We would like to thank Helmut Scheu for the opportunity to conduct our research in the pigpen at Hofgut Neum&#252;hle.

All procedures were approved by the local ethics committee (#23177-07/G10-1-010/G 15-15-011) and followed the European and the German national regulations (European Communities Council Directive, 86/609/ECC; Tierschutzgesetz).
All animal procedures were performed in accordance with the Medical Center of the Johannes Gutenberg-University Mainz animal care committee&#39;s regulations.
1. Setup
<ol>
	<li>Prior to the experiment, check for any line noise and find an adequate location for the set-up and the antenna. Line noise is visible as a 60 or 50 Hz sine wave. 
	NOTE: The placement of the antenna and especially the distance between the transmitter and the receiver depends on the transmission strength of the system. The system used here is adjustable. It was adjusted to a relatively low power, with an approximately 3 m transmission range. Additionally, the metallic fences in the pigpen can dampen the signal and cause interferences. In this case, it is necessary to place the antenna inside the metal cage.</li>
	<li>Use a cable drum to supply the set-up with line power. Connect the laptop, the receiver unit, and the analog-to-digital converter (if necessary) for the specific telemetry system that is being used. 
	NOTE: The telemetry system used here sent digital data to the receiver. This might be different for other systems.</li>
	<li>Place the electrodes, the adhesives, the Q-tips, and the wipes, as well as the mixing blocks, on a separate table.</li>
	<li>Prepare the electrodes with short cables. To do so, cut the electrodes and solder them again with a length as short as possible, depending on the size of the animal. The cables must be long enough to connect the desired recording positions on the head with the telemetric EEG unit, transmitting the data. Cables that are too long must be recoiled and covered with skin adhesive silicone elastomer. Longer cables that must be recoiled make the silicone patch bigger and heavier.</li>
</ol>
2. Piglet
<ol>
	<li>Catch a piglet by grabbing it at the leg or at the thorax. Hold it and be aware of any defecation or urination.</li>
	<li>If necessary, mark the piglet with a number.</li>
	<li>Wrap the piglet in a towel. The piglet will calm down. Be aware of overheating the piglet.</li>
	<li>Hold the piglet with one hand at the body or forearm. Use the other hand to hold the snout. Be aware of overheating the piglet and make sure it is free to breathe properly.</li>
</ol>
3. Electrodes
<ol>
	<li>Have a second person attach the electrodes.</li>
	<li>Clean the skin from dirt with water or ethanol. If necessary, shave the head.</li>
	<li>Remove any dead skin cells with an abrasive EEG gel and a Q-tip. Remove the abrasive gel afterward. Alternatively, use sandpaper.</li>
	<li>Fix the self-adhesive electrodes at the desired location. Place the ground electrode above the cerebellum (between the ears) and the reference electrode on the nose. Place the recording electrode at the desired location. 
	NOTE: In this case, a unipolar recording was performed, because the reference was placed at a neutral position (nose). There is no standardized system available for piglets until now. Here, a parietal recording position was used (between the eye and the ear) on the right brain hemisphere.</li>
	<li>Connect the cables to the telemetry unit. Turn the unit on. Depending on the telemetry system used, this might be a magnetic switch or a radio frequency wake-up signal.</li>
	<li>Cover the telemetry unit and all cables as well as all electrodes with two-component skin adhesive silicone rubber (see Table of Materials). By mixing equal amounts of both components, the curing time will be in the range of 1 min. Eyes and eyelashes should not be covered with the rubber.</li>
	<li>Wait until the silicone rubber is completely cured.</li>
	<li>Place the piglet back in the pigpen.</li>
	<li>Observe the piglet to see if it is showing signs of discomfort over a longer period of time (several minutes).</li>
</ol>
4. Measurement
<ol>
	<li>Wait until the piglet has recovered and starts to synchronize its behavior with that of its siblings (feeding, playing, sleeping), usually after 30 s (Figure 1).</li>
	<li>Wait for sleep phases, if desired. The recording time depends on the specific scientific question. Here, 10 min recording sessions were used.</li>
	<li>If the telemetry unit is covered by more than 2 other piglets, the signal might be too low for the receiver. Gently push the piglets away if they are sleeping on top. Be aware of the sow; it might react aggressively.</li>
	<li>Start the recording with the data acquisition software (see Table of Materials).</li>
</ol>
5. Finish
<ol>
	<li>After the recording (usually several hours), catch the piglet again as has been described in step 2. Be aware of the sow; it might react aggressively.</li>
	<li>Gently lift the silicone rubber at one edge. Then, remove the whole patch of silicone rubber containing the electrodes and the telemetry unit. Be careful with the piglet&#39;s eyes.</li>
	<li>Place the piglet back in the pigpen.</li>
</ol>

