required, remove the brain from the skull and process the tissue appropriately. 
	NOTE: Depending on the intended staining methods, transcardial perfusion may be necessary. For post-mortem verification of the electrode position, a standard Nissl staining is sufficient in most cases to visualize the electrode trajectory in e.g. coronal brain sections. Further approaches to facilitate histological target verification include the use of electrically induced lesions to the brain tissue by applying electrical current via the recording electrodes or the application of biocompatible dye prior to electrode insertion16,17.</li>
</ol>

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<li>Mahmud, M., Vassanelli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Processing+and+Analysis+of+Multichannel+Extracellular+Neuronal+Signals%3A+State-of-the-Art+and+Challenges%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">Processing and Analysis of Multichannel Extracellular Neuronal Signals: State-of-the-Art and Challenges.</a> Front Neurosci. 10, 248(2016).</li>
<li>Hadar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+neural+oscillations+and+elevated+dopamine+levels+in+the+reward+pathway+during+alcohol+relapse%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Altered neural oscillations and elevated dopamine levels in the reward pathway during alcohol relapse.</a> Behav Brain Res. 316, 131-135 (2017).</li>
<li>Voget, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+local+field+potential+activity+and+serotonergic+neurotransmission+are+further+characteristics+of+the+Flinders+sensitive+line+rat+model+of+depression%5BTitle%5D)+AND+%22Behav+Brain+Res%22%5BJournal%5D)">Altered local field potential activity and serotonergic neurotransmission are further characteristics of the Flinders sensitive line rat model of depression.</a> Behav Brain Res. 291, 299-305 (2015).</li>
</ol>

The authors have nothing to disclose.

In the present study, the method is demonstrated how to record extracellular electrophysiological signals simultaneously from multiple sites of a given network using the example of the hyperdirect cortico-basal ganglia pathway that connects the M1 with the STN and SNr in rodents.
A critical step in the recording of small subcortical structures such as the STN is the precisely guided insertion of the recording electrodes into the target. In the presented method, taking care of two crucial steps...

Recent cumulative evidence on different neuropsychiatric disorders such as Parkinson&#180;s disease (PD) and schizophrenia strongly suggests that their pathophysiology is based on a critical dysfunction of extended neuronal circuits that often involve cortical and subcortical structures1,2,3. According to this theory, the clinical manifestations of the diseases arise as a consequence of an impaired information processing capacity of a network of cells instead of single cells or specific neuronal elements1,2,3. In order to enhance the understanding of this complex group of neuropsychiatric diseases and to find new treatment options, it is mandatory to characterize the neuronal dynamics of those disordered networks in human patients and in animal models in great detail. An excellent method to study large-scale networks in living subjects is multi-site electrophysiological recordings of extracellular potentials4. Using this method, it is possible to simultaneously assess local field potentials (LFPs), that primarily represent the temporal summation of excitatory and inhibitory postsynaptic currents and multi-unit activity (MUA), that is generated by presynaptic potentials5. The recording of extracellular potentials has several advantages over alternative methods to study networks, e.g., functional magnetic resonance imaging and calcium imaging, because it provides a higher temporal and spatial resolution and because it is not dependent on genetically engineered organisms5. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied.
In vivo electrophysiological recordings can be performed in awake as well as in anesthetized animals6. Both methods are accompanied by specific pros and cons. Studies in awake animals allow the recording of brain signals during the performance of defined behavioral tasks, but are prone to movement related and other artifacts7,8. Recordings in anesthetized animals on the other hand offer the opportunity to assess LFPs and MUA with a minimum of artifacts at highly defined cortical synchronization states, but the results also differ to some extent to what can be found in awake subjects9,10,11.
In recent years, it has been demonstrated that the sampling of LFPs is especially useful to delineate pathological changes of network activity. A prominent example of this is research on the pathophysiology of PD in human patients and animal models of the disease, where it could be shown that enhanced beta oscillations in the cortico-basal ganglia loop are linked to parkinsonian motor symptoms12,13. As a consequence of this line of research, it is currently investigated if beta oscillations could be used as an online feedback biomarker for closed-loop deep brain stimulation14,15.
In the present study, a detailed description of acute multi-site in vivo electrophysiological recordings of LFPs and MUA in rats anesthetized with urethane is provided. It is demonstrated how a complete network, such as the hyperdirect cortico-basal ganglia pathway can be characterized electrophysiologically using standard and customized electrodes and how those electrodes can be build. It is especially emphasized how a precise targeting of basal ganglia nuclei can be achieved by combining stereotactic surgery together with the online registration of MUAs.

