We gratefully acknowledge support from the NIH (NS047073 to H.J.C.). We also are grateful to Catherine Kehl for her early experiments with Kwik-Sil, which encouraged us to use it for our application.

1. Electrode Fabrication
<ol>
<li>To create the electrode, use scissors to cut a piece of enamel-coated 0.001 inch diameter stainless steel wire about 2 feet long. Attach a small ball of putty to each end of the wire, fold the wire in half, and spin the ends to create a twisted pair, called a differential electrode.</li>
<li> Tape both ends of the wire to prevent unraveling. Make four differential electrodes, using a different color of tape for each differential electrode for identification purposes (Figure 1A).</li>
<li> Twist two of the differential electrodes together, leaving two inches untwisted on one end and 1 inch on the other end. Repeat with the remaining pair of differential electrodes (Figure 1B).</li>
<li> Twist both sets of two differential electrodes together, making sure that the ends remain untwisted. Hold up the electrode assembly and apply household silicone glue at the junction of the four differential electrodes, leaving the ends free. Hang and allow the glue to dry for at least 4 hours (Figure 1C).</li>
<li> To insulate the branches, lay the electrode assembly down on a flat, movable, surface such a piece of wood. Fan out the ends and secure them with tape. Use a small piece of plastic to elevate the electrode assembly just before it branches, and secure that with tape.</li>
<li> At a dissecting microscope, use forceps to coat the branches with silicone glue. The 1-inch long ends will be inside the animal, so they should have a very thin coat of glue. Allow the glue to dry for at least 3 hours.</li>
<li> Shorten the 1-inch long end of one differential electrode to two centimeters. On each wire, remove 1cm of silicone and enamel. Curl one bare wire into a hook for recording. Bend the other wire up and away from the hook; this will serve as the ground wire. Repeat for the remaining three electrodes.</li>
<li> Select one of the four electrodes on the 2 inch end. Use forceps to scrape silicone and enamel off, exposing 1cm of bare stainless steel wire on each of the 2 wires. Solder a gold connector to each of the two wires. Use a piece of lab tape to secure the end and maintain a separation between the ground and recording pins. Repeat for the remaining three electrodes.
</li>
<li> Attach a 1-2cm ball of the silicone glue to the cable, about 1.5 inches from the hook end to anchor the electrodes within the animal. Allow to dry for at least one hour (Figure 1D). <img src="/files/ftp_upload/1791/1791fig1.jpg" alt="Figure 1" /> Figure 1. Electrode Assembly Fabrication. (A) Single differential electrode consisting of a folded and twisted wire with its ends secured with tape. (B) Two differential electrodes twisted together, leaving untwisted space at the ends. Different colored tape or another marking should be used to identify the ends. (C) All four differential electrodes combined into a single electrode assembly forming a single cable with separate branches of the individual electrodes on each end, marked with distinct colors of tape. (D) Final form of electrode assembly. On the left, there are curled hooks for recordings and ground wires for each differential electrode. On the right, gold connector pins have been soldered to the wires for connection to the amplifier. An anchoring ball of silicone glue has also been placed towards the left side of the assembly.</li>
</ol>
2. Preparation of Animal For Implantation
<ol>
<li> Select a healthy animal that weighs about 350-450g. In response to small pieces of seaweed, the animal should generate bites at intervals of between 3 to 5 seconds. The complete surgery with electrode implantation should take about an hour.</li>
<li> To anesthetize the animal, inject it with 30% w/v isotonic MgCl2. To prevent the loss of hemolymph and MgCl2, the injection site can be coated in superglue before removing the needle. <img src="/files/ftp_upload/1791/1791fig2.jpg" alt="Figure 2" /> Figure 2. Schematic diagram showing how the animal is pinned for electrode implantation, and the location of the incision (indicated by a bar). </li>
<li> Lay the anesthetized animal out in a dissection tray with its foot down. Facing the animal head on, shift the gill area to your left such that the animal is partially on its side, leaning away. Pin down the anterior tentacles (Figure 2).</li>
<li> Place the tray on the stage of a dissecting microscope. Prop up the tray at about a 20 degree angle to elevate the head and collect the hemolymph in the tail, away from the incision.</li>
<li> Using dissection scissors, start an incision slightly to your right of the eyespot and extend it 1 to 1.5cm anteriorly. Use retractors attached to the edges of the tray to hold the incision open (Figure 2; note bar near eyespot indicating incision).</li>
<li> To expose the buccal mass, use forceps to lift the clear membrane underneath the skin and use scissors to cut an opening matching the main incision. Tuck the edges of the membrane into the retractors. </li>
</ol>
3. Electrode Implantation
<ol>
<li>Place the electrode assembly on a micromanipulator and move it over the animal's incision.</li>
<li> The radular nerve is most difficult to access, and should be approached first. There are two options for accessing the radular nerve.</li>
