Name: simultaneous recordings of cortical local field potentials and electrocorticograms in response to nociceptive laser stimuli from freely moving rats
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats at JoVE.co

This work was supported by CAS Key Laboratory of Mental Health, Institute of Psychology,&#160;the National Natural Science Foundation of China (31671141 and 31822025), the 13th Five-year Informatization Plan of the Chinese Academy of Sciences (XXH13506), and the Scientific Foundation project of the Institute of Psychology, Chinese Academy of Sciences (Y6CX021008).

Adult male Sprague-Dawley rats (weighing 400 - 450 g) were used in the experiment. All surgical and experimental procedures followed the Guide for Care and Use of Laboratory Animals of the National Institutes of Health. The procedures were approved by the Research Ethics Committee at the Institute of Psychology, Chinese Academy of Sciences.
1. Electrode Implantation
<ol>
	<li>Anesthetize the rat in a chamber with 5% isoflurane and an air flow rate of 1 L/min before the surgery.</li>
	<li>Usi...

<ol>
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In the present study, we described a technique to concurrently record ECoGs and cortical LFP responses elicited by nociceptive laser stimuli from freely moving rats. The results showed that LEP responses could be clearly detected after the onset of laser stimuli in both ECoG and LFP signals. The simultaneous recording of ECoG and cortical LFP signals will enable scientists to investigate their relationship for better understanding the contribution of neuronal activities to the LEP components.
...

EEG is a technique to record electrical potentials and oscillatory brain activities generated by the synchronized activities of thousands of neurons in the brain. It is popularly used in many basic studies and clinical applications1,2. For instance, EEG responses to intense laser heat pulses (i.e., LEPs) are widely adopted to investigate the peripheral and central processing of nociceptive sensory input3,4,5. In humans, LEPs mainly consist of three distinct deflections: the early component (N1) that is somatotopically organized and likely to reflect the activity of the primary somatosensory cortex (S1)6, and the late components (N2 and P2) that are centrally distributed and more likely to reflect the activity&#160;of the secondary somatosensory cortex, insula, and anterior cingulate cortex7,8. In previous studies9,10, we demonstrated that rat LEPs, sampled using ECoG (a type of intracranial EEG) from electrodes placed directly on the exposed surface of the brain, also consist of three distinct deflections (i.e., somatotopically organized N1 and the centrally distributed N2 and P2). The polarity, order, and topography of the rat LEP components are similar to human LEPs11. However, due to the limited spatial resolution of the scalp EEG and subdural ECoG recordings12, as well as the inaccurate nature of EEG source analysis techniques13, the detailed contribution of the neural activities to the LEP components is much debated. For example, it is unclear if and the extent to which S1 contributes to the early part of the cortical response (N1) elicited by laser stimuli6.
Different from the recording technique at the macroscopic level, direct intracranial recordings using microwire arrays aided by a stereotaxic apparatus and microdrives14,15 could measure neural activities (e.g., LFPs) of specific regions. LFPs mainly reflect the summation of inhibitory or excitatory postsynaptic potentials of local neuronal populations16. Since LFP-sampled neural activities reflect neuronal processes occurring within hundreds of micrometers around the recording electrode, this recording technique is widely used to investigate the information processing in the brain at the mesoscopic level. However, it only focuses on precise local changes of brain activities and cannot answer the question of how signals from multiple regions are integrated (e.g., how LEP components are integrated at multiple brain regions).
It is worth noting that the simultaneous recording of an ECoG and cortical LFPs from freely moving rats could facilitate the investigation of cortical information processing at both macroscopic and mesoscopic levels. In addition, this methodology provides an excellent opportunity to investigate the extent to which the neural activities of the predefined brain regions contribute to the LEPs. Indeed, several previous studies have assessed the coherence between spikes, cortical LFP, and ECoG signals17,18 and demonstrated that the LFP19,20 adjacent to the EEG electrode contributes to the formation of stimulus-related brain responses. However, the existing technique is usually used to record brain responses from anesthetized animals due to the lacking of a protective shell to prevent the electrodes from being damaged by the collision. In other words, the technique that could build the bridge of electrocortical signals at the mesoscopic (cortical LFP) and macroscopic (EEG and ECoG) levels in freely moving rats is still lacking.
To address this issue, we developed a technique that could record an ECoG and cortical LFPs in multiple brain regions simultaneously from freely moving rats. This technique helps establish the direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, thus facilitating the investigation of nociceptive information processing in the brain.