<table><tbody><tr><td>Ag/AgCl custom epidural electrodes</td><td>Goodfellow GmbH&lt;br/&gt; D-61213 Bad Nauheim, Germany&lt;br/&gt; info@goodfellow.com</td><td>Product-ID AG005127 for 99.99% silver wire</td><td>Ag/AgCl electrodes will allow for better signal quality, but may only be used in acute experiments. Possible replacement: Stainless steel electrodes</td></tr><tr><td>Stereotaxic holder with acrylic block</td><td>David Kopf Instruments,&lt;br/&gt; 7324 Elmo Street, Tujunga, CA 91042, USA</td><td>Product ID Model 1770 Standard Electrode Holder</td><td>Make sure the acrylic block has recesses which suit the electrode setup for the desired target. Acrylic blocks can easily be modified with a file to obtain the desired configuration. Possible replacement: Self-constructed electrode holders</td></tr><tr><td>Tungsten microwire electrodes 1.5 M&amp;Omega; impedance</td><td>Microprobes.com&lt;br/&gt; 18247-D Flower Hill Way&amp;nbsp; Gaithersburg, Maryland, 20879 USA</td><td>Product-ID WE3ST31.5A5-250um</td><td>The 1.5 M&amp;Omega; is necessary to record MUA and LFP at the same time. Possible replacement: Microelectrodes of different materials can be used. The electrodes have to be straight, robust and as thin as possible.</td></tr><tr><td>Rat alignment tool</td><td>David Kopf Instruments,&lt;br/&gt; 7324 Elmo Street, Tujunga, CA 91042, USA</td><td>Product ID Model 944 Rat Alignment Tool</td><td>Allows the exact orientation of the brain to match stereotaxic atlases. Possible replacement: Stereotaxic holder with a cannula</td></tr><tr><td>Two-component dental acrylic</td><td>Associated Dental Products Ltd.&lt;br/&gt; Kemdent Works, Purton, Swindon&lt;br/&gt; Wiltshire, SN5 4HT, United Kingdom</td><td>Simplex Rapid Powder Clear 225g, Product code: ACR803; Simplex Rapid Liquid 150ml, Product code: ACR920</td><td>Depending in the electrodes used, superglue might be an easy alternative, if the electrodes are small and lightweight. Possible replacement: Superglue (Cyanacrylate-based)</td></tr><tr><td>Faraday cage</td><td>Self-construction</td><td></td><td>A proper Faraday cage will be the best protection from electromagnetic artifacts, but everything which can be formed into a box shape or applied to a frame and is made of conductive material may help. Possible replacement: Aluminum foil or copper mesh</td></tr><tr><td>Electrophysiological setup with recording software and online spike-sorting capabilities</td><td>OmniPlex&lt;sup&gt;&amp;reg;&lt;/sup&gt; Neural Data Acquisition System&lt;br/&gt; Plexon Inc&lt;br/&gt; 6500 Greenville Avenue, Suite 700&lt;br/&gt; Dallas, Texas 75206&lt;br/&gt; USA</td><td></td><td>Offline sorting software is a potential alternative, multiple scripts and softwares can be found for free in the open source community.</td></tr></tbody></table>

acute in vivo electrophysiological recordings of local field potentials and multi-unit activity from the hyperdirect pathway in anesthetized rats

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

With the herein used recording electrodes, it is possible to sample LFPs from the primary motor cortex, the subthalamic nucleus and the substantia nigra pars reticulata and MUA from the STN and SNr. Initially, LFPs and multi-unit activity are recorded together in a broad-band signal. Thereafter, LFPs and MUAs are separated by bandpass filters (0.05-250 Hz for LFPs and 300-4,000 Hz for MUA).
For the correct targeting of subcortic...