<li> The first option, which is preferable, is to look under the buccal ganglion between BN1 and BN2. If a branch of the radular nerve is visible, use fine-tipped forceps bent into a hook to lift out the branch (Figure 3, loop of RN under ganglion labeled 1).</li>
<li> If a branch of the radular nerve is not accessible above the muscle, a second option is to access it via a small incision in I2 next to the connective tissue at the base of the buccal ganglion (Figure 3, RN beneath muscle labeled 2). The incision in I2 must go through two layers of the I2 muscle. Hold onto the edge of the muscle incision with straight forceps and use the hooked forceps to pull out an RN branch.</li>
<li> While holding the nerve with the hooked forceps, use a different set of forceps to manipulate the electrode hook onto the nerve.</li>
<li> Use the micromanipulator to raise the hook so that the nerve is lifted up away from the buccal mass and clear of any other nerves or tissue. Do not overstretch the nerve.</li>
<li> Dry the electrode using a Kim-Wipe or suction off the excess fluid with a syringe. Water tension can cause the nerve to collapse on itself, or a water seal to form under the nerve. In either case, the area should be completely dried before glue is applied.</li>
<li> Apply a small dab of superglue where the base of the hook is in contact with the bottom of the nerve. Apply a drop of Aplysia saline or hemolymph to set the superglue. When the glue is wet nothing should be moved as getting glue between the wire and nerve would block the electrical connection.</li>
<li> Dry the hook again. Mix Kwik-Sil glue on an ice pack.</li>
<li> Use the tip of a pin to apply a small amount of the Kwik-Sil glue, completely covering any exposed wire of the hook, but not the ground wire.</li>
<li> Allow the glue to set for about 5 minutes, readjusting the glue if it pulls away from sections of the hook. The glue is set when it no longer sticks when touched with the pin (Figure 4 shows the final configuration).</li>
<li> Once the radular nerve electrode is set, the I2 muscle can be accessed. In the area between BN1 and BN2, use the hooked forceps to hook and to separate 2 bands of the I2 muscle from the rest of the muscle by gently pulling the bands upwards. Attach the electrode using the same procedure.</li>
<li> Buccal nerves 2 and 3 are easily accessed and electrodes are attached with the same procedure.</li>
<li> Once all the electrodes are attached, slide the electrode assembly into the animal up to the silicone anchor. </li>
<li> Suture the incision closed around the protruding electrode cable, keeping the anchor inside the animal. Superglue the stitches to ensure a good seal.</li>
<li> Insert the gold connector tips at the other end of the electrode into a small block of Styrofoam. Return the animal to the main tank in an aerated isolation container for recovery overnight.</li>
</ol>
<img src="/files/ftp_upload/1791/1791fig3.jpg" alt="Figure 3" /> Figure 3. A diagram of the Aplysia feeding apparatus known as the buccal mass. Shown are the buccal nerves (BN1, BN2, BN3), the radular nerve (RN) that exits the bottom of the buccal ganglion, then plunges beneath the muscle, and the location on the I2 muscle where the electrode is attached.
<img src="/files/ftp_upload/1791/1791fig4.jpg" alt="Figure 4" /> Figure 4. Schematic diagram of single electrode attached to a nerve (or muscle) and glued in place. Note that the de-insulated region of the curled hook is in direct contact with the nerve (or muscle), anchored in place with superglue, and electrically insulated from the fluid medium using Kwik-Sil. Note also that the deinsulated tip of the ground electrode is exposed to the fluid medium.
4. Recording
<ol>
<li> Acclimate the animal in an aerated container while attached to the recording equipment for at least 1.5 hours. Monitor the signal while the animal is acclimating to verify that all electrodes are working and that noise levels are low.</li>
<li> Use a video camera to record feeding behavior while using the computer to record signals from the neural and muscular electrodes. Synchronize the video and electrode recordings using a square wave signal that controls a digital counter in the camera view while it is simultaneously recorded as an additional channel on the computer in Axoscope.</li>
<li> Feeding behaviors are elicited by the techniques described in Morton and Chiel 1993.</li>
</ol>
Although invertebrate animals do not require formal approval by an institutional animal use and care committee, we have ensured that all treatments of Aplysia minimize harm and suffering to the animal, and that all surgical techniques are done while the animal is fully anesthetized.
Representative Results
<img src="/files/ftp_upload/1791/1791fig5.jpg" alt="Figure 5" /> 
Figure 5. (A) Simultaneous four channel recordings from a muscle (the protractor muscle I2), and from three buccal nerves (the radular nerve, RN, buccal nerve 2, BN2, and buccal nerve 3, BN3) during a swallowing response. (B) A still frame from the simultaneous video recording of the freely behaving animal as it swallows a precut seaweed strip (0.5 cm in width), which has been marked at 0.5 cm intervals with white bands so that its movements can be readily visualized as the animal feeds.