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			<td height="21" style="height:21px;width:200px;">Male Sprague-Drawley rats</td>
			<td style="width:148px;">Vital River</td>
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			<td height="21" style="height:21px;width:200px;">Isoflurane</td>
			<td style="width:148px;">RWD Life Science</td>
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			<td></td>
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			<td height="42" style="height:42px;width:200px;">Small animal isoflurane anaesthetic system</td>
			<td style="width:148px;">RWD Life Science</td>
			<td style="width:73px;"></td>
			<td style="width:259px;">Including the anesthesia gas mask for rats</td>
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			<td height="21" style="height:21px;width:200px;">Stereotaxic apparatus</td>
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			<td height="63" style="height:63px;width:200px;">The apparatus with combined ECoG and LFP electrodes</td>
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			<td style="width:259px;">The apparatus is home-made, which assembles the ECoG and depth wire electrodes to a connector module</td>
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			<td height="108" style="height:108px;width:200px;">3D-printed protective shell</td>
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			<td style="width:259px;">The texture of shell is polylactic, and the shell is home-made and contains three parts: a base, a wall and a cap. The wall is covered by copper tapers to construct as a Faraday cage</td>
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			<td height="42" style="height:42px;width:200px;">Tungsten wires (diameter: 50 mm)</td>
			<td style="width:148px;">California Fine Wires Company</td>
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			<td style="width:259px;">The electrodes for cortical LFP recording</td>
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			<td height="42" style="height:42px;width:200px;">&#160;Stainless steel screws 
			(diameter: 0.6 mm)</td>
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			<td style="width:259px;">The electrodes for ECoG recording</td>
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			<td height="21" style="height:21px;width:200px;">Electric cranial drill</td>
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			<td height="42" style="height:42px;width:200px;">&#160;Drill bit (diameter: 0.5 mm)</td>
			<td style="width:148px;">RWD Life Science</td>
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			<td style="width:259px;">The drill is used for drilling the holes of ECoG screws</td>
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			<td height="42" style="height:42px;width:200px;">&#160;Drill bit (diameter: 0.2 mm)</td>
			<td style="width:148px;">RWD Life Science</td>
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			<td style="width:259px;">The drill is used for drilling the holes of depth wires&#160;</td>
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			<td height="21" style="height:21px;width:200px;">Dental arylic powder</td>
			<td style="width:148px;">SNC dental</td>
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			<td height="33" style="height:33px;width:200px;">Paraffin</td>
			<td style="width:148px;">Fisher Scientific</td>
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			<td rowspan="2" style="width:259px;">The mixture is used for seal the craniotomy to ensure the following movement of micro-wire arrays</td>
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			<td height="30" style="height:30px;width:200px;">Mineral Oil</td>
			<td style="width:148px;">Fisher Scientific</td>
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			<td height="42" style="height:42px;width:200px;">Electrocoagulator&#160;</td>
			<td style="width:148px;">Bovie medical Corporation</td>
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			<td height="21" style="height:21px;width:200px;">RHD2132 Amplifier Boards&#160;</td>
			<td style="width:148px;">Intan Technologies</td>
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			<td style="width:259px;">A 32-channel headstage</td>
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			<td height="21" style="height:21px;width:200px;">RHD2000 systerm</td>
			<td style="width:148px;">Intan Technologies</td>
			<td style="width:73px;"></td>
			<td style="width:259px;">The data acquisition systerm</td>
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			<td height="70" style="height:70px;width:200px;">Infrared neodymium yttrium aluminum perovskite (Nd:YAP) laser generator</td>
			<td style="width:148px;">Electronical Engineering</td>
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			<td height="21" style="height:21px;width:200px;">Matlab R2016b</td>
			<td style="width:148px;">The MathWorks&#160;</td>
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simultaneous recordings of cortical local field potentials and electrocorticograms in response to nociceptive laser stimuli from freely moving rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

In the representative experiment, the electrophysiological data from five rats were recorded. The laser stimuli were delivered to the right forepaw of each rat for 20 times with &#62;40 s interstimulus intervals. The laser-evoked brain responses were recorded using both ECoG screws and depth wires, and the depth wires were implanted in bilateral primary somatosensory cortices (S1) and primary motor cortices (M1).
As summarized i...

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

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.

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...