<ol>
	<li>Conrad, M. S., Sutton, B. P., Dilger, R. N., Johnson, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+three-dimensional+magnetic+resonance+imaging-based+averaged+brain+collection+of+the+neonatal+piglet+(Sus+scrofa).">An in vivo three-dimensional magnetic resonance imaging-based averaged brain collection of the neonatal piglet (Sus scrofa).</a> PLoS ONE. 9 (9), e107650 (2014).</li><li>de Camp, N. V., Hense, F., Lecher, B., Scheu, H., Bergeler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+preterm+cortical+development+using+non+invasive+clinical+EEG.">Models for preterm cortical development using non invasive clinical EEG.</a> Translational Neuroscience. 8, 211-224 (2017).</li><li>Lapray, D., Bergeler, J., Dupont, E., Thews, O., Luhmann, H. J., Barculo, D., Daniels, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+telemetric+system+for+recording+brain+activity+in+small+animals.">A novel telemetric system for recording brain activity in small animals.</a> Telemetry: Research, Technology and Applications. , 195-203 (2009).</li><li>Kim, D., Yeon, C., Kim, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+experimental+validation+of+a+dry+non-+invasive+multi-channel+mouse+scalp+EEG+sensor+through+visual+evoked+potential+recordings.">Development and experimental validation of a dry non- invasive multi-channel mouse scalp EEG sensor through visual evoked potential recordings.</a> Sensors. 17, 326 (2017).</li><li>Moshayedi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+glial+cell+mechanosensitivity+and+foreign+body+reactions+in+the+central+nervous+system.">The relationship between glial cell mechanosensitivity and foreign body reactions in the central nervous system.</a> Biomaterials. 35, 3919-3925 (2014).</li><li>Barrese, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+mode+analysis+of+silicon-based+intracortical+microelectrode+arrays+in+non-human+primates.">Failure mode analysis of silicon-based intracortical microelectrode arrays in non-human primates.</a> Journal of Neural Engineering. 10, 066014 (2013).</li><li>Hellstr&#246;m-Westas, L., Ros&#233;n, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroencephalography+and+brain+damage+in+preterm+infants.">Electroencephalography and brain damage in preterm infants.</a> Early Human Development. 81, 255-261 (2005).</li><li>Lloyd, R. O., Goulding, R. M., Filan, P. M., Boylan, G. 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The authors have nothing to disclose.

A critical step in the protocol is the adequate skin contact with the electrodes, especially the ground electrode, to achieve stable recordings with low noise. Furthermore, since piglets are very agile, it is important to cover the whole system with silicone rubber to protect the electrodes and the telemetry unit. Furthermore, if the experiments are conducted in a stable with a slatted floor, be cautious with small devices or connectors.
In case of an inadequate grip of the self-adhesive hydro...

Piglets are an emerging model system for neuroscience1. In order to strengthen translational research, we invented a method to record non-invasive, clinical EEGs from unrestrained piglets2 (Figure 1 and Figure 2). Two prerequisites for a translational use of EEG recordings, regarding EEG patterns associated with cortical maturation, are a non-invasive methodology, comparable to the clinical setting, and the abstinence of sedatives or anesthesia. The one-channel telemetry system3 in combination with self-adhesive electrodes can be fixed in about 5 min. Afterward, the piglets will recover quickly from the handling procedure and synchronize their feeding and sleeping behavior to that of the other piglets and the sow.
Even though there are already attempts to use non-invasive EEG recordings from sedated animals4, most electroencephalography studies from animals are conducted with invasive approaches. These methods have side effects regarding inflammatory processes around the implanted electrodes5,6 and, in most cases, they require a social separation of the animals due to the external components of the implanted EEG system. Hence, the translation of these data to the clinical context is difficult. The need for translational approaches is becoming clear by the fact that it is still not known how a &#34;normal&#34; brain maturation during the early cortical development is represented by clinical, non-invasive electroencephalography7. This knowledge gap is caused by technical challenges associated with EEG recordings from preterm babies8. In animal model systems, patterns of early cortical development are better accessible, since most animals are born with a &#34;preterm brain&#34; in comparison to human cortical development9. Besides conserved patterns of cortical development across species2, it has recently been shown that EEG recordings from preterm babies can also predict the individual clinical outcome during later life10,11. The method described here is especially useful for the translational aspects of developmental neuroscience.

<table border="0" cellpadding="0" cellspacing="0" width="867">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Disposable adhesive 
			surface silver/silver chloride electrodes</td>
			<td style="width:309px;">Spes 
			Medica S.r.l., Genova, Italy</td>
			<td style="width:119px;"></td>
			<td style="width:223px;">Self adhesive hydrogel electrode</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Abralyt HiCl</td>
			<td style="width:309px;">Easycap GmbH</td>
			<td style="width:119px;"></td>
			<td>Abrasive cream</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Body Double fast</td>
			<td style="width:309px;">Smooth On Inc.</td>
			<td style="width:119px;"></td>
			<td>Skin adhesive silicone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Telemetry system</td>
			<td style="width:309px;"></td>
			<td style="width:119px;"></td>
			<td>Internal development</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Picolog 1216</td>
			<td style="width:309px;">Pico Technology</td>
			<td style="width:119px;"></td>
			<td>AD converter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laptop</td>
			<td style="width:309px;">Panasonic</td>
			<td style="width:119px;"></td>
			<td>Rugged laptop</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Receiver</td>
			<td style="width:309px;"></td>
			<td style="width:119px;"></td>
			<td>Internal development</td>
		</tr>
	</tbody>
</table>

noninvasive eeg recordings from freely moving piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

We were able to record typical EEG patterns associated with non-REM sleep, like spindle bursts or delta brushes, from freely moving piglets (Figure 1 and Figure 2). We were mostly interested in representative patterns during non-REM sleep, but phases of REM-like sleep12 with a very low amplitude have also been recorded (Figure 3). The physiology and the amount of REM sleep dif...

Watch this Scientific Journal Video about Noninvasive EEG Recordings from Freely Moving Piglets at JoVE.com

Noninvasive EEG Recordings from Freely Moving Piglets

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

noninvasive-eeg-recordings-from-freely-moving-piglets

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7.2K Views.  Free University Berlin. Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

Video: Noninvasive EEG Recordings from Freely Moving Piglets

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

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

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

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.

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...