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

The overall goal of this experiment is to demonstrate how to perform acute in vivo electrophysiological recordings of local field potentials and multi-unit activity from the cortico-basal ganglia loop of anesthetized rats. This method can help answer key questions in the neuroscience field such as how pathological oscillations contribute to the complex pathophysiology in neuropsychiatric diseases. The main advantage of this technique is that it can be used to record extracellular potentials from various cortical and subcortical targets of the brain simultaneously.The implications of this technique extend toward therapy of different neuropsychiatric diseases because local field potentials can be potentially used as a biomarker for treatments such as closed-loop stimulation. Though this method can provide insight into the pathophysiology of the basal ganglia, it can also be applied to other brain areas. To begin this procedure, prepare a five centimeter length of silver wire with a diameter of 200 micrometers and remove any coating if necessary.Next, hold the wire tip downwards in a flame until the tip starts to melt and forms a ball with a diameter of one millimeter. Then cut off the electrode to a total length of 15 millimeters from the beginning of the ball-shaped tip to the wire end. Solder a precision connector to the end of the wire which fits the electrophysiological recording system.Then cover the soldering point from the wire end to the connector with conductive silver varnish to improve conductivity. After the conductive varnish has dried, cover the soldering point with a one to three millimeter heat shrinking tube. Carefully flatten the ball-shaped tip to half the thickness using a watchmaker's hammer.Subsequently, put on examination gloves and use a lint-free cleaning cloth with 100%ethanol to remove any dirt and grease. Then place the electrodes in a 15 milliliter centrifuge tube or a cell culture tube and fill it up with household chlorine bleach until the ball-shaped tip is fully covered. After 23 minutes, take the electrodes out and flush them generously with distilled water.The electrode surface should appear homogeneously dark purple in the successful application of silver chloride. Following that, leave the electrodes to dry in air. Take the electrodes with fine tweezers after they have been fully dried.Then use a fine paintbrush to apply liquid electrical insulation onto the electrodes. Start from the wire directly behind the electrode tip and cover everything up to heat shrinking tube. Afterward, let the insulation dry for at least two hours.In this step, loosely fix the first pair of electrodes to the acrylic block of the holder with pieces of adhesive tape. The electrodes should protrude from the acrylic block by approximately 12 millimeters. Next, carefully fix the second bipolar electrode next to the first electrode.For targeting the structures of the hyper direct pathway, the distance between the electrode pairs should be two millimeters. Under the microscope, adjust the second bipolar electrode by sliding it carefully to a position in which the most ventral tip is approximately 200 micrometers recessed compared to the first electrode. Press on the adhesive tape and then secure the electrodes with the holder's metal clamp.After anesthetizing the animal, shave its head to achieve a clean surgical field. Fix the animal in the stereotaxic frame and then disinfect the incision site and the surrounding area with appropriate surgical disinfectant. Subsequently, perform a two centimeter long incision on the scalp in the sagittal direction with a scalpel.Use the scalpel to slightly scrape off the skull aponeurosis and disinfect the skull with 3%hydrogen peroxide. Then use cotton buds soaked in 3%hydrogen peroxide to remove any remaining tissue. Next, adjust the incisor bar until the top of the skull is leveled which means the bregma and lambda as stereotaxic reference points are in the same plane.Under the microscope, use a standard stereotaxic rat alignment tool to calibrate the designated tip to the bregma and adjust the incisor bar until the designated points for the bregma and lambda on the tool touch the skull at the same time. Using a stereotaxic holder with cannula, calibrate it to the bregma and then calculate the position of all drill holes on the skull. Then mark the positions of the holes to be drilled either by carefully scratching the skull or by using a surgical color marker.Then using a micro drill, carefully drill all the holes with a diameter of about one millimeter. For the STN and SNr, drill a common hole approximately two millimeters by three millimeters. Take one fine cannula and bend the tip to form two hooks.Then use the hooks to remove any debris from the drill holes and carefully cut and remove the dura mater in the common STN/SNr hole. After that, flush the drill holes with physiological saline. Apply a drop of physiological saline every 15 minutes to the drill holes to prevent the brain and dura from drying out.Then take a micro drill and matching stainless steel micro screw, drill a hole and screw in a micro screw between the drill holes of the reference epidural electrodes above the cerebellum. Next, guide the electrode tips with fine tweezers and slide them directly below the skull bone into the drill holes. Fix all epidural electrodes with two components stencil acrylic.Make sure not to cover the bregma point nor affect the common STN/SNr hole. Then insert the prepared holder with the tungsten micro wire electrodes into the stereotaxic frame. Calibrate the most ventral electrode to the bregma which is intended to target the STN.Adjust to the calculated position above the common STN/SNr hole and lower the electrodes down to the brain. Make sure that the tungsten micro wire electrodes go inside the brain smoothly. In this procedure, set up the recording software such as band-pass filter and amplify the raw data signal.Then use an online LFP and spike filter with appropriate settings. For all filters, use a Butterworth-type filter. Set up a spike threshold for online spike sorting.Most recording software allows for the setting up of a spike threshold which is an amplitude value above the threshold that a signal is marked as a spike by the software. Next, slowly lower the tungsten micro wire electrodes to one millimeter above the STN and wait for the signal to stabilize if necessary. For the electrophysiological mapping, advance the electrodes ventrally in the steps of 100 micrometers.At each step, evaluate the firing pattern, firing rate and shape of spikes by comparing those with the typical examples given in figure two of the accompanying manuscript. Once the electrodes are in the desired structures, set up online filtering and spike sorting and then start the recording of the data. Here is a representative 600-second LFP recording of the primary motor cortex.Time periods with high frequency low amplitude activity corresponding to the activated state and the time periods with the slower rhythm and higher amplitude corresponding to the slow wave activity state can be differentiated. And this is the corresponding time frequency plot over an interval of 600 seconds illustrating the zero to 20 Hertz relative power of the LFPs. Once mastered, this technique can be done in two hours if it's performed properly.After watching this video, you should have a good understanding of how to perform extracellular in vivo electrophysiological recordings from the cortico-basal ganglia loop of anesthetized rats.

