This work was supported by Kaken-hi (17H05939; 17H05551), the Nakatomi Foundation, and the Suzuken Memorial Foundation.

All of the procedures involving animal subjects were performed according to the NIH guidelines for the care and use of animals.
1. Preparation of the Integrative Micro-Drive Array
<ol>
	<li>Create a micro-drive array for cortical LFP recordings as described elsewhere1,2,3. Leave at least 6 metal holes open on an electrode interface board (EIB) for use as ECG/EMG/BR channels that are connected to bioflex wires as described in 1.2.</li>
	<li>Cut a bioflex wire into 6 pieces with lengths of 5.0 cm. Peel off the polytetrafluoroethylene (PTFE) coating of both ends of all the wire pieces with lengths of ~5.0 mm. Connect one end of each of the wire pieces to one of the open metallic holes (ECG/EMG/BR channels) on the EIB with a gold pin.</li>
	<li>Cut an enamel wire into two 5.0-cm pieces. Solder one end of each of these wires to the ground/reference (g/r) channels on the EIB (Figure 3, see also previous papers12,13).</li>
	<li>For the preparation of ECG electrodes, cut a bioflex wire into two 16-cm pieces. Peel off the PTFE coating of the ends of these wire pieces at lengths of ~5.0 mm on one end (short end) and ~15 mm on the other end (long end).</li>
	<li>Form a wire ring with a diameter of 2.0 mm by bending the long end of the wire, and fixing the shape of the ring by soldering.</li>
	<li>For the preparation of EMG electrodes, cut a bioflex wire into 2 pieces with lengths of 8 cm. Peel off the PTFE coating from both ends of these wire pieces with lengths of ~5.0 mm.</li>
	<li>For the preparation of BR electrodes, cut a bioflex wire into 2 pieces with lengths of 6.0 cm. Peel off the enamel coating of both ends of these wire pieces with lengths of ~5.0 mm. Solder one end of each of these wire pieces to the head of a stainless- steel screw (stem diameter: 1.0 mm, stem length: 4.0 mm).</li>
	<li>For the preparation of ground/reference (gr) electrodes, cut an enamel wire into 2 pieces with lengths of 6.0 cm. Peel off the enamel coating of both ends of these wire pieces with lengths of ~5.0 mm. Solder one end of each of these wire pieces to the head of a stainless- steel screw (stem diameter: 1.4 mm, stem length: 3.0 mm).</li>
	<li>Gas sterilize all electrodes and stainless screws&#160;and keep these in a clean space.</li>
</ol>
2. Implantation of the ECG/EMG Electrodes
NOTE: Perform all surgical steps with aseptic technique using sterilized gloves and autoclaved instruments. For all steps involving the creation of an incision, sterilize the skin with 70% ethanol before, and cover the incision with surgical drapes.
<ol>
	<li>Fix an anesthetized (1.0-3.0% isoflurane gas) rat on its back on a flat heat pad. Give buprenorphine as an analgesic. Place veterinary ointment on the rat&#39;s eyes to prevent dryness.&#160;Use betadine to clean the surface of the skin.&#160;</li>
	<li>Make an incision of ~2.0 cm in the medial chest area. Expose the intercostal muscles by separating the chest muscles. Suture the rings of the ECG electrodes to the intercostal muscles.</li>
	<li>Fix the stomach of the animal on the heat pad. Make an incision of ~1.0 cm in the dorsal neck area.</li>
	<li>Insert the ECG electrodes subcutaneously through the chest incision. Slide the ends to the dorsal neck area, and pull them out from the neck incision. Suture the chest incision.</li>
	<li>Insert one end of each of the EMG electrodes subcutaneously to a length of ~2.0 cm through the neck incision. Fix the EMG electrodes to the neck muscle by suturing.</li>
</ol>
3. Implantation of the Integrative Micro-Drive Array and the BR Electrodes
<ol>
	<li>Fix the rat on a stereotaxic device. Make an incision of ~3.0 cm on the head along the midline from the point between the eyes to the neck area. Expose the skull.</li>
	<li>Make two circular craniotomies with diameters of 0.7-1.0 mm above the olfactory bulb 11.0 mm anterior and 1 mm bilateral to bregma with a high-speed drill. Implant two BR electrodes in the skull until the tips of the screw stems are attached to the brain surface.</li>
	<li>Make two circular craniotomies with diameters of 0.7-1.0 mm above the frontal cortex 2.7 mm anterior and 2.7 mm bilateral to bregma. Implant two g/r electrodes in the skull until the tip of the screw stem is attached to the brain surface.</li>
	<li>Make six to eight holes with diameters of 1.0 mm in the area surrounding the large craniotomy. Implant anchor screws (stem diameter: 1.4 mm, stem length: 3.0 mm) in the skull.</li>
	<li>Make a large circular craniotomy with a diameter of ~2.0 mm above the hippocampus 3.8 mm posterior and 2.5 mm bilateral to bregma. Place the integrative micro-drive array such that the cannula tip of the drive array is located above the large craniotomy</li>
	<li>Fill the gap space between the cannula tip and the brain surface with ~100 &#181;L of two solutions, i.e., 0.5% (by mass) sodium alginate and 10% (by mass) calcium chloride. 
	NOTE: This process forms a transparent gel in ~5 min, after the two solutions are mixed on the skull.</li>
	<li>Cover the cannula, BR electrodes, g/r electrodes, and anchor screws with dental cement with a thickness of 0.5 cm. Be careful NOT to cover the open ends of BR and g/r electrodes with the cement at this step.</li>
	<li>Solder the open ends of the ECG, EMG, BR and g/r electrodes to the individual wire tips that were previously connected to the EIB (see the steps 1.2 and 1.3).</li>
	<li>Cover the bottom part of the integrative micro-drive array, and all electrode wires, with dental cement. Ensure that all the electrode wires are completely covered so that the rat cannot scratch them out after the implantation.&#160;</li>
	<li>After regaining sufficient consciousness to maintain sternal recumbency, return the animal to its transparent Plexiglas home cag, and keep it on its own with free access to water and food.&#160;After the surgery, treat the animal with antibiotics (gentamicin).</li>
	<li>After the surgery, monitor the animals with&#160;daily observation. Check that they walk properly, and that they do not squeak when the experimenter touches the micro-drive array.&#160; &#160;</li>
</ol>
4. In Vivo Recordings
NOTE: All signals are amplified, sampled at 2 kHz, and band-pass filtered (0.1 - 500 Hz) except for unit activities (sampled at 30 kHz and band-pass filtered (500 - 6 kHz)).
<ol>
	<li>Connect the EIB of the integrative micro-drive array to the headstage of a recording device.</li>
	<li>Advance the tetrodes by turning the screws for a few weeks after surgery. Once the tetrodes are adjacent to the target brain areas, settle the tetrodes into the areas over a period of several days for stable recordings.</li>
	<li>Monitor the electrical signals while the animal freely moves in a recording chamber.</li>
</ol>

