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).
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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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Research

JoVE Journal

Behavior

Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior

We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 &deg;C and 60 &deg;C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.

Use of the Operant Orofacial Pain Assessment Device OPAD to Measure Changes in Nociceptive Behavior

We present a user-friendly, high-throughput operant system for the evaluation of pain behaviors in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) can assess pain through a reward/conflict paradigm thus providing a more humane way of testing. This protocol will yield more clinically relevant and translational data from rodents.

We present an operant  system for the detection of pain in awake, conscious rodents. The Orofacial  Pain Assessment Device (OPAD) assesses pain behaviors ...

Irrelevant Stimuli and Action Control: Analyzing the Influence of Ignored Stimuli via the Distractor-Response Binding Paradigm

Selection tasks in which simple stimuli (e.g. letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are frequently used in the research on human action control. One important question in these settings is how distractor stimuli, competing with the target stimulus for a response, influence actions. The distractor-response binding paradigm can be used to investigate this influence. It is particular useful to separately analyze response retrieval and distractor inhibition effects. Computer-based experiments are used to collect the data (reaction times and error rates). In a number of sequentially presented pairs of stimulus arrays (prime-probe design), participants respond to targets while ignoring distractor stimuli. Importantly, the factors response relation in the arrays of each pair (repetition vs. change) and distractor relation (repetition vs. change) are varied orthogonally. The repetition of the same distractor then has a different effect depending on response relation (repetition vs. change) between arrays. This result pattern can be explained by response retrieval due to distractor repetition. In addition, distractor inhibition effects are indicated by a general advantage due to distractor repetition. The described paradigm has proven useful to determine relevant parameters for response retrieval effects on human action.

The distractor-response binding paradigm is described. It can be used to shed light on the influence irrelevant stimuli, competing with targets for a response, can have on human action. Both response retrieval effects and distractor inhibition effects can be analyzed within the paradigm.

Selection tasks in which simple stimuli (e.g. letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are ...

Cortical Source Analysis of High-Density EEG Recordings in Children

EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and signal processing make it possible to exploit information from other imaging modalities like structural MRI that provide high spatial resolution to overcome this constraint1. This is especially useful for investigations that require high resolution in the temporal as well as spatial domain. In addition, due to the easy application and low cost of EEG recordings, EEG is often the method of choice when working with populations, such as young children, that do not tolerate functional MRI scans well. However, in order to investigate which neural substrates are involved, anatomical information from structural MRI is still needed. Most EEG analysis packages work with standard head models that are based on adult anatomy. The accuracy of these models when used for children is limited2, because the composition and spatial configuration of head tissues changes dramatically over development3.&#160;
In the present paper, we provide an overview of our recent work in utilizing head models based on individual structural MRI scans or age specific head models to reconstruct the cortical generators of high density EEG. This article describes how&#160;EEG recordings are acquired, processed, and analyzed with pediatric populations at the London Baby Lab, including laboratory setup, task design, EEG preprocessing, MRI processing, and EEG channel level and source analysis.&#160;

In recent years, there has been increasing interest in estimating the cortical sources of scalp measured electrical activity for cognitive neuroscience experiments. This article describes how high density EEG is acquired and how recordings are processed for cortical source estimation in children from the age of 2 years at the London Baby Lab.

EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and ...

Training Rats to Voluntarily Dive Underwater: Investigations of the Mammalian Diving Response

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a parasympathetically-induced bradycardia and a sympathetically-induced alteration of vascular resistance that maintains blood flow to the heart, brain and exercising muscles. While many of the metabolic and cardiorespiratory aspects of the diving response have been studied in marine animals, investigations of the central integrative aspects of this brainstem reflex have been relatively lacking. Because the physiology and neuroanatomy of the rat are well characterized, the rat can be used to help ascertain the central pathways of the mammalian diving response. Detailed instructions are provided on how to train rats to swim and voluntarily dive underwater through a 5 m long Plexiglas maze. Considerations regarding tank design and procedure room requirements are also given. The behavioral training is conducted in such a way as to reduce the stressfulness that could otherwise be associated with forced underwater submergence, thus minimizing activation of central stress pathways. The training procedures are not technically difficult, but they can be time-consuming. Since behavioral training of animals can only provide a model to be used with other experimental techniques, examples of how voluntarily diving rats have been used in conjunction with other physiological and neuroanatomical research techniques, and how the basic training procedures may need to be modified to accommodate these techniques, are also provided. These experiments show that voluntarily diving rats exhibit the same cardiorespiratory changes typically seen in other diving animals. The ease with which rats can be trained to voluntarily dive underwater, and the already available data from rats collected in other neurophysiological studies, makes voluntarily diving rats a good behavioral model to be used in studies investigating the central aspects of the mammalian diving response.

Detailed instructions are provided on how to train rats to voluntarily dive underwater through a 5 m long Plexiglas maze. Because the brains of rats have been very well characterized, voluntarily diving rats may help elucidate the central pathways of the mammalian diving response.

Underwater submergence produces autonomic changes that are observed in virtually all diving animals. This reflexly-induced response consists of apnea, a ...

Place and Response Learning in the Open-field Tower Maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

Event-related Potentials During Target-response Tasks to Study Cognitive Processes of Upper Limb Use in Children with Unilateral Cerebral Palsy

Unilateral Cerebral Palsy (CP) is a neurodevelopmental disorder that is a very common cause of disability in childhood. It is characterized by unilateral motor impairments that are frequently dominated in the upper limb. In addition to a reduced movement capacity of the affected upper limb, several children with unilateral CP show a reduced awareness of the remaining movement capacity of that limb. This phenomenon of disregarding the preserved capacity of the affected upper limb is regularly referred to as Developmental Disregard (DD). Different theories have been postulated to explain DD, each suggesting slightly different guidelines for therapy. Still, cognitive processes that might additionally contribute to DD in children with unilateral CP have never been directly studied. The current protocol was developed to study cognitive aspects involved in upper limb control in children with unilateral CP with and without DD. This was done by recording event-related potentials (ERPs) extracted from the ongoing EEG during target-response tasks asking for a hand-movement response. ERPs consist of several components, each of them associated with a well-defined cognitive process (e.g., the N1 with early attention processes, the N2 with cognitive control and the P3 with cognitive load and mental effort). Due to its excellent temporal resolution, the ERP technique enables to study several covert cognitive processes preceding overt motor responses and thus allows insight into the cognitive processes that might contribute to the phenomenon of DD. Using this protocol adds a new level of explanation to existing behavioral studies and opens new avenues to the broader implementation of research on cognitive aspects of developmental movement restrictions in children.

Several children with unilateral Cerebral Palsy seem to disregard the preserved capacity of their affected upper limb. This Developmental Disregard is extensively described in the literature but the involved cognitive processes have not been studied. To study underlying cognitive factors of upper limb control, an event-related potential protocol was developed.

Unilateral Cerebral Palsy (CP) is a neurodevelopmental disorder that is a very common cause of disability in childhood. It is characterized by unilateral ...

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

A series of basic methods to enable the study of the reproductive ecology of fish kept in aquaria are described. These are useful protocols for collecting fish using SCUBA, transporting live fish, and observing the reproductive behavior of wild-caught fish kept in aquaria.

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...