Name: noninvasive eeg recordings from freely moving piglets
Uploaded: 2018-07-13T15:00:00
Description: Watch this Scientific Journal Video about Noninvasive EEG Recordings from Freely Moving Piglets at JoVE.com

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
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	<li>Prior to the experiment, check for any line noise and find an a...

<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. 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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.

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	<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 ...

This method can help answer key questions in the neuroscience field about cortical development. The main advantage of this technique is that the animals do not need to be sedated. In general individuals new to this method will struggle because the animals are handled when they are awake and the experimenter must work quickly.Before beginning the experiment, find an adequate location for the setup. Use a cable drum to supply the setup with line power, and connect the laptop, receiver unit, and analog to digital converter as necessary for the specific telemetry system being used. Place the electrodes, adhesives, cotton swabs, wipes, mixing blocks, sandpaper and abralate on a separate space, and cut and solder the electrodes to as short a length as possible, according to the size of the animal.The cables must be long enough to connect from the recording electrode positions on the animal's head with the telemetric EEG unit for the data transmission. When all of the materials are ready, catch a piglet at the leg or thorax taking care to be aware of any potential defecation or urination. Mark the piglet with a number as necessary, and wrap the animal in a towel to calm it down.Holding the piglet with one hand at the body or forearm and using the other hand to control the snout, have an assistant clean the animal's skin, using an appropriate tool to remove any dead skin as necessary. Place the recording electrodes at the appropriate experimental locations. The ground electrode above the cerebellum and the reference electrode on the snout.When all of the electrodes have been placed, connect the cables to the telemetry unit and turn it on. Cover the telemetry unit, cables and electrodes with two component skin adhesive silicone rubber, when the silicone rubber is completely cured, place the piglet back into the pen and monitor the animal for several more minutes for any signs of discomfort. Before beginning the experiment check for any line noise, the piglet should recover and begin synchronizing its behavior with its siblings after about 30 seconds.Wait for a sleep phase and begin the recording, gently moving any sleeping siblings from the telemetry unit as necessary. At the end of the recording period, catch the piglet again without aggravating the sow, and gently lift the silicone rubber at one edge, then remove the whole patch of silicone rubber containing the electrodes and the telemetry unit, being careful with the piglet's eyes, and returning the piglet to the pen. Using the telemetry unit as demonstrated, EEG patterns associated with non-REM sleep, like spindle bursts or delta brushes can be captured from freely moving piglets as well as phases of REM-like sleep with very low amplitude.A good quality recording can also be achieved during lactation. The message is also potentially useful for the assessment of animal welfare. After its development this technique paved the way for researchers in the field of developmental neuroscience to explore EEG patterns in awake animals.

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

Research

JoVE Journal

Neuroscience

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 ...

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.

Recording Single Neurons&#039; Action Potentials from Freely Moving Pigeons Across Three Stages of 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 ...

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 ...

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 ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...

Extracellular Electrophysiological Recording in the Motor Cortex of Mice

Multichannel Extracellular Recording in Freely Moving Mice

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

Author Spotlight: Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice 

The protocol describes the methodology of extracellular recording in the motor cortex (MC) to reveal extracellular electrophysiological properties in freely moving conscious mice, as well as the data analysis of local field potentials (LFPs) and spikes, which is useful for evaluating the network neural activity underlying behaviors of interest.

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by ...

Cerebrospinal Fluid and Blood Withdrawal from Rats with Concurrent EEG Recording

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats During EEG Recordings

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Author Spotlight: Obtaining High-Quality CSF and Blood Samples for Epilepsy Biomarker Discovery 

The protocol shows repeated cerebrospinal fluid and blood collections from epileptic rats performed in parallel with continuous video-electroencephalogram (EEG) monitoring. These are instrumental for exploring possible links between changes in various body fluid molecules and seizure activity.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many ...

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...