<ol>
	<li>Kloosterman, F., 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=Micro-drive+Array+for+Chronic+in+vivo+Recording:+Drive+Fabrication.">Micro-drive Array for Chronic in vivo Recording: Drive Fabrication.</a> JoVE. (26), e1094 (2009).</li><li>Nguyen, D. 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=Micro-drive+Array+for+Chronic+in+vivo+Recording:+Tetrode+Assembly.">Micro-drive Array for Chronic in vivo Recording: Tetrode Assembly.</a> Journal of Visualized Experiments : JoVE. (26), e1098 (2009).</li><li>Jog, M. S., 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=Tetrode+technology:+advances+in+implantable+hardware,+neuroimaging,+and+data+analysis+techniques.">Tetrode technology: advances in implantable hardware, neuroimaging, and data analysis techniques.</a> J Neurosci Methods. 117 (2), 141-152 (2002).</li><li>Fenske, S., 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=Comprehensive+multilevel+in+vivo+and+in+vitro+analysis+of+heart+rate+fluctuations+in+mice+by+ECG+telemetry+and+electrophysiology.">Comprehensive multilevel in vivo and in vitro analysis of heart rate fluctuations in mice by ECG telemetry and electrophysiology.</a> Nat Protoc. 11 (1), 61-86 (2016).</li><li>Rossi, S., 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+effect+of+aging+on+the+specialized+conducting+system:+a+telemetry+ECG+study+in+rats+over+a+6+month+period.">The effect of aging on the specialized conducting system: a telemetry ECG study in rats over a 6 month period.</a> PLoS One. 9 (11), 112697 (2014).</li><li>Cesarovic, N., Jirkof, P., Rettich, A., Arras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+radiotelemetry+transmitters+yielding+data+on+ECG,+heart+rate,+core+body+temperature+and+activity+in+free-moving+laboratory+mice.">Implantation of radiotelemetry transmitters yielding data on ECG, heart rate, core body temperature and activity in free-moving laboratory mice.</a> JoVE. (57), (2011).</li><li>Zeredo, J. L., Kumei, Y., Shibazaki, T., Yoshida, N., Toda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+biting+behavior+induced+by+acute+stress+in+the+rat.">Measuring biting behavior induced by acute stress in the rat.</a> Behav Res Methods. 41 (3), 761-764 (2009).</li><li>Young, G. A., Khazan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromyographic+power+spectral+changes+associated+with+the+sleep-awake+cycle+and+with+diazepam+treatment+in+the+rat.">Electromyographic power spectral changes associated with the sleep-awake cycle and with diazepam treatment in the rat.</a> Pharmacol Biochem Be. 19 (4), 715-718 (1983).</li><li>Oishi, Y., 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=Polygraphic+Recording+Procedure+for+Measuring+Sleep+in+Mice.">Polygraphic Recording Procedure for Measuring Sleep in Mice.</a> JoVE. (107), e53678 (2016).</li><li>Chaput, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory-phase-related+coding+of+olfactory+information+in+the+olfactory+bulb+of+awake+freely-breathing+rabbits.">Respiratory-phase-related coding of olfactory information in the olfactory bulb of awake freely-breathing rabbits.</a> Physiol Behav. 36 (2), 319-324 (1986).</li><li>Ravel, N., Pager, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+patterning+of+the+rat+olfactory+bulb+unit+activity:+Nasal+versus+tracheal+breathing.">Respiratory patterning of the rat olfactory bulb unit activity: Nasal versus tracheal breathing.</a> Neurosci Lett. 115 (2-3), 213-218 (1990).</li><li>Okada, S., Igata, H., Sakaguchi, T., Sasaki, T., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+device+for+the+simultaneous+recording+of+cerebral,+cardiac,+and+muscular+electrical+activity+in+freely+moving+rodents.">A new device for the simultaneous recording of cerebral, cardiac, and muscular electrical activity in freely moving rodents.</a> J Pharmacol Sci. 132 (1), 105-108 (2016).</li><li>Sasaki, T., Nishimura, Y., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Recordings+of+Central+and+Peripheral+Bioelectrical+Signals+in+a+Freely+Moving+Rodent.">Simultaneous Recordings of Central and Peripheral Bioelectrical Signals in a Freely Moving Rodent.</a> Biol Pharm Bull. 40 (5), 711-715 (2017).</li></ol>