<ol>
	<li>Lu, H., Chestek, C. A., Shaw, K. M., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+extracellular+stimulation+of+individual+neurons+in+ganglia.">Selective extracellular stimulation of individual neurons in ganglia.</a> J. Neural Eng. 5, 287-309 (2008).</li><li>Morton, D. W., Chiel, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+buccal+nerve+activity+that+distinguishes+ingestion+from+rejection+can+be+used+to+predict+behavioral+transitions+in+Aplysia.">In vivo buccal nerve activity that distinguishes ingestion from rejection can be used to predict behavioral transitions in Aplysia.</a> J Comp. Physiol. A. 172, 17-32 (1993).</li><li>Warman, E. N., Chiel, H. 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+new+technique+for+chronic+single+extracellular+recording+in+freely+behaving+animals+using+pipette+electrodes.">A new technique for chronic single extracellular recording in freely behaving animals using pipette electrodes.</a> J. Neurosci. Methods. 57, 161-169 (1995).</li><li>Ye, H., Morton, D. W., Chiel, H. J., J, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nbsp;Neuromechanics+of+multifunctionality+during+rejection+in+Aplysia+californica.">nbsp;Neuromechanics of multifunctionality during rejection in Aplysia californica.</a> J. Neurosci. 26, 10743-10755 (2006).</li></ol>

A key innovation that has made these recordings successful is combining the electrodes into one cable. Two electrode recordings have been used before, but expanding to four individual electrodes resulted in the formation of surgical adhesions that often prevented the animal from making normal feeding movements. Combining the electrodes into one cable reduces the bulk of the electrodes inside the animal, which in turn reduces the immune response and allows the animal to move more freely.
The o...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Enamel coated stainless steel wire</td>
<td>&nbsp;</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>0.001D, coating h</td>
</tr>
<tr>
<td>Household Silicone II Glue</td>
<td>&nbsp;</td>
<td>GE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kwik-Sil Adhesive</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Duro Qwik-Gel superglue</td>
<td>&nbsp;</td>
<td>Henkel corp.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>A-M Systems model 1700 amplifier</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>&nbsp;</td>
<td>Filter settings:100-1000Hz nerves,10-1000Hz I2 muscle</td>
</tr>
<tr>
<td>Axoscope 10.1</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Gold Connector Pins</td>
<td>&nbsp;</td>
<td>Bulgin</td>
<td>SA3148/1</td>
<td>&nbsp;</td>
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<td>Gold Connector Sockets</td>
<td>&nbsp;</td>
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electrode fabrication and implantation in aplysia californica for multi-channel neural and muscular recordings in intact, freely behaving animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Watch this Scientific Journal Video about Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals at JoVE.com

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

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13.0K Views.  Case Western Reserve University. A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Video: Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

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 ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

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 ...