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This video demonstrates the detailed procedure for preparing an anesthetized rat for electrophysiological recordings of brain activity from multiple sites. The scalp is dissected to access the skull, and holes are drilled at precise locations to install and secure electrodes. Tungsten microwire electrodes are then inserted into the targeted location to record the data.

In Vivo Electrophysiological Recordings of Brain Activities from Multiple Sites in Rats

This video demonstrates the procedure for concurrent recordings of co-localized electroencephalography (EEG) and local field potentials (LFPs) in an anesthetized rat. The process involves preparing the rat and attaching stimulating electrodes to its whisker pads. An EEG spider electrode is placed on the rat's skull for surface EEG measurements. At the same time, a multi-channel microelectrode is inserted into a specific brain region through a burr hole in the skull to record neural activity...

Concurrent Electroencephalographic and Local Field Potential Recordings in an Anesthetized Rat

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Here, we present a method for assessing the activation and block of sensory axons in peripheral nerves in acute, anesthetized, in vivo rat experiments using two types of spinal electrophysiological measurements that can discriminate the activity of nerve fiber subtypes: Local Field Potentials and Wide Dynamic Range single neuron recordings.

Electrophysiological Methods to Assess Peripheral Pain Block in an Anesthetized Rat

Learn acute in vivo electrophysiological recordings of local field potentials and multi-unit activity from the hyperdirect pathway in anesthetized rats. This method enables simultaneous cortical and subcortical recordings for neuroscience research.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats (Video) | JoVE Journal

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

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