The authors have nothing to disclose.

For understanding how the brain modulates peripheral activity levels, and vice versa, large-scale recording methods to simultaneously capture electrical biosignals from multiple body areas are necessary. This study described a surgical procedure, and a recording system for monitoring cerebral local field potentials, heart rates, the magnitude of muscle construction and respiratory rates, which have been improved on a recording system that is used for extracellular recordings in brain tissue. This system collects electric...

The central nervous system controls body states in response to various environmental changes, and this control is typically represented as changes in heart rate, breathing rate, and muscle contractions. However, few studies have tested how such peripheral physiological factors are associated with cortical activity. To address this issue, a large-scale recording method for monitoring electrical biosignals from both central and peripheral tissues is necessary. In the cerebral cortex, local field potential (LFP) signals are extracellularly recorded by electrodes that are inserted into the cortical tissues1,2,3. To simultaneously record multiple LFP signals from the cortical regions of small mammals, such as rats and mice, a number of studies have developed various types of custom-made electrode assemblies that are termed micro-drives. A conventional micro-drive is composed of metal screws attached to the middle parts of the electrodes (which are typically tetrodes), a core body that accommodates the screws and electrodes, and an electrical interface board (EIB) that accommodates metal holes to connect the open ends of the electrodes (Figure 1, Figure 2, and Figure 3). This electrode assembly enables the operator to control the depth of many electrodes inserted into the brain over the course of days to weeks, and allows the conducting of long-term chronic recordings of neuronal activity as the animal is challenged with various behavioral tasks. In the peripheral organs, heartbeat signals are recorded as electrocardiograms (ECGs) by a pair of electrodes that are implanted on or around the heart area4,5,6, and skeletal muscle signals are recorded as electromyograms (EMGs) with electrodes that are inserted into the muscle tissue7,8,9. The relationship between electrical signals of the olfactory bulb and breathing (BR) rhythm has been studied with single unit recordings10,11. In conventional recording systems, these signals from different tissues have been captured by independent recording devices, which means that an additional experimental system is required to precisely synchronize these multiple devices for simultaneous recordings of brain-body signals. This system was developed to overcome this issue. In this system, all electrical signals recorded from the peripheral organs, including ECGs, EMGs, and electrical signals from the olfactory bulb that reflect the breathing rhythm, are integrated into a single micro-drive array1,2,3, here termed an integrative micro-drive array. This system requires only one multi-channel recording device, and is applicable to any conventional micro-drive array. The advantages of this technique are that it does not require any special devices or trigger signals to match the recording time of multiple devices, and it allows for more convenient data processing, since all of the signals are recorded as similar data types. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs. This paper describes the procedures associated with the technique and presents representative datasets obtained from a rat.