This method helps establish the direct relationship of electrocortical signals recorded from both ECoG and the depths of wires to answer key questions about the neuronal contributions of laser-evoke potentials. The main advantage of this technique is that electrode implantation can be protected by polylactic shell, thus the recording can be applied to our free moving rats. This method can be applied in recording brain responses evoked by a stimuli of different sensations or brief features of psychiatric diseases which promote the investigation of their respective neuromagnetisms.Begin this procedure with anesthetization of the rat as detailed in the text protocol. Then, use a stereotaxic apparatus to fix the head of the rat with it's nose placed into the anesthetic mask. Surgical tolerance is achieved when then rat fails to respond to toe pinching.Apply ophthalmic ointment to the eyes to avoid corneal drying. Shave the top of the rat's scalp using a standard shaver. Then, sterilize the scalp using the medical iodophor disinfectant solution and 75%alcohol to remove the iodine.Inject 2%lidocaine into the scalp for local analgesia. Then, administer atropine to inhibit respiratory hypersecreation. Make a midline incision of approximately two to three centimeters on the scalp using a scalpel.Cut and remove part of the scalp along the midline and expose the cranium. Mark the locations of the ECoG electrodes based on the predefined stereotaxic coordinates. Also, mark the locations of the reference and ground electrodes on the midline.Drill holes for the ECoG screws using an electric cranial drill on the skull at the marked sights without destroying the dura. Drive a stainless screw, which connects to the insulation coated copper wire, into the hole for an approximate one millimeter depth. Avoid penetrating the underlying dura.Place a protective shell base on the cranium. Fix the base with it's adjacent screws on the cranium using dental acrylic. Mark the locations of the depth wire electrodes based on the predefined stereotaxic coordinates.Now, drill small holes on the skull around the mark sights for wire implantation and carefully remove the bone flap to expose the dura. Wash the craniotomy frequently with normal saline. Using a needle, lift and cut the dura without damaging the pia mater, vessels and the surface of the neocortex.Lower the depth wire electrodes to the surface of the neocortex. Then, slowly penetrate the brain to the target depth. Seal the craniotomy with a mixture of wax and paraffin oil to ensure that the depth wire electrodes can be moved for subsequent experimental manipulations.Fix the electrode apparatus using dental acrylic on the skull. Weld each copper wire that connects to the ECoG screw to the corresponding channel on the connector module. Assemble the protective shell wall to the base and weld the reference and ground electrodes to the corresponding channels.Fix the cap to the protective shell using tapes to avoid contamination. After injecting the rat with penicillin, single house the rat in a temperature and humidity controlled cage after the surgery as detailed in the text protocol. Place the rat in the behavior chamber for at least one hour before the experiment to ensure that the rat acclimatizes to the recording environment.Gently connect the recording head stage with the electrode module to avoid scaring the rat and damaging the electrode module. Set up the laser generator. Connect the optic fiber and adjust the spot size of the laser according to the equipment operators manual.Now, connect the digital output from the trigger generator to the digital input port of the recording board. Set the video camera beneath the corner of the experimental chamber to continuously record the nociceptive behaviors of the rat when it's paw receives nociceptive laser stimulation. Deliver ongoing white noise via a loud speaker at the top of the chamber.Collect the electrophysiological data from both the ECoG and the depth wire electrodes using the recording system according to the equipment operators manual. Now, deliver the laser pulses to the plantar of the rat's forepaw through the gaps in the bottom of the chamber by first positioning the laser generator. Then, press the switch to deliver laser stimulation.Shown here is the raw electrophysiological data from bilateral primary motor cortices, bilateral primary somatosensory cortices and ECoG. A clear laser-evoked potential, or LEP response, is detectable after the onset of the laser stimulus. Shown here is the group level averaged LEP wave forms from six electrodes of five rats.The LEP responses consist of a dominant negative deflection called N1 Wave. Note the the N1 Wave of bilateral primary somatosensory cortices shows a shorter latency and higher amplitude than that of bilateral primary motor cortices. This figure shows wavelet transformed coherence between LEPs sampled from the ECoG and depth wires in bilateral S1 and M1, with the laser delivered on the plantar of the rat's right forepaw.Note that the left S1 and M1 showed a higher coherence than the right S1 and M1 at the gamma frequency band. After it's development this technique will pave the way for researchers to concurrently recall both ECoG and intercortical local fill potentials in free moving rats to expose the neural contributions of laser-evoked potentials.

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