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			<td height="40" style="height:40px;width:307px;">FEP Hookup Wire Stranded Stainless Steel&#160;</td>
			<td style="width:324px;">Cooner Wire Company, Chatsworth, CA</td>
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			<td height="40" style="height:40px;">EIB-36-PTB</td>
			<td>Neuralynx, Inc., Bozeman, MT</td>
			<td>EIB-36-PTB</td>
			<td>EIB</td>
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			<td height="40" style="height:40px;">Cereplex&#160; M</td>
			<td>Blackrock&#160; Microsystems, Salt Lake City, UT</td>
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			<td>Digital headstage</td>
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			<td height="40" style="height:40px;">Cereplex Direct&#160;</td>
			<td>Blackrock&#160; Microsystems, Salt Lake City, UT</td>
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			<td>Data acquisition system</td>
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			<td height="40" style="height:40px;">UEW polyurethane magnet wire</td>
			<td>Oyaide.com, Tokyo, Japan</td>
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			<td>Enamel wire</td>
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			<td height="40" style="height:40px;">SD-102</td>
			<td>Narishige, Tokyo, Japan</td>
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			<td>Minitor Co.,Ltd, Tokyo, Japan</td>
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			<td>High-peed drill Power Supply&#160;</td>
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			<td>Shofu Inc., Kyoto, Japan</td>
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			<td style="width:331px;">Dental cement</td>
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			<td>Biomachinery, Chiba, Japan</td>
			<td>TK-7</td>
			<td>Anesthetizer&#160;</td>
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			<td>Sigma-Aldrich, St. Louis, MO</td>
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			<td>Analgesic</td>
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			<td height="40" style="height:40px;">Isoflurane</td>
			<td>DS Pharma Animal Health, Osaka, Japan&#160;</td>
			<td>Isoflu 250mL</td>
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			<td height="40" style="height:40px;">Vaseline, White&#160;</td>
			<td>Wako Pure Chemical Industries, Ltd., Osaka, Japan</td>
			<td>224-00165&#160;</td>
			<td>Vet ointment&#160;</td>
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			<td height="40" style="height:40px;">&#160;Sodium alginate</td>
			<td>Nacalai tesque, Kyoto, Japan</td>
			<td>31131-85</td>
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			<td height="40" style="height:40px;">Calcium Chloride Dihydrate</td>
			<td>Wako Pure Chemical Industries, Ltd., Osaka, Japan</td>
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			<td>MonotaRO, Hyogo, Japan</td>
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			<td>Stainless steel screw for BR electrodes</td>
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			<td height="40" style="height:40px;width:307px;">Stainless steel screw M1.4&#215;3.0</td>
			<td>MonotaRO, Hyogo, Japan</td>
			<td>42617687</td>
			<td>Stainless steel screw for g/r electrodes and anchors</td>
		</tr>
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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 method can simultaneously capture bioelectrical signals from multiple organs that represent the neuronal activity of the brain, heart rate, breathing rhythm, and skeletal muscle contractions (Figure 1). Figure 4 provides representative recording data from a freely moving rat that was freely foraging in a rectangular box (25 &#215; 40 cm2). The example dataset includes typical behavioral transition...

Watch this Scientific Journal Video about Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat at JoVE.com

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

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 ...

simultaneous-recordings-cortical-local-field-potentials

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.8K Views.  The University of Tokyo. 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.

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

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

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 ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

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 ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

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

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

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 ...

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

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

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

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

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

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.

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 ...

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 ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

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

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Chapters in this video

Single-unit In vivo Recordings from the Optic Chiasm of Rat

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

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

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

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

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

Noninvasive EEG Recordings from Freely Moving Piglets